Iimfundiso ezihlukahlukeneko zokuvikela iintjalo zinikela ngesinqophiso esiqakathekileko sokuhlathulula amaphetheni wokugaya ukudla kweentjalo, kodwana ukubikezela kwazo okuqakathekileko kusazokuhlolwa. Lapha, sisebenzise ukuhlaziywa kwe-tandem mass spectrometry (MS/MS) ukuhlola ngendlela ehlelekileko i-metabolome yeentjalo ezibuthakathaka zegwayi ukusuka eentjalweni ngazinye ukuya emihlobeni kanye nemihlobo ehlobene khulu, begodu sasebenza inani elikhulu leemfundiso ze-mass spectrometric ezisekelwe kuma-spectra we-compound elwazini. Ihlaka lokuhlola ukubikezela okuqakathekileko kwethiyori yokuzivikela okuhle (OD) kanye nethagethi ehambako (MT). Ilwazi le-metabolomics yeentjalo lihambisana nethiyori ye-OD, kodwana liphikisana nokubikezela okukhulu kwethiyori ye-MT malungana nokuguquguquka kwe-metabolomics okubangelwa ziinlwana ezidla iintjalo. Ukusuka esikalini esincani ukuya esikalini esikhulu, itshwayo le-jasmonate lakhonjwa njengesiqunto esikhulu se-OD, ngesikhathi itshwayo le-ethylene linikela ngokulungiswa okuhle kwempendulo ekhethekileko ye-herbivore ebhalwe yi-MS/MS molecular network.
Ama-metabolites akhethekileko anezakhiwo ezihlukahlukeneko ngiwo ahlanganyela khulu ekutjhugulukeni kweentjalo ebhodulukweni, khulukhulu ekuvikeleni izitha (1). Ukuhlukahluka okukarisako kwe-metabolism ekhethekileko etholakala eentjalweni kukhuthaze amatjhumi weminyaka yokurhubhulula okujulileko ngezinto ezinengi eziphathelene nemisebenzi yemvelo, begodu kwakha irhelo elide leemfundiso zokuvikela iintjalo, okungukuthi ukuziphendukela kwemvelo nokuthuthukiswa kwemvelo kokusebenzisana kweentjalo nezinambuzane. Irhubhululo elihlolwako linikela isiqondiso esiqakathekileko (2). Kodwana, iimfundiso zokuvikela iintjalo azange zilandele indlela ejwayelekileko yokucabanga okucatshangelwako, lapho ukubikezela okuqakathekileko bekusezingeni elifanako lokuhlaziywa (3) begodu kwahlolwa ngokulinga ukuthuthukisa umjikelezo olandelako wokuthuthukiswa kwethiyori (4). Ukulinganiselwa kwezobuchwepheshe kukhawulela ukubuthelelwa kwedatha emikhakheni ethileko ye-metabolic begodu kungafaki ukuhlaziywa okupheleleko kwama-metabolites akhethekileko, ngaleyo ndlela kuvimbele ukumadanisa hlangana kwemikhakha okuqakathekileko ekuthuthukisweni kwethiyori (5). Ukutlhogeka kwedatha epheleleko ye-metabolomics kanye nemali ejayelekileko yokumadanisa ukugeleza komsebenzi wendawo ye-metabolic hlangana namaqembu weentjalo ahlukileko kuphazamisa ukuvuthwa kwezesayensi kwensimu.
Iintuthuko zakamuva emkhakheni we-tandem mass spectrometry (MS/MS) metabolomics zingahlathulula ngokuzeleko amatjhuguluko we-metabolic ngaphakathi naphakathi kwemihlobo yehlelo elithileko, begodu zingahlanganiswa neendlela zokubala ukubala ukufana kwesakhiwo hlangana nemihlanganisela eyinkimbinkimbi. Ilwazi langaphambilini lekhemistri (5). Ukuhlanganiswa kwethekhnoloji ethuthukileko ekuhlaziyeni nekhomphyutheni kunikela ihlaka elitlhogekako lokuhlolwa kwesikhathi eside kweenbikezelo ezinengi ezenziwe ziimfundiso zemvelo nezemvelo zokuhlukahluka kwe-metabolic. UShannon (6) wethula ithiyori yelwazi kokuthoma esihlokweni sakhe esiqakathekileko ngo-1948, wabeka isisekelo sokuhlaziywa kwelwazi ngeembalo, ebegade isetjenziswe emikhakheni eminengi ngaphandle kokusetjenziswa kwayo kwasekuthomeni. Ku-genomics, ithiyori yelwazi isetjenziswe ngepumelelo ukulinganisa ilwazi elilondoloziweko lokulandelana (7). Ecwaningweni le-transcriptomics, itiyori yelwazi ihlaziya amatjhuguluko woke we-transcriptome (8). Ecwaningweni elidlulileko, sisebenzise ihlelo leembalo lethiyori yelwazi ku-metabolomics ukuhlathulula ubuchwepheshe be-metabolic bezinga lezicubu eentjalweni (9). Lapha, sihlanganisa ukugeleza komsebenzi okusekelwe ku-MS/MS nohlaka lwezibalo lwethiyori yelwazi, ebonakala ngokuhlukahluka kwe-metabolic ngemali evamileko, ukumadanisa ukubikezela okuqakathekileko kwethiyori yokuvikela iintjalo ye-metabolome ebangelwa ziinlwana ezidla utjani.
Iinhlaka zethiyori zokuzivikela kweentjalo zivamise ukufaka hlangana begodu zingahlukaniswa zibe ziinkoro ezimbili: lezo ezilinga ukuhlathulula ukusabalaliswa kwama-metabolites akhethekileko weentjalo ngokuya ngemisebenzi yokuzivikela, njengokuzivikela okuhle (OD) (10), umnqopho ohambako (MT) (11) ) Nethiyori yokubukeka (12), lokha abanye bafuna ukuhlathulula ukuthi ukutholakala kwemithombo kuthinta njani ukukhula kweentjalo kanye nokubuthelela kwezinto ezikhethekileko, ezifana nekhabhoni: umbono wokulinganisa izakhamzimba (13), umbono wezinga lokukhula (14 ), kanye nombono wokulinganisa ukukhula nokuhlukana (15). Amasethi amabili wemibono asezingeni elihlukileko lokuhlaziywa (4). Kodwana, iimfundiso ezimbili ezibandakanya imisebenzi yokuzivikela ezingeni lokusebenza zibusa ingcoco malungana nokuzivikela kweentjalo: ithiyori ye-OD, ecabanga bona iintjalo zitjala imali ekuzivikeleni kwazo kwamakhemikhali okubizako kwaphela lokha nazitlhogekako, isibonelo, lokha nazidliwa Lokha isilwana sotjani nasihlaselako, ngalokho-ke, ngokuya ngethuba lokuhlaselwa kwesikhathi esizako, umsebenzi wokuzivikela uklanyelwe (10); umbono we-MT uphakamisa bona ayikho i-eksisi yokutjhuguluka kwe-metabolite, kodwana i-metabolite itjhuguluka ngokungakahleleki, lokho kwenza bona kube nethuba lokuvimbela "umnqopho wokunyakaza" wokuhlasela iinlwana ezidla utjani. Ngamanye amagama, imibono emibili le yenza ukubikezela okuphikisanako ngokulungiswa kabutjha kwe-metabolic okwenzeka ngemva kokuhlaselwa ziinlwana ezidla utjani: ubudlelwano hlangana nokubuthelela kwe-metabolites okuqondileko ngomsebenzi wokuzivikela (OD) kanye nokutjhuguluka kwe-metabolic okungakaqondiswa (MT) (11 ).
Imibono ye-OD ne-MT ayihlanganisi amatjhuguluko abangelwa ku-metabolome kwaphela, kodwana nemiphumela ye-ekholoji neyokuziphendukela kwemvelo yokubuthelela ama-metabolites la, njengeendleko zokutjhuguluka kanye neenzuzo zamatjhuguluko we-metabolic endaweni ethile ye-ekholoji (16). Nanyana zombili iimcabango ziqaphela umsebenzi wokuzivikela wama-metabolites akhethekileko, okungabiza namkha kungabizi, ukubikezela okuqakathekileko okuhlukanisa i-OD ne-MT hypotheses kulele ekuqondisweni kwamatjhuguluko we-metabolic abangelwako. Ukubikezela kwethiyori ye-OD kuthole ukutjhejwa okukhulu khulu bekube nje. Iimhloliso lezi zifaka hlangana ukufunda imisebenzi yokuzivikela eqondileko nofana engaqondileko yezicubu ezihlukahlukeneko zamakhompawundi athileko ema-greenhouse kanye nobujamo bemvelo, kanye namatjhuguluko esigabeni sokukhula kweentjalo (17-19). Kodwana, bekube nje, ngebanga lokutlhogeka kokugeleza komsebenzi kanye nohlaka lwezibalo lokuhlaziywa okupheleleko kwephasi loke kokuhlukahluka kwe-metabolic yanoma ngisiphi isilwana, ukubikezela umehluko omkhulu hlangana namathiyori amabili (okutjho, indlela yokutjhuguluka kwe-metabolic) kusazokuhlolwa. Lapha, sinikela ngokuhlaziywa okunjalo.
Olunye lwezinto eziqakatheke khulu zama-metabolites akhethekileko weentjalo kukuhlukahluka kwawo okukhulu kwesakhiwo kiwo woke amazinga ukusuka eentjalweni ezizodwa, iintjhaba ukuya emihlobeni efanako (20). Amatjhuguluko amanengi wobunengi ema-metabolites akhethekileko angabonwa esikalini senani labantu, ngesikhathi umehluko oqinileko wekhwalithi uvame ukugcinwa ezingeni lemihlobo (20). Ngalokho-ke, ukuhlukahluka kwe-metabolic yeentjalo yinto ekulu yokuhlukahluka kokusebenza, okutjengisa ukutjhuguluka eendaweni ezihlukileko, khulukhulu lezo ndawo ezinamathuba ahlukileko wokuhlaselwa ziinlwanyana ezikhethekileko kanye nezilwane ezidla utjani ezijayelekileko (21). Solo kwango-Athikili ka-Fraenkel (22) eqakathekileko malungana neenzathu zokuba khona kwama-metabolites akhethekileko weentjalo, ukusebenzisana neenunwana ezihlukahlukeneko bekuthathwa njengeengcindezi eziqakathekileko zokukhetha, begodu ukusebenzisana lokhu kukholelwa bona kwabumba iintjalo ngesikhathi sokuziphendukela kwemvelo. Indlela yokugaya ukudla (23). Umehluko phakathi kwemihlobo ehlukileko yama-metabolites akhethekileko kungatjengisa ukulingana komzimba okuhlobene nokuzivikela kweentjalo okubunjwako nokukhuthazwako emaqhingeni wokudla utjani, njengombana imihlobo emibili le ivamise ukuhlobana kumbi (24). Nanyana kungaba lisizo ukugcina ukuzivikela okuhle ngaso soke isikhathi, amatjhuguluko we-metabolic ngesikhathi esifaneleko ahlotjaniswa nokuzivikela anikela ngeenzuzo ezicacileko ekuvumeleni iintjalo bona zabele iinsetjenziswa eziqakathekileko kezinye iinsetjenziswa zomzimba (19, 24), begodu zibalekele isidingo sokusebenzisana. Umonakalo wesibambiso (25). Ukungezelela, ukuhlelwa kabutjha kwama-metabolites akhethekileko abangelwa ziinlwanyana ezidla utjani kungabangela ukusabalala okubhubhisako esiqhemeni (26), begodu kungatjengisa ukufundwa okunqophileko kwamatjhuguluko wemvelo amakhulu esiginali ye-jasmonic acid (JA), engagcinwa esiqhemeni. Amatshwayo we-JA aphezulu naphasi kuthengiselana hlangana nokuzivikela eenlwaneni ezidla utjani kanye nokuncintisana nemihlobo ethileko (27). Ukungezelela, iindlela ezikhethekileko zokwenza i-metabolite zizokulahleka msinyana begodu zitjhuguluke ngesikhathi sokuziphendukela kwemvelo, okuzokwenza bona kube nokusabalala okungakalungi kwe-metabolic hlangana nemihlobo ehlobene khulu (28). Ama-polymorphism la angasungulwa msinyana ngokuphendula ukutjhuguluka kwamaphetheni weenlwana ezidla iintjalo (29), okutjho bona ukutjhuguluka kwemiphakathi yeenlwana ezidla iintjalo kuyinto eqakathekileko etjhukumisa ukuhlukahluka kwe-metabolic.
Lapha, sirarulule imiraro elandelako. (I) Isinambuzane esidla utjani siyihlela njani kabutjha i-metabolome yesitjalo? (Ii) Ngiziphi iingcenye ezikulu zolwazi lokusebenza kwe-metabolic okungalinganiswa ukuhlola ukubikezela kwethiyori yokuzivikela yesikhathi eside? (Iii) Ukuthi kufanele na ukuhlela kabutjha i-metabolome yesitjalo ngendlela ehlukileko kumhlaseli, nangabe kunjalo, ngiyiphi indima edlalwa yi-hormone yesitjalo ekwenzeni ukuphendula okuthileko kwe-metabolic, begodu ngiziphi i-metabolites ezifaka isandla ekuzivikeleni kwemihlobo ekhethekileko? (Iv) Njengombana ukubikezela okwenziwe ziimfundiso ezinengi zokuzivikela kungadluliselwa kiwo woke amazinga wezicubu zezinto eziphilako, sibuze bona ukuphendula kwe-metabolic okubangelwa kuyafana kangangani ukusuka ekumadaniseni kwangaphakathi ukuya ekumadaniseni hlangana nemihlobo? Ukuze sifikelele lokhu, sifunde ngendlela ehlelekileko i-metabolome yamakari we-tobacco nicotine, okuyisitjalo esiyimodeli ye-ekholoji ene-metabolism ekhethekileko, begodu isebenza kuhle malungana nama-larvae weenlwanyana ezimbili ezidla utjani, i-Lepidoptera Datura (Ms) ( Inolaka khulu, edliwa khulu) Ku-Solanaceae kanye ne-Spodopteral (Storalist), mhlobo wama-leaf worm "umhlobo", eneentjalo ezibambako ze-Solanaceae nezinye iinlwana zeminye imihlobo nemindeni Ukudla kweentjalo. Sihlaziye i-MS/MS metabolomics spectrum begodu sikhiphe iinhlathululo zeembalo zethiyori yolwazi ukumadanisa ithiyori ye-OD ne-MT. Yenza ibalazwe elikhethekileko ukuveza ubunikazi bama-metabolites aqakathekileko. Ukuhlaziywa kwadluliselwa ebantwini bendabuko be-N. nasi kanye nemihlobo yegwayi ehlobene khulu ukuragela phambili nokuhlaziywa kokuhlukahluka hlangana nokuvezwa kwamahomoni weentjalo nokufakwa kwe-OD.
Ukuze sithathe imephu epheleleko nge-plasticity nesakhiwo se-metabolome yamakari wegwayi elidla iintjalo, sisebenzise ukuhlaziywa okwakhiwe ngaphambilini nokusebenza kokubala ukubuthelela ngokupheleleko nokuhlukanisa idatha ephezulu ye-MS/MS spectra evela eentjalweni ( 9). Indlela engahlukaniswako (ebizwa ngokuthi yi-MS/MS) ingakha ama-spectra wekhompawundi angatlhogekiko, angasetjenziselwa koke ukuhlaziywa kwezinga lekhompawundi okuchazwe lapha. Ama-metabolites weentjalo ahlukanisiweko ngemihlobo ehlukileko, ahlanganisa amakhulu ukuya eenkulungwaneni zama-metabolites (ama-500-1000-s/MS/MS lapha). Lapha, sicabangela iplastiki ye-metabolic ngohlaka lwethiyori yelwazi, begodu silinganise ukuhlukahluka nobuchwepheshe be-metabolome ngokuya nge-Shannon entropy yokusabalalisa imvamisa ye-metabolic. Ngokusebenzisa ifomula esetjenziswe ngaphambilini (8), sibale isethi yeenkomba ezingasetjenziselwa ukulinganisa ukuhlukahluka kwe-metabolome (inkomba ye-Hj), ukukhetheka kwephrofayili ye-metabolic (inkomba ye-δj) kanye nokukhethekileko kwe-metabolic ye-metabolite eyodwa (inkomba ye-Si). Ukungezelela, sisebenzise i-Relative Distance Plasticity Index (RDPI) ukulinganisa ukuthonywa kwe-metabolome yeenlwana ezidla utjani (Umfanekiso 1A) (30). Ngaphakathi kohlaka lwezibalo, siphatha i-MS/MS spectrum njengeyunithi yolwazi oluyisisekelo, begodu sisebenza ubunengi be-MS/MS ibe yimephu yokusabalalisa imvamisa, bese sisebenzisa i-Shannon entropy ukulinganisa ukuhlukahluka kwe-metabolome kiyo. Ukukhetheka kwe-metabolome kulinganiswa ngobukhulu obuphakathi be-MS/MS spectrum eyodwa. Ngalokho-ke, ukwanda kobunengi bamaklasi we-MS/MS ngemva kokufakwa kwe-herbivore kutjhugululwa kube yi-spectral inducibility, i-RDPI kanye nokukhethekileko, okutjho bona, ukwanda kwe-δj index, ngombana kukhiqizwa ama-metabolites akhethekileko begodu kukhiqizwa inkomba ephezulu ye-Si. Ukwehliswa kwenkomba yokuhlukahluka kwe-Hj kutjengisa bona inani le-MS/MS eyakhiweko liyancipha, nofana ukusabalala kwefrikhwensi yephrofayili kutjhuguluka ngendlela engafaniko, ngesikhathi kunciphisa ukungaqiniseki kwayo koke. Ngokubala i-Si index, kungenzeka ukuveza bona ngiyiphi i-MS/MS ebangelwa ziinlwana ezidla utjani, ngokuphambene nalokho, ngiyiphi i-MS/MS engaphenduli ekungenisweni, okuyinkomba eqakathekileko yokuhlukanisa ukubikezela kwe-MT ne-OD.
(A) Iinhlathululo zezibalo ezisetjenziselwa ukudla iintjalo (H1 ukuya ku-Hx) MS/MS idatha-inducibility (RDPI), ukuhlukahluka (Hj index), ukukhethekile (δj index) kanye metabolite specificity (Si index). Ukunyuka kwezinga lokukhethekileko (δj) kutjengisa bona, ngokwesilinganiso, kuzokukhiqizwa ama-metabolites amanengi adla iintjalo, ngesikhathi ukwehla kokuhlukahluka (Hj) kutjengisa ukwehla kokukhiqizwa kwama-metabolites nofana ukusabalala okungalingani kwama-metabolites kumephu yokusabalalisa. Inani le-Si lihlola bona ingabe i-metabolite iqondene nobujamo obuthileko (lapha, edla utjani) nofana igcinwe ezingeni elifanako. (B) Umdwebo womqondo wokubikezela kwethiyori yokuzivikela ngokusebenzisa i-eksisi yethiyori yolwazi. Ithiyori ye-OD ibikezela bona ukuhlaselwa kweenlwana ezidla iintjalo kuzokwandisa ama-metabolites wokuzivikela, ngaleyindlela kukhuphule i-δj. Ngesikhathi esifanako, i-Hj yehla ngombana iphrofayili ihlelwe kabutjha ukuya ekunciphiseni ukungaqiniseki kwelwazi lokugaya ukudla. Ithiyori ye-MT ibikezela bona ukuhlaselwa kweenlwana ezidla utjani kuzokubangela amatjhuguluko angasiwo aqondileko ku-metabolome, ngaleyo ndlela kukhulise i-Hj njengesitjengiso sokungaqiniseki kwelwazi lokugaya ukudla begodu kubangele ukusabalala okungakahleleki kwe-Si. Siphinde saphakamisa imodeli ehlangeneko, i-MT engcono khulu, lapho amanye ama-metabolites anamazinga aphezulu wokuzivikela azokukhuphuka khulu (inani eliphezulu le-Si), lokha amanye atjengisa iimpendulo ezingakahleleki (inani eliphasi le-Si).
Ngokusebenzisa iinhlathululi zethiyori yolwazi, sihlathulula ithiyori ye-OD ukubikezela bona amatjhuguluko we-metabolite akhethekileko abangelwa ziinlwana ezidla iintjalo ebujameni obungakaqalwa azokurholela (i) ekukhuphukeni kwe-metabolic specificity (Si index) eqhuba i-metabonomic specificity (δj index) Ukwanda) kwamaqembu athileko we-metabolite akhethekileko anenani eliphezulu lokuzivikela, kanye (ii) nokwehla kwe-metabolite (Hj) ukutjhuguluka kokusabalalisa imvamisa yokugaya ukudla ukuya ekusabalalisweni okukhulu komzimba we-leptin. Ezingeni le-metabolite eyodwa, ukusabalala okuhlelekileko kwe-Si kulindelekile, lapho i-metabolite izokukhuphula inani le-Si ngokuya ngenani layo lokuzivikela (Umfanekiso 1B). Ngalendlela, sihlathulula ithiyori ye-MT ukubikezela bona ukuthaba kuzokurholela (i) ematjhugulukweni angasiwo aqondileko kuma-metabolites azokurholela ekwehleni kwe-δj index, kanye (ii) nokwanda kwe-Hj index ngebanga lokukhuphuka kokungaqiniseki kwe-metabolic. Nofana ukungahleleki, okungalinganiswa nge-Shannon entropy ngendlela yokuhlukahluka okujayelekileko. Malungana nokwakheka kwe-metabolic, ithiyori ye-MT izokubikezela ukusabalala okungakahleleki kwe-Si. Nasitjheja bona amanye ama-metabolite angaphasi kwemibandela ethileko ngaphasi kwemibandela ethileko, begodu eminye imibandela ayikho ngaphasi kwemibandela ethileko, begodu inani lawo lokuzivikela liya ngobujamo obuthileko, sibuye saphakamisa imodeli yokuzivikela ehlangeneko, lapho i-δj ne-Hj basabalaliswe kabili eceleni kwe-Si Increase kizo zoke iinqophiso, amaqembu athileko we-metabolite kwaphela, anamanani wokuzivikela aphezulu, azokukhuphuka khulukhulu lokha amanye ama-Siure azokuba nokusabalalisa okungakahleleki (Umfanekiso a 1B).
Ukuze sihlole ukubikezela kwethiyori yokuzivikela ehlathululwe kabutjha ku-eksisi yomchazi wethiyori yolwazi, siphakamise iinbungu ezidla iintjalo ezidla iintjalo (uMs) nofana ezijayelekileko (Sl) emaqabungeni we-Nepenthes pallens (Umfanekiso 2A). Ngokusebenzisa ukuhlaziywa kwe-MS/MS, sibuyise ama-599 we-MS/MS spectra angasidingeki (ifayela ledatha S1) ukusuka eentweni ze-methanol zezicubu zamakari ezibuthelwe ngemva kokudla isibungu. Ukusebenzisa iinkomba ze-RDPI, Hj, kanye ne-δj ukubona ngeso lengqondo ukuhlelwa kabutjha kokuqukethwe kwelwazi kumafayela wokuhlela we-MS/MS kuveza amaphethini athakazelisako (Umfanekiso 2B). Umkhuba jikelele kukobana, njengombana kuchaziwe mhlathululi welwazi, njengombana amabhungane aragela phambili nokudla amakari, izinga lokuhlelwa kabutjha kwe-metabolic liyakhula ngokukhamba kwesikhathi: ama-awa ama-72 ngemva kokuthi isilwane esidla utjani sidlile, i-RDPI ikhuphuka khulu. Nayiqathaniswa nokulawula okungalimalanga, i-Hj yehliswe khulu, okwabangelwa kukhuphuka kwezinga lokukhetheka kwephrofayili ye-metabolic, eyalinganiswa nge-δj index. Umkhuba lo obonakalako uyahambisana nokubikezela kwethiyori ye-OD, kodwana akuhambisani nokubikezela okukhulu kwethiyori ye-MT, ekholelwa bona amatjhuguluko angahlelekile (ongakaqondi) emazingeni we-metabolite asetjenziswa njengendlela yokuzivikela (Umfanekiso 1B). Nanyana ukukhiqizwa komlomo (OS) nokuqukethwe kokudla kweenlwana ezimbili ezidla utjani kuhlukile, ukudla kwazo okunqophileko kwabangela amatjhuguluko afanako eenqophisweni ze-Hj ne-δj ngesikhathi sokuvuna sama-awa ama-24 kanye nama-awa ama-72. Umehluko wenzeka emahoreni ama-72 we-RDPI. Nakuqathaniswa nalokho okubangelwa kukudla kwe-Ms, ukugaya ukudla okupheleleko okubangelwa kukudla kwe-Sl bekuphezulu.
(A) Umklamo wokuhlola: iimfarigi ezijayelekileko (S1) nofana ingcwethi (Ms) ezidla utjani ziphakelwa ngamakari ahlanjululweko weentjalo ze-pitcher, kuthi ekudleni utjani okulingisiweko, i-OS ye-Ms (W + OSMs) isetjenziselwa ukusingatha ukubhoboza kwesilonda samaqabunga ajwayelekileko. S1 (W + OSSl) izibungu nofana amanzi (W + W). Isilawuli (C) liqabunga elingakalimazi. (B) Ukuthonywa (i-RDPI nayiqathaniswa netjhadi lokulawula), ukuhlukahluka (inkomba ye-Hj) kanye nenkomba yokukhethekileko (inkomba ye-δj) ebalwe imephu ekhethekileko ye-metabolite (599 MS/MS; ifayela ledatha S1). Iinkwekwezi zitjengisa umehluko oqakathekileko hlangana nokudla okuqondileko kweenlwana ezidla iintjalo kanye nesiqhema esilawulako (I-t-test yomfundi ene-t-test ehlangeneko, *P<0.05 kanye ne-***P<0.001). ns, akukaqakatheki. (C) Inkomba yokusombulula isikhathi ye-main (ibhokisi eliluhlaza okwesibhakabhaka, i-amino acid, i-organic acid netjhukela; ifayela ledatha S2) kanye ne-metabolite spectrum ekhethekileko (ibhokisi elibomvu 443 MS/MS; ifayela ledatha S1) ngemva kokwelatjhwa kwe-herbivory. Ibhendi yombala ibhekisela esikhathini sokuqiniseka esingu-95%. Inkanyezi itjengisa umehluko oqakathekileko hlangana nokwelatjhwa nokulawula [ukuhlaziywa kwe-quadratic of variance (ANOVA), kulandele umehluko oqakathekileko ka-Tukey (HSD) wokumadanisa okunengi ngemva kwesikhathi, *P<0.05, **P<0.01 Begodu *** P <0.001]. (D) Ukukhetheka kwama-scatter plots kanye namaphrofayili akhethekileko we-metabolite (amasampula aphindaphindiweko anemithi ehlukileko).
Ukuhlola bona ukulungiswa kabutjha okubangelwa ziinlwana ezidla iintjalo ezingeni le-metabolome kubonakala na ematjhugulukweni wezinga lama-metabolites ngamunye, sathoma ngokudzimelela ema-metabolites afundwe ngaphambilini emakarini we-Nepenthes pallens ngokujamelana ne-herbivore. Ama-phenolic amides ziinhlanganisela ze-hydroxycinnamamide-polyamine ezibuthelela ngesikhathi senqubo yokudla utjani wezinambuzane begodu zaziwa ngokuthi zinciphisa ukusebenza kwezinambuzane (32). Siphenye ama-precursors we-MS/MS ahambelanako begodu sadweba amajika wawo we-kinetic (Umfanekiso S1). Akumangalisi, ama-phenol derivatives angabandakanyeki ngqo ekuzivikeleni ezilwaneni ezidla utjani, njenge-chlorogenic acid (CGA) kanye ne-rutin, alawulwa phasi ngemva kokudla iintjalo. Ngokuphikisako, iinlwana ezidla utjani zingenza ama-phenol amides abe namandla khulu. Ukudla okuragela phambili kweenlwana ezimbili ezidla utjani kwabangela bona kube nokuvusa okufanako kwama-phenolamides, begodu iphetheni le beyibonakala khulu ekuhlanganisweni kwe-de novo yama-phenolamides. Isimanga esifanako sizokubonwa lokha nakuhlolwa indlela ye-17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), ekhiqiza inani elikhulu lama-acyclic diterpenes anemisebenzi ephumelelako yokulwisana nokudla kweentjalo (33), lapho uNksz Feeding nge-Sl abangela ukuvezwa okufanako (Umfanekiso S1)).
Ububi obungenzeka bokuhlolwa kokudla okuqondileko kweenlwana ezidla iintjalo ngumehluko wezinga lokusetjenziswa kwamakari kanye nesikhathi sokudla kweenlwana ezidla iintjalo, okwenza kube budisi ukuqeda imiphumela ekhethekileko yeenlwana ezidla iintjalo ezibangelwa ziinlonda neenlwana ezidla iintjalo. Ukuze sirarulule ngcono ukukhetheka kwemihlobo yeenlwana ezidla iintjalo zokuphendula kwe-metabolic yamakari, salingisa ukudla kwe-Ms ne-Sl larvae ngokusebenzisa msinyana i-OS esanda kubuthelelwa (OSM ne-OSS1) ekubhobozeni okujayelekileko kwe-W kwezikhundla zamakari ezingaguqukiko. Ikambiso le ibizwa ngokuthi yi-W + OS treatment, begodu yenza bona ukungeniswa kube sezingeni elifanako ngokuthi kubekwe isikhathi esifaneleko sokuthoma kwependulo ebangelwa silwane esidla utjani ngaphandle kokubangela imiphumela edidako yomehluko esilinganisweni nofana inani lokulahlekelwa zicubu (Umfanekiso 2A) (34). Ngokusebenzisa ukuhlaziywa kwe-MS/MS kanye nephayiphi yokubala, sibuyise ama-spectra we-MS/MS ama-443 (ifayela ledatha S1), ebegade ahlangana nama-spectra ebegade ahlanganiswe ngaphambilini ukusuka ekuhlolweni kokudla okunqophileko. Ukuhlaziywa kwethiyori yelwazi yesethi yedatha ye-MS/MS kutjengise bona ukuhlela kabutjha ama-metabolome akhethekileko wamaqabunga ngokulingisa iinlwana ezidla utjani kutjengise ukukhuthazwa okukhethekileko kwe-OS (Umfanekiso 2C). Ngokukhethekileko, nayiqathaniswa nokwelatjhwa kwe-OSS1, i-OSM yabangela ukuthuthukiswa kokukhethekileko kwe-metabolome emahoreni ama-4. Kuqakathekile ukutjheja bona nayiqathaniswa nesethi yedatha yokulinga yokudla okuqondileko kwe-herbivore, ukunyakaza kwe-metabolic okubonwa esikhaleni esinemikhakha emibili kusetjenziswa i-Hj ne-δj njengama-coordinate kanye nokuqondiswa kokukhethekile kwe-metabolome ngokuphendula ekwelapheni okulingisiweko kwe-herbivore ngokukhamba kwesikhathi Kukhuphuke ngokufanako (Umfanekiso 2D). Ngesikhathi esifanako, salinganisa okuqukethwe kwama-amino acid, ama-organic acid kanye netjhukela (ifayela ledatha S2) ukuphenya bona ukwanda okuqothelweko kobungcweti be-metabolome kungenxa yokuhlelwa kabutjha kwe-metabolism yekhabhoni ephakathi ngokuphendula ezilwaneni ezidla utjani ezilingisako (Umfanekiso S2). Ukuhlathulula ngcono iphetheni le, siragele phambili nokutjheja ukubuthelela kwe-metabolic kinetics ye-phenolamide kanye ne-17-HGL-DTG ekhulunywe ngaphambilini. Ukufakwa kwe-OS okukhethekileko kweenlwana ezidla utjani kutjhugululwa kube yiphetheni yokuhlela kabutjha okuhlukileko ngaphakathi kokugaya ukudla kwe-phenolamide (Umfanekiso S3). Ama-phenolic amides ane-coumarin kanye nama-caffeoyl moieties akhethwa khulu yi-OSS1, lokha ama-OSM abangela ukufakwa okuthileko kwama-ferulyl conjugates. Ngendlela ye-17-HGL-DTG, ukungeniswa kwe-OS okuhlukileko ngemikhiqizo ye-malonylation ne-dimalonylation kwatholakala (Umfanekiso S3).
Okulandelako, safunda nge-OS-induced transcriptome plasticity sisebenzisa isethi yedatha ye-time-course microarray, elingisa ukusetjenziswa kwama-OSM ukwelapha amakari wesitjalo se-rosette eenlwaneni ezidla iintjalo. I-kinetics yokuthatha isampula ihlangana ne-kinetics esetjenziswe esifundweni se-metabolomics (35). Nakuqathaniswa nokuhlelwa kabutjha kwe-metabolome lapho iplastiki ye-metabolic ikhuphuka khona ngokukhamba kwesikhathi, sibona ukuqhuma kokutlolwa kwesikhatjhana emaqabungeni abangelwa yi-Ms, lapho ukutlolwa kwe-transcriptome inducibility (RDPI) nokukhetheka (δj) ku-1 Kwaba nokwanda okukhulu kwama-awa, nokuhlukahluka (Hj) ngesikhathesi, ukuvezwa kwe-BMP1, kwehliswe khulu ngokukhululeka ukukhethekile kwe-transcriptome (Umfanekiso S4). Imindeni yamajini we-metabolic (njenge-P450, i-glycosyltransferase, kanye ne-BAHD acyltransferase) ihlanganyela ehlelelweni lokuhlanganisa ama-metabolites akhethekileko avela kumayunithi wesakhiwo avela e-metabolism yokuthoma, ngokulandela imodeli yokuqala ephezulu ekhulunywe ngayo. Njengesibonelo, indlela ye-phenylalanine yahlaziywa. Ukuhlaziywa kwaqinisekisa bona amajini asemqoka ekusebenzeni kwe-phenolamide abangelwa khulu yi-OS eenlwaneni ezidla iintjalo naziqathaniswa neentjalo ezingakhangwako, begodu ahambisana khulu namaphetheni wokuvezwa kwawo. Isici sokutlola i-MYB8 kanye namajini wesakhiwo i-PAL1, i-PAL2, i-C4H kanye ne-4CL ngaphezulu kwendlela le itjengise ukuthoma kwangaphambilini kokutlola. Ama-acyltransferases adlala indima ekuhlanganisweni kokugcina kwe-phenolamide, njenge-AT1, DH29, kanye ne-CV86, atjengisa iphetheni yokulawulwa kwesikhathi eside (Umfanekiso S4). Okubonwako okungenhla kutjengisa bona ukuthoma kwangaphambilini kokukhethekile kwe-transcriptome nokuthuthukiswa kwakamuva kokukhethekile kwe-metabolomics yindlela ehlangeneko, okungenzeka kubangelwa lihlelo lokulawula elihambisanako eliqalisa ukuphendula okunamandla kokuzivikela.
Ukuhlelwa kabutjha kokutjengisa amahomoni weentjalo kusebenza njengesendlalelo esilawulako esihlanganisa ilwazi lokudla iintjalo ukuhlela kabutjha umzimba weentjalo. Ngemva kokulingiswa kweenlwana ezidla iintjalo, salinganisa ukubuthelela kweenkoro eziqakathekileko zamahomoni weentjalo begodu sabona ukuvezwa kwesikhathi hlangana nazo [i-Pearson correlation coefficient (PCC)> 0.4] (Umfanekiso 3A). Njengombana bekulindelwe, amahomoni weentjalo ahlobene nokwenziwa kwezinto eziphilako ahlotjaniswa ngaphakathi kwenethiwekhi yokuvezwa kwamahomoni weentjalo. Ukungezelela, ukukhetheka kwe-metabolic (i-Si index) kuvezwe kule nethiwekhi ukuveza amahomoni weentjalo abangelwa mithi ehlukileko. Iindawo ezimbili ezikulu zokuphendula okukhethekileko kweenlwana ezidla iintjalo zidwetshwe: enye isesiqhemeni se-JA, lapho i-JA (ifomu yayo esebenzako yezinto eziphilako i-JA-Ile) nezinye iinhlobo ze-JA zitjengisa amaphuzu aphezulu we-Si; enye yi-ethylene (ET). I-Gibberellin itjengise ukwanda okulinganiselweko kokukhethekileko kweenlwana ezidla iintjalo, lokha amanye amahomoni weentjalo, afana ne-cytokinin, i-auxin, kanye ne-abscisic acid, bekane-induction specificity ephasi yeenlwana ezidla iintjalo. Nakuqathaniswa nokusebenzisa i-W + W yodwa, ukukhuliswa kwenani eliphezulu le-JA derivatives ngokusebenzisa i-OS (W + OS) kungatjhugululwa kube yinkomba eqinileko yama-JA. Ngokungakalindeleki, i-OSM ne-OSS1 enokuqukethwe okuhlukileko kwe-elicitor zaziwa ngokuthi zibangela ukubuthelela okufanako kwe-JA ne-JA-Ile. Ngokungafani ne-OSS1, i-OSM ikhuthazwa ngokukhethekileko begodu ngamandla ma-OSM, ngesikhathi i-OSS1 ingakhulisi ukuphendula kwamanceba we-basal (Umfanekiso 3B).
(A) Ukuhlaziywa kwenethiwekhi yokuvezwa ngokubambisana okusekelwe ekubalweni kwe-PCC kokulingisa ukubuthelela kwe-kinetics yamahomoni weentjalo abangelwa ziinlwana ezidla iintjalo. I-node ijamele i-hormone yesitjalo yinye, begodu ubukhulu be-node bujamele inkomba ye-Si eqondene ne-hormone yesitjalo hlangana nokwelatjhwa. (B) Ukubuthelela kwe-JA, i-JA-Ile kanye ne-ET emakarini okubangelwa mithi ehlukileko etjengiswa mibala ehlukileko: i-apricot, W + OSM; oluhlaza okwesibhakabhaka, W + OSSl; omnyama, W + W; grey, C (ukulawula) . Iinkwekwezi zitjengisa umehluko oqakathekileko hlangana nokwelapha nokulawula (i-ANOVA eneendlela ezimbili elandelwa yi-Tukey HSD post hoc multiple comparison, *** P <0.001). Ukuhlaziywa kwethiyori yelwazi ye-(C)697 MS/MS (ifayela ledatha S1) ku-JA biosynthesis kanye nokukhubazeka kwe-perception spectrum (irAOC kanye ne-irCOI1) kanye (D)585 MS/MS (ifayela ledatha S1) ku-ETR1 ngesiginali ye-ET ekhubazekileko. Iinkwekwezi zitjengisa umehluko oqakathekileko hlangana nokwelatjhwa kwe-W+OS nokulawula okungalimalanga (i-ANOVA yeendlela ezimbili elandelwa yi-Tukey HSD ngemva kokumadanisa okunengi, *P<0.05, **P<0.01 kanye ne-***P<0.001). (E) Amagrafu ahlakazekileko wokuphikisa okusakazekileko kokukhethekileko. Imibala ijamele imihlobo ehlukileko etjhugululwe ngokwezakhi zofuzo; amatshwayo ajamele iindlela ezihlukileko zokwelapha: unxantathu, W + OSS1; unxande, W + OSM; isiyingi C
Okulandelako, sisebenzisa umhlobo otjhugululwe ngokwezakhi zofuzo we-Nepenthes (irCOI1 kanye ne-sETR1) emagadangweni aqakathekileko we-JA ne-ET biosynthesis (irAOC ne-irACO) kanye nokubonwa (irCOI1 ne-sETR1) ukuhlaziya ukusebenza kwamahomoni weentjalo ezimbili eenlwaneni ezidla iintjalo Igalelo lokuhlela kabutjha. Ngokuvumelana nokuhlolwa kwangaphambilini, siqinisekise ukufakwa kwe-herbivore-OS eentjalweni ezingenalutho (EV) (Umfanekiso 3, C ukuya ku-D) kanye nokwehla okupheleleko kwe-Hj index okubangelwa yi-OSM, lokha inkomba ye-δj ikhuphuka. Ipendulo izwakala khulu ukudlula ipendulo ebangelwe yi-OSS1. Igrafu enemigqa emibili esebenzisa i-Hj ne-δj njengamakhodini itjengisa ukuhlukaniswa okuthileko (Umfanekiso 3E). Umkhuba obonakalako khulu kukobana emihlobeni engenaso isiginali ye-JA, ukuhlukahluka kwe-metabolome nokutjhuguluka okukhethekileko okubangelwa ziinlwana ezidla iintjalo kuqedwa ngokupheleleko (Umfanekiso 3C). Ngokuphikisako, ukubonwa kwe-ET okuthulileko eentjalweni ze-sETR1, nanyana umphumela woke ematjhugulukweni wokugaya ukudla kweentjalo ungaphasi khulu kunalowo we-JA, wehlisa umehluko we-Hj ne-δj hlangana nokuvusa kwe-OSM ne-OSS1 (Umfanekiso 3D noMfanekiso S5). . Lokhu kutjengisa bona ngaphezu komsebenzi omkhulu wokudluliselwa kwesiginali ye-JA, ukudluliselwa kwesiginali ye-ET nakho kusebenza njengokulungisa okuhle kwempendulo ye-metabolic yeenlwana ezidla iintjalo. Ngokuvumelana nomsebenzi wokulungisa, bekunganatjhuguluko ekuthomeni kwe-metabolome eentjalweni ze-sETR1. Ngakelinye ihlangothi, nayiqathaniswa neentjalo ze-sETR1, iintjalo ze-irACO zibangele ama-amplitude afanako wokutjhuguluka kwe-metabolic okubangelwa ziinlwana ezidla iintjalo, kodwana zitjengise amaphuzu ahluke khulu we-Hj ne-δj hlangana neentjhijilo ze-OSM ne-OSS1 (Umfanekiso S5).
Ukuze sikhombe ama-metabolites akhethekileko anegalelo eliqakathekileko ekuphenduleni kwemihlobo ethileko yeenlwana ezidla iintjalo begodu silungise ukukhiqizwa kwazo ngokusebenzisa amatshwayo we-ET, sisebenzise indlela ye-MS/MS eyakhiwe ngaphambilini. Indlela le ithembele endleleni yokuhlanganisa kabili ukubuyisela umndeni we-metabolic ukusuka eencenyeni ze-MS/MS [umkhiqizo wedothi ovamileko (NDP)] kanye nesikolo sokufana ngokuya ngokulahlekelwa okungathathi ihlangothi (NL). Isethi yedatha ye-MS/MS eyakhiwe ngokuhlaziywa kwemigqa ye-ET transgenic yakhiqiza i-585 MS/MS (ifayela ledatha S1), eyararululwa ngokuyihlanganisa ibe ngamamojula alikhomba we-MS/MS (M) (Umfanekiso 4A) . Amanye wamamojula la agcwele ama-metabolites akhethekileko: isibonelo, i-M1, i-M2, i-M3, i-M4 ne-M7 inothile ngama-phenol ahlukahlukeneko (M1), ama-flavonoid glycosides (M2), ama-acyl sugars (M3 kanye ne-M4), kanye ne-17-HGL-DTG (M7). Ukungezelela, ilwazi elikhethekileko lokugaya ukudla (i-Si index) ye-metabolite eyodwa kumojuli ngayinye liyabalwa, begodu ukusabalala kwayo kwe-Si kungabonwa ngokuzwakalako. Ngamafuphi, ama-MS/MS spectra atjengisa ukudla okuphezulu kweentjalo kanye nokukhethekileko kwe-genotype abonakala ngamanani aphezulu we-Si, begodu iimbalo ze-kurtosis zitjengisa ukusabalala kweboya ekhoneni lomsila wangakwesokudla. Okhunye ukusabalalisa okunjalo kwe-colloid kwatholakala ku-M1, lapho i-phenol amide itjengise khona i-Si fraction ephezulu khulu (Umfanekiso 4B). I-17-HGL-DTG ekhulunywe ngaphambilini yokudla utjani ku-M7 itjengise isikoro se-Si esilinganiselwe, okutjengisa izinga elilinganiselweko lokulawula okuhlukileko hlangana nemihlobo emibili ye-OS. Ngokuphikisako, inengi lama-metabolites akhethekileko akhiqizwa ngokusemthethweni, afana ne-rutin, i-CGA, kanye ne-acyl sugars, ziphakathi kwamaphuzu we-Si aphasi khulu. Ukuze kuhlolisiswe ngcono ubudisi besakhiwo nokusabalala kwe-Si hlangana nama-metabolites akhethekileko, kwakhiwa inethiwekhi yamamolekhyuli yemojuli ngayinye (Umfanekiso 4B). Ukubikezela okuqakathekileko kwethiyori ye-OD (efingqwe kuMfanekiso 1B) kukobana ukuhlelwa kabutjha kwama-metabolites akhethekileko ngemva kokudla utjani kufanele kurholele ekutjhugulukeni kwendlela eyodwa kuma-metabolites anenani eliphezulu lokuzivikela, khulukhulu ngokukhulisa ukukhetheka kwawo (ngokuphikisana nokusabalala okungakahleleki) Indlela) I-metabolite yokuzivikela ebikezelwe yithiyori ye-MT. Inengi lama-phenol abuthelelwe ku-M1 ahlotjaniswa nokwehla kokusebenza kwezinambuzane (32). Lokha nakumadanisa amanani we-Si kuma-metabolites we-M1 hlangana namakari akhuthaziweko kanye namakari ahlanganisako weentjalo zokulawula i-EV emahoreni ama-24, sibone bona ukukhetheka kwe-metabolic yama-metabolites amanengi ngemva kwezinambuzane ezidla utjani kunomkhuba omkhulu wokukhuphuka (Umfanekiso 4C). Ukwanda okukhethekileko kwenani le-Si kwatholakala kwaphela kuma-phenolamides avikelako, kodwana akukho ukwanda kwenani le-Si okwatholakala kwamanye ama-phenols kanye nama-metabolites angaziwako ahlala ndawonye kule module. Le yimodeli ekhethekileko, ehlobene nethiyori ye-OD. Ukubikezela okukhulu kwamatjhuguluko wokugaya ukudla okubangelwa ziinlwana ezidla utjani kuyafana. Ukuhlola bona ukukhetheka kwe-phenolamide spectrum kwabangelwa yi-OS-specific ET, sadweba inkomba ye-metabolite Si begodu sabangela inani lokuvezwa okuhlukileko hlangana ne-OSM ne-OSS1 kuma-genotypes we-EV ne-sETR1 (Umfanekiso 4D ). Ku-sETR1, umehluko obangelwa yi-phenamide hlangana kwe-OSM ne-OSS1 wehliswe khulu. Indlela yokuhlanganisa kabili nayo yasetjenziswa kudatha ye-MS/MS ebuthelelwe emihlobeni ene-JA enganeleko ukuthola amamojula amakhulu we-MS/MS ahlobene nokukhethekileko kwe-metabolic elawulwa yi-JA (Umfanekiso S6).
(A) Imiphumela yokuhlanganisa ye-585 MS/MS esekelwe esiqhemeni esabelwanako (ukufana kwe-NDP) nokulahlekelwa okungathathi ihlangothi okwabelwanako (ukufana kwe-NL) kuphumela ekutheni imojuli (M) ivumelane nomndeni wekhompawundi eyaziwako, nofana ngokubunjwa kwe-Metabolite okungaziwako nofana okungasebenzi kuhle. Eduze kwemojuli ngayinye, ukusatshalaliswa kwe-metabolite (MS/MS) okukhethekileko (Si) kutjengiswa. (B) Inethiwekhi yamamolekhyuli: Amanothi ajamele i-MS/MS namaphethelo, i-NDP (ebomvu) kanye ne-NL (eluhlaza okwesibhakabhaka) amaphuzu we-MS/MS (ukusika,> 0.6). Inkomba yokukhetheka kwe-metabolite (Si) efakwe umbala ngokuya ngemojuli (ngesinceleni) begodu ifakwe kunethiwekhi yamamolekhyuli (kwesokudla). (C) Imodyuli M1 yesitjalo se-EV esisesimweni esibunjwako (sokulawula) nesibangelweko (esidla utjani esilingisako) emahoreni ama-24: umdwebo wenethiwekhi yamamolekhyuli (inani le-Si lisayizi ye-node, i-phenolamide yokuzivikela ivezwe ngombala oluhlaza okwesibhakabhaka). (D) Umdwebo wenethiwekhi yamamolekhyuli we-M1 womlayini we-spectrum sETR1 onokukhubazeka kwe-EV ne-ET: ikhompawundi ye-phenolic emelelwa yi-node yesiyingi esiluhlaza, kanye nomehluko oqakathekileko (inani le-P) hlangana nokwelatjhwa kwe-W + OSM kanye ne-W + OSS1 njengobukhulu be-Node. CP, N-caffeoyl-tyrosine; CS, N-caffeoyl-spermidine; FP, i-N-ferulic acid ester-uric acid; FS, N-ferulyl-spermidine; CoP, N', N "-Coumarolyl-tyrosine; DCS, N', N"-dicaffeoyl-spermidine; I-CFS, N', N”-caffeoyl, feruloyl-spermidine; i-Lycium barbarum ku-wolfberry Son; u-Nick. O-AS, i-O-acyl sugar.
Siragele phambili nokwandisa ukuhlaziywa ukusuka ku-genotype eyodwa ye-Nepenthes ukuya ebantwini bemvelo, lapho amatjhuguluko aqinileko we-intraspecific emazingeni we-JA we-herbivorous kanye namazinga we-metabolite athileko sele achazwe ngaphambilini ebantwini bemvelo (26). Sebenzisa isethi yedatha le ukuvala amagciwani ama-43. Ama-germplasm la anemihlobo eli-123 yeentjalo ezivela ku-N. pallens. Iintjalo lezi zithathwe embewini ebuthelelwe eendaweni ezihlukahlukeneko zendabuko e-Utah, Nevada, Arizona, ne-California (Umfanekiso S7), sibale ukuhlukahluka kwe-metabolome (lapha okubizwa ngezinga labantu) ukuhlukahluka kwe-β) kanye nokukhethekileko okubangelwa yi-OSM. Ngokuya ngeemfundo zangaphambilini, sibone amatjhuguluko amanengi wokugaya ukudla e-Hj ne-δj axes, okutjengisa bona ama-germplasms anomehluko omkhulu ekuphenduleni kwawo kokugaya ukudla eenlwaneni ezidla iintjalo (Umfanekiso S7). Ihlangano le ikhumbuza ukutjheja kwangaphambilini malungana nobubanzi obutjhugulukako bama-JAs abangelwa ziinlwana ezidla utjani, begodu igcine inani eliphezulu khulu esiqhemeni sinye (26, 36). Ngokusebenzisa i-JA ne-JA-Ile ukuhlola ukuhlobana kwezinga elipheleleko hlangana ne-Hj ne-δj, sithole bona kunokuhlobana okuhle hlangana ne-JA kanye ne-metabolome β ukuhlukahluka kanye nenkomba yokukhethekileko (Umfanekiso S7). Lokhu kuveza bona ukuhlukahluka okubangelwa ziinlwana ezidla iintjalo ekuthonyweni kwe-JAs okutholwe ezingeni labantu kungabangelwa ziindlela eziqakathekileko zokugaya ukudla ezibangelwa kukhethwa kweenlwanyana ezidla iintjalo.
Iimfundo zangaphambilini zitjengise bona imihlobo yegwayi ihluke khulu ngohlobo nokuthembela okuhlobene nokuzivikela kwe-metabolic. Kukholelwa bona amatjhuguluko la ekudlulisweni kwesiginali yokulwisana nokudla iintjalo kanye namakghono wokuzivikela alawulwa mfutho wenani leenunwana, umjikelezo wokuphila kweentjalo, kanye neendleko zokukhiqiza ukuzivikela endaweni lapho umhlobo othileko ukhula khona. Sifunde ukuvumelana kokuvuselelwa kwe-metabolome yamakari okubangelwa ziinlwana ezidla iintjalo zeenhlobo ezisithandathu ze-Nicotiana ezivela e-North America ne-South America. Imihlobo le ihlobene khulu ne-Nepenthes North America, okuthiwa ngu-Nicolas Bociflo. La, N. nicotinis, Nicotiana n. utjani obubuthakathaka, i-Nicotiana tabacum, i-linear tobacco, i-tobacco (i-Nicotiana spegazzinii) kanye ne-tobacco leaf tobacco (i-Nicotiana obtusifolia) (Umfanekiso 5A) (37). Iinhlobo ezisithandathu zalezi, okufaka hlangana i-N. please, ziintjalo ezikhula qobe mnyaka ze-petunia clade, begodu i-obtusifolia N. ziintjalo ezihlala isikhathi eside ze-sister clade Trigonophyllae (38). Ngemva kwalokho, ukufakwa kwe-W + W, W + OSM kanye ne-W + OSS1 kwenziwa emihlobeni elikhomba le ukufunda ukuhlelwa kabutjha kwe-metabolic yezinga lemihlobo yokudla kwezinambuzane.
(A) Umuthi we-bootstrap phylogenetic osekelwe ekwenzekeni okukhulu [kwe-nuclear glutamine synthesis (38)] kanye nokusabalala kwendawo kwemihlobo elikhomba ye-Nicotiana ehlobene khulu (imibala ehlukileko) (37). (B) Iplothi yokuhlakazeka yokuhlukahluka okukhethekileko kwamaphrofayili we-metabolic wemihlobo elikhomba ye-Nicotiana (939 MS/MS; ifayela ledatha S1). Ezingeni lemihlobo, ukuhlukahluka kwe-metabolome kuhlotjaniswa kumbi nezinga lokukhethekileko. Ukuhlaziywa kokuhlobana kwezinga lemihlobo hlangana nokuhlukahluka kwe-metabolic nokukhetheka kanye nokubuthelela kwe-JA kutjengiswe kuMfanekiso 2. S9. Umbala, imihlobo ehlukileko; unxantathu, W + OSS1; unxande, W + OSM; (C) I-Nicotiana JA ne-JA-Ile dynamics ihlelwe ngokuya nge-OS excitation amplitude (two-way ANOVA kanye ne-Tukey HSD ngemva kokumadanisa okunengi, * P <0.05, ** P <0.01 kanye * ** Ukumadanisa i-W + OS ne-W + W, P <0.001). Ibhokisi lebhokisi (D) lokuhlukahluka kanye (E) nokukhethekileko komhlobo ngamunye ngemva kokulingisa i-herbivorous kanye ne-methyl JA (MeJA). Inkanyezi itjengisa umehluko oqakathekileko hlangana ne-W + OS ne-W + W nofana i-lanolin kanye ne-W (Lan + W) nofana i-Lan kanye ne-MeJA (i-Lan + MeJa) kanye nokulawula kwe-Lan (ukuhlaziywa kweendlela ezimbili zokuhlukahluka, kulandelwe yi-Tukey's HSD post hoc multiple comparison , *P<0.05, **P<0.01 kanye ne-***P<0.01).
Ngokusebenzisa indlela ye-dual cluster, sithole amamojula ayi-9 we-939 MS/MS (ifayela ledatha S1). Ukwakheka kwe-MS/MS ehlelwe kabutjha ngemithi ehlukileko kuyahluka khulu hlangana namamojula ahlukileko hlangana nemihlobo (Umfanekiso S8). Ukubona ngelihlo lengqondo i-Hj (ebizwa lapha njenge-γ-diversity yezinga lemihlobo) kanye ne-δj kuveza bona imihlobo ehlukileko ihlangana ibe maqembu ahlukileko khulu esikhaleni sokugaya ukudla, lapho ukwahlukana kwezinga lemihlobo kuvamise ukubonakala khona ukudlula ukuthaba. Ngaphandle kwe-N. linear ne-N. obliquus, zitjengisa imiphumela ehlukileko (Umfanekiso 5B). Ngokuphikisako, imihlobo efana ne-N. purpurea ne-N. obtusifolia inokuphendula okungabonakaliko kwe-metabolic ekwelatjhweni, kodwana i-metabolome ihluke khulu. Ukusabalala okuthileko kwemihlobo yokuphendula kwe-metabolic kwabangela ukuhlobana okungakalungi hlangana nokukhethekileko nokuhlukahluka kwe-gamma (PCC = -0.46, P = 4.9×10-8). Amatjhuguluko abangelwa yi-OS emazingeni we-JAs ahlotjaniswa kuhle nokukhethekileko kwe-metabolome, begodu ahlotjaniswa kumbi nokuhlukahluka kwe-gamma ye-metabolic etjengiswa mhlobo ngamunye (Umfanekiso 5B noMfanekiso S9). Kuqakathekile ukutjheja bona imihlobo ebizwa ngokuthi “yimihlobo yokuphendula kwesiginali” kuMfanekiso 5C, njenge-Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, kanye ne-Nepenthes attenuated, yabangela amatshwayo aqakathekileko emizuzwini ema-30. Ukuqubuka kwe-OS-specific JA kanye ne-JA-Ile kwamuva nje, lokha amanye amagciwane abizwa ngokuthi "awaphenduli ngesiginali", afana ne-Nepenthes mills, i-Nepenthes powdery kanye ne-N. obtusifolia atjengisa ukufakwa kwe-JA-Ile Edge ngaphandle kokukhetheka kwe-OS (Umfanekiso 5C). Ezingeni lokugaya ukudla, njengombana kutjhiwo ngehla, nge-Nepenthes ehlanjululweko, izinto eziphendula isignali zitjengise ukukhetheka kwe-OS begodu zandisa khulu i-δj, lokha nazinciphisa i-Hj. Umphumela lo wokuthoma we-OS akhenge utholakale emihlobeni ehlukaniswe njengemihlobo engasabeli ngesiginali (Umfanekiso 5, D no-E). Ama-metabolites akhethekileko we-OS abelana kanengi hlangana nemihlobo ephendula isignali, begodu amaqoqo wesignali la ahlangana nemihlobo eneempendulo zesignali ezibuthakathaka, lokha imihlobo eneempendulo ezibuthakathaka zesignali itjengisa ukuthembelana okuncani (Umfanekiso S8 ). Umphumela lo utjengisa bona ukufakwa kwe-OS okukhethekileko kwama-JAs kanye nokuhlelwa kabutjha kwe-OS okukhethekileko kwe-metabolome engezansi kuhlanganiswe ezingeni lemihlobo.
Okulandelako, sisebenzise i-lanolin paste ene-methyl JA (MeJA) ukwelapha iintjalo ukuhlola bona iindlela zokuhlanganisa zivinjelwe na ngokutholakala kwe-JA esetjenziswa yi-JA yangaphandle, ezakuba se-cytoplasm yeentjalo. Ukuqedwa msinyana yi-JA. Sithole umukghwa ofanako wokutjhuguluka kancanikancani ukusuka emihlobeni ephendula isignali ukuya emihlobeni engaphenduli isignali ebangelwa kunikelwa okuragela phambili kwe-JA (Umfanekiso 5, D no-E). Ngamafuphi, ukwelatjhwa nge-MeJA kwahlela kabutjha ama-metabolome we-linear nematodes, i-N. obliquus, i-N. aquaticus, i-N. pallens, kanye ne-N. mikimotoi, okurholele ekukhuphukeni okukhulu kwe-δj nokwehla kwe-Hj. I-N. purpurea itjengise ukwanda kwe-δj kwaphela, kodwana ingasi i-Hj. I-N. obtusifolia, ebegade itjengiswe ngaphambilini bona ibuthelela amazinga aphasi khulu we-JA, nayo ayiphenduli kuhle ekwelatjhweni kwe-MeJA malungana nokuhlelwa kabutjha kwe-metabolome. Imiphumela le itjengisa bona ukukhiqizwa kwe-JA nofana ukudluliselwa kwesiginali kuvinjelwe ngokomzimba emihlobeni engaphenduli isiginali. Ukuhlola umbono lo, sifunde imihlobo emine (N. pallens, N. mills, N. pink kanye ne-N. microphylla) ebangelwa yi-W + W, W + OSMs kanye ne-W + OSS1 Transcriptome (39). Ngokuvumelana nephetheni yokuvuselelwa kwe-metabolome, imihlobo le ihlukaniswe kuhle esikhaleni se-transcriptome, hlangana nayo i-N. attenuated itjengise i-RDPI ephezulu khulu ebangelwa yi-OS, kuthi i-N. gracilis inephasi khulu (Umfanekiso 6A). Kodwana, kwatholakala bona ukuhlukahluka kwe-transcriptome okubangelwa yi-N. oblonga bekuphasi khulu hlangana nemihlobo emine, ngokuphambene nokuhlukahluka okukhulu kwe-metabonomic ye-N. oblonga ebegade kutjengiswa ngaphambilini emihlobeni elikhomba. Iimfundo zangaphambilini zitjengise bona isethi yamajini ahlobene namatshwayo wokuzivikela wokuthoma, kufaka hlangana amatshwayo we-JA, ahlathulula ukuqakatheka kweempendulo zokuzivikela zokuthoma ezibangelwa ziinthombe ezihlobene nokudla utjani emhlobeni we-Nicotiana (39). Ukumadanisa iindlela zokuthumela i-JA hlangana nemihlobo emine kuveze iphetheni ekarisako (Umfanekiso 6B). Inengi lamajini endleleni le, afana ne-AOC, i-OPR3, i-ACX kanye ne-COI1, itjengise amazinga aphezulu wokungeniswa kwemihlobo emine. Kodwana, ijini eliqakathekileko, i-JAR4, itjhugulula i-JA ibe yifomu yayo esebenzako ye-JA-Ile, begodu izinga lokutlolwa kwayo liphasi khulu, khulukhulu ku-N. mills, Nepenthes pieris kanye ne-N. microphylla. Ukungezelela, umtlolo welinye ijini i-AOS kwaphela ongatholakali ku-N. bifidum. Amatjhuguluko la ekuvezweni kwezakhi zofuzo angaba nesibopho se-phenotypes embi khulu ebangelwa kukhiqizwa kwe-JA ephasi emihlobeni ye-anergic yesiginali kanye nokungeniswa kwe-N. gracilis.
(A) Ukuhlaziywa kwethiyori yelwazi yokuhlelwa kabutjha kweempendulo zokutlola zokuthoma zemihlobo emine yegwayi ehlobene khulu ethathwe ngemva kwemizuzu ema-30 ngemva kokuthonywa kokudla utjani. I-RDPI ibalwa ngokumadanisa amakari abangelwa yi-OS ye-herbivore nokulawula isilonda. Imibala itjengisa imihlobo ehlukileko, begodu amatshwayo atjengisa iindlela ezihlukileko zokwelapha. (B) Ukuhlaziywa kokuvezwa kwezakhi zofuzo eendleleni zokutjengisa i-JA hlangana nemihlobo emine. Indlela ye-JA eyenziwe lula itjengiswa eduze kweplothi yebhokisi. Imibala ehlukileko itjengisa iindlela ezihlukileko zokucubungula. Inkanyezi itjengisa bona kunomehluko oqakathekileko hlangana nokwelatjhwa kwe-W + OS nokulawula kwe-W + W (nge-t-test ye-Student yomehluko ngababili, *P<0.05, **P<0.01 kanye ne-***P<0.001). I-OPDA, i-12-oxophytodienoic acid; I-OPC-8: 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
Engcenyeni yokugcina, sifunde bona ukulungiswa kabutjha kwe-metabolome yeentjalo ezihlukileko kungajamelana njani neenlwana ezidla utjani. Irhubhululo langaphambilini beligandelela umhlobo we-Nicotiana. Ukujamelana kwabo nama-Ms kanye nama-larvae kuhluka khulu (40). Lapha, sifunde ukuhlobana hlangana nemodeli le nobujamo babo bokugaya ukudla. Ngokusebenzisa imihlobo emine yegwayi engenhla, nokuhlola ukuhlobana hlangana nokuhlukahluka nokukhethekileko kwe-metabolome okubangelwa ziinlwana ezidla iintjalo nokujamelana kweentjalo ku-Ms ne-Sl, sithole ukujamelana, ukuhlukahluka nokukhetheka kwe-generalist Sl Zoke kuhlobana kuhle, ngesikhathi ukuhlobana hlangana nokujamelana namakhosikazi weengcweti nokukhethekile kubuthakathaka, begodu ukuhlukahlukana akukaqakatheki no-Sl (Umfanekiso 10). Malungana nokujamelana ne-S1, kokubili i-N. chinensis kanye ne-N. gracilis, ebegade itjengiswe ngaphambilini bona itjengisa amazinga wokudlulisa isignali ye-JA kanye ne-metabolome plasticity, beyineempendulo ezihluke khulu ekufakweni kwe-herbivore, begodu nayo itjengise ukujamelana okuphezulu okufanako. Ubulili.
Eminyakeni ematjhumi asithandathu edlulileko, ithiyori yokuvikela iintjalo inikele ngehlelo lethiyori, elisekelwe lapho abarhubhululi babikezele khona inani elikhulu lokuziphendukela kwemvelo nemisebenzi yama-metabolites akhethekileko weentjalo. Inengi leemfundiso azilandeli ikambiso ejayelekileko yokuphetha okuqinileko (41). Baphakamisa ukubikezela okuqakathekileko (3) ezingeni elifanako lokuhlaziywa. Lokha ukuhlolwa kweenbikezelo eziqakathekileko kuvumela bona imibono ethileko ihlaziywe, lokhu kuzokwenza bona umkhakha uqhubekele phambili. Sekelwa, kodwana ulahle abanye (42). Esikhundleni salokho, ithiyori etja yenza ukubikezela emazingeni ahlukileko wokuhlaziya begodu yengeza isendlalelo esitjha sokucabangela okuhlathululako (42). Kodwana, iimfundiso ezimbili eziphakanyiswe ezingeni lokusebenza, i-MT ne-OD, zingachazwa lula njengeenbikezelo eziqakathekileko zamatjhuguluko akhethekileko wokugaya ukudla abangelwa ziinlwana ezidla utjani: ithiyori ye-OD ikholelwa bona amatjhuguluko “esikhaleni” esikhethekileko sokugaya ukudla aqondiswe khulu. Ithiyori ye-MT ikholelwa bona amatjhuguluko la azokuba angaqondi begodu abekwe ngokungakahleleki esikhaleni se-metabolic, begodu athambekele ekubeni nama-metabolites aphezulu wokuzivikela. Ukuhlolwa kwangaphambilini kwe-OD ne-MT ukubikezela kuhloliwe kusetjenziswa isethi encani yamakhompawundi "wokuzivikela" we-priori. Iimhloliso ezidzimelele ema-metabolite zivimbela ikghono lokuhlaziya ubukhulu nendlela yokuhlelwa kabutjha kwe-metabolome ngesikhathi sokudla utjani, begodu azivumeli ukuhlolwa ngaphakathi kohlaka lwezibalo olufanako ukutlhoga ukubikezela okuqakathekileko okungathathwa njengokupheleleko. Lapha, sisebenzise ithekhnoloji etja ye-metabolomics esekelwe ku-MS yekhomphyutha begodu senza ukuhlaziywa kwe-MS ye-deconvolution ngemali ejwayelekileko yeenhlathululo zethiyori yolwazi ukuhlola umehluko phakathi kwalokhu okubili okuphakanyiswe ezingeni le-metabolomics yephasi loke. Ukubikezela okuqakathekileko kwalethiyori. Ithiyori yelwazi isetjenziswe emikhakheni eminengi, khulukhulu emkhakheni werhubhululo lokuhlukahlukana kwezinto eziphilako nokugeleza kwezakhamzimba (43). Kodwana, ngokwazi kwethu, lokhu kusetjenziswa kokuthoma okusetjenziselwa ukuhlathulula isikhala selwazi lokugaya ukudla kweentjalo nokurarulula imiraro yemvelo ehlobene nokutjhuguluka kwesikhatjhana kokugaya ukudla ngokuphendula iinkomba zebhoduluko. Khulukhulu, ikghono lendlela le lilele ekghoneni layo lokumadanisa amaphetheni ngaphakathi naphakathi kwemihlobo yeentjalo ukuhlola bona iinlwana ezidla iintjalo zitjhuguluke njani ukusuka emihlobeni ehlukileko ukuya emaphetheni wokuziphendukela kwemvelo phakathi kwemihlobo emazingeni ahlukileko wokuziphendukela kwemvelo. Ukugaya ukudla.
Ukuhlaziywa kweengcenye ezikulu (i-PCA) kutjhugulula isethi yedatha ehlukileko ibe yindawo yokunciphisa ubukhulu ukwenzela bona umukghwa omkhulu wedatha uchazwe, njeke kuvamise ukusetjenziswa njengendlela yokuhlola ukuhlaziya isethi yedatha, njenge-deconvolution metabolome. Kodwana, ukwehliswa kobukhulu kuzokulahlekelwa yingcenye yelwazi eliqukethwe kusethi yedatha, begodu i-PCA angeze yakghona ukunikela ilwazi elilinganiselweko ngeempawu ezihlobene khulu nethiyori yezemvelo, ezifana nokuthi: iinlwana ezidla iintjalo zilungisa njani ukuhlukahluka emikhakheni ekhethekileko (ngokwesibonelo, ukunotha, ukusabalalisa) Nobunengi) ama-metabolites? Ngimaphi ama-metabolites aqagela ubujamo obubangelwa silwane esidla utjani? Ngokombono wokukhethekileko, ukuhlukahluka nokuthonywa, okuqukethwe kwelwazi lephrofayili ye-metabolite yamakari iyabola, begodu kutholakala bona ukudla iinlwana ezidla utjani kungenza bona i-metabolism ethileko isebenze. Ngokungalindelekileko, sabona bona, njengombana kuchaziwe eenkomba zethiyori yelwazi ezisetjenzisiweko, ubujamo bokugaya ukudla bunokuhlangana okukhulu ngemva kokuhlaselwa kweenlwana ezimbili ezidla utjani (i-generalist u-Sl) kanye nesazi se-Solanaceae uNksz. Nanyana ukuziphatha kwazo kokudla nokugxila kwazo kuhluke khulu. I-Fatty acid-amino acid conjugate (FAC) initiator ku-OS (31). Ngokusebenzisa i-OS ye-herbivore ukwelapha amanceba wokubhoboza ajwayelekileko, ukwelapha okulingisiweko kwe-herbivore nakho kutjengise umukghwa ofanako. Ikambiso ejwayelekileko yokulingisa ukuphendula kweentjalo ekuhlaselweni ziinlwana ezidla iintjalo iqeda izinto eziphazamisako ezibangelwa matjhuguluko ekuziphatheni kokudla kweenlwana ezidla iintjalo, okurholela ekulimaleni okuhlukahlukeneko ngeenkhathi ezihlukileko (34). I-FAC, eyaziwa njengembangela ekulu ye-OSM, yehlisa i-JAS nezinye iimpendulo zamahomoni weentjalo ku-OSS1, ngesikhathi i-OSS1 yehlisa amahlandla amanengi (31). Kodwana, i-OSS1 yabangela amazinga afanako wokubuthelela kwe-JA nayiqathaniswa ne-OSM. Sekutjengiswe ngaphambilini bona ipendulo ye-JA ku-Nepenthes ephasi izwela khulu ku-OSM, lapho i-FAC ingagcina umsebenzi wayo ngitjho nanyana ihlanjululwe nge-1:1000 ngamanzi (44). Ngalokho-ke, nayimadaniswa ne-OSM, nanyana i-FAC ku-OSS1 iphasi khulu, yanele ukubangela ukuqubuka kwe-JA okwaneleko. Iimfundo zangaphambilini zitjengise bona amaphrotheyini afana ne-porin (45) nama-oligosaccharides (46) angasetjenziswa njengeenkomba zamamolekhyuli ukuvusa iimpendulo zokuvikela iintjalo ku-OSS1. Kodwana, akukacaci bona ama-elicitors la ku-OSS1 anesibopho sokubuthelela i-JA ebonwe esifundweni samanje.
Nanyana kuneemfundo ezimbalwa ezihlathulula ukuhlukahluka kweminwe yokugaya ukudla okubangelwa kusetjenziswa kweenlwana ezidla iintjalo ezihlukahlukeneko nofana i-JA nofana i-SA (i-salicylic acid) (47), akekho umuntu ophazamise ukuphazamiseka kwemihlobo yeenlwana ezidla iintjalo enethiwekhi yotjani beentjalo kanye nemiphumela yayo eentweni ezithileko. ilwazi lomuntu mathupha. Ukuhlaziywa lokhu kubuye kwaqinisekisa bona ukuhlobana kwenethiwekhi yamahomoni yangaphakathi namanye amahomoni weentjalo ngaphandle kwama-JA kubumba ukuhlelwa kabutjha kwe-metabolic okubangelwa ziinlwana ezidla utjani. Ngokukhethekileko, sithole bona i-ET ebangelwe yi-OSM beyikhulu khulu kunaleyo ebangelwa yi-OSS1. Imodi le ihambisana nokuqukethwe okunengi kwe-FAC ku-OSM, okubujamo obutlhogekako nobanele bokuvusa ukuqhuma kwe-ET (48). Ngokwesimo sokuthintana hlangana kweentjalo neenlwana ezidla utjani, umsebenzi wokutjengisa we-ET ekuguquguqukeni kwe-metabolite ekhethekileko yeentjalo usaphazanyiswa begodu uqale isiqhema sinye kwaphela. Ukungezelela, iimfundo ezinengi zisebenzise ukusetjenziswa kwangaphandle kwe-ET nofana ama-precursors wayo nofana iinqabo ezihlukahlukeneko ukufunda ukulawulwa kwe-ET, hlangana nalokho ukusetjenziswa kwamakhemikhali angaphandle kuzokukhiqiza imiphumela eminengi engakaqakatheki. Ngokwazi kwethu, irhubhululo leli lijamele ukuhlolwa kokuthoma okukhulu okuhlelekileko kwendima ye-ET ekusetjenzisweni kwe-ET ukukhiqiza nokubona iintjalo ezitjhugululweko ezikhubazekileko ukuhlanganisa amandla we-metabolome yeentjalo. Ukufakwa kwe-ET okukhethekileko kwe-herbivore kungatjhugulula ukuphendula kwe-metabolome. Okuqakatheke khulu kukutjhugululwa kwe-ET biosynthesis (ACO) kanye namajini wokubona (ETR1) okuveze ukubuthelela kwe-phenolamides okukhethekileko kwe-herbivore. Sekutjengiswe ngaphambilini bona i-ET ingalungisa ukubuthelela kwe-nicotine okubangelwa yi-JA ngokulawula i-putrescine N-methyltransferase (49). Kodwana, ukusuka embonweni wemitjhini, akucaci bona i-ET ilungisa njani ukungeniswa kwe-phenamide. Ngaphezu komsebenzi wokudlulisa isignali ye-ET, ukutjhuguluka kwe-metabolic kungatjhugululwa ku-S-adenosyl-1-methionine ukulawula ukutjala imali kuma-polyaminophenol amides. I-S-adenosyl-1-methionine yi-ET begodu yi-Common intermediate ye-polyamine biosynthetic pathway. Indlela isignali ye-ET elawula ngayo izinga le-phenolamide idinga ukufundwa okunabileko.
Isikhathi eside, ngebanga lenani elikhulu lama-metabolites akhethekileko wesakhiwo esingaziwako, ukutjheja okukhulu kwemikhakha ethileko ye-metabolic akukghoni ukuhlola ngokuqinileko amatjhuguluko wesikhatjhana wokuhlukahluka kwe-metabolic ngemva kokusebenzisana kwezinto eziphilako. Njenganje, ngokuya ngokuhlaziywa kwethiyori yelwazi, umphumela omkhulu wokutholwa kwe-MS/MS spectrum ngokusekelwe kuma-metabolites angathathi ihlangothi kukobana iinlwana ezidla iintjalo ezidla nofana ezilingisa iinlwana ezidla iintjalo ziragela phambili nokwehlisa ukuhlukahluka kwe-metabolic ye-metabolome yamakari ngesikhathi zikhuphula izinga lokukhetheka kwazo. Lokhu kukhuphuka kwesikhatjhana kokukhethekile kwe-metabolome okubangelwa ziinlwana ezidla iintjalo kuhlotjaniswa nokwanda okusebenzisanako kokukhethekile kwe-transcriptome. Isici esifaka isandla khulu ekukhetheni okukhulu kwe-metabolome (ekunenani eliphezulu le-Si) yi-metabolite ekhethekileko enomsebenzi wokudla utjani owawuvezwe ngaphambilini. Imodeli le ihambisana nokubikezela kwethiyori ye-OD, kodwana ukubikezela kwe-MT okuhlobene nokungahleleki kokuhlela kabutjha kwe-metabolome akuhambisani. Kodwana, idatha le ihambisana nokubikezela kwemodeli ehlangeneko (i-MT engcono khulu; Umfanekiso 1B), ngombana amanye ama-metabolites angakavezwa anemisebenzi yokuzivikela engaziwako angase alandele ukusabalalisa okungakahleleki kwe-Si.
Iphetheni eqakathekileko ebanjwe lirhubhululo leli kukuthi ukusuka ezingeni lokuziphendukela kwemvelo okuncani (isitjalo sinye nesibalo segwayi) ukuya esigabeni esikhulu sokuziphendukela kwemvelo (imihlobo yegwayi ehlobene khulu), amazinga ahlukileko wokuziphendukela kwemvelo “asekuzivikeleni okuhle khulu” Kunomehluko oqakathekileko emandleni weenlwana ezidla utjani. U-Moore nabanye. (20). Abatloli laba akhenge bafingqe Iinkambiso zokubuthelela idatha ye-metabolome ngezinga elikhulu nazo azivezi bona kungabalwa njani ukuhlukahluka kwe-metabolic ukusuka kuledatha. Kulesi sifundo, ukulungiswa okuncani kokuhlukaniswa okusebenzako kwaka-Whittaker kuzokucabangela ukuhlukahluka kwe-α-metabolic njengokuhlukahluka kwe-MS/MS spectra esitjalweni esinikelweko, kanye nokuhlukahluka kwe-β-metabolic njenge-basic intraspecific metabolism yesiqhema seentjhaba Isikhala, kanye nokuhlukahluka kwe-γ-metabolic kuzokuba singezelelo sokuhlaziywa kwemihlobo efanako.
Isiginali ye-JA iqakathekile ekuphenduleni okuhlukahlukeneko kwe-herbivore. Kodwana, kunokutlhogeka kokuhlolwa okuqinileko kwegalelo lokulawulwa kwangaphakathi kwe-JA biosynthesis ekuhlukahlukeni kwe-metabolome, nokuthi ingabe isignali ye-JA yindawo ejwayelekileko yokuhlukahluka kwe-metabolic okubangelwa kugandeleleka esikalini esiphezulu se-macroevolutionary kusese budisi. Sitjheje bona ubujamo bokudla utjani be-Nepenthes bubangela ukukhetheka kwe-metabolome begodu ukuhlukahluka kokukhetheka kwe-metabolome ngaphakathi kwenani lemihlobo ye-Nicotiana kanye hlangana nemihlobo ye-Nicotiana ehlobene khulu kuhlotjaniswa kuhle nokutjengisa i-JA. Ukungezelela, lokha isiginali ye-JA nayikhubazekileko, ukukhetheka kwe-metabolic okubangelwa yi-genotype eyodwa ye-herbivore kuzokukhanselwa (Umfanekiso 3, C no-E). Njengombana amatjhuguluko we-metabolic spectrum weentjhaba ze-Nepenthes ezibuthakathaka ngokwemvelo zinengi khulu, amatjhuguluko ekuhlukahlukeni kwe-β metabolic nokukhethekile kilokhu kuhlaziywa kungenzeka kubangelwe khulu kukuthaba okunamandla kwemikhakha yamakhompawundi ane-metabolite. Amaklasi wamakhompawundi la abusa ingcenye yephrofayili ye-metabolome begodu arholela ekuhlobaneni okuhle namatshwayo we-JA.
Ngombana iindlela ze-biochemical zemihlobo yegwayi ehlobene nayo zihluke khulu, ama-metabolites akhonjwa ngokukhethekileko esigabeni sekhwalithi, ngalokho-ke kuyahlaziywa khulu. Ukucubungula kwethiyori yelwazi kwephrofayili ye-metabolic ebanjiweko kuveza bona ukufakwa kweenlwana ezidla iintjalo kukhuphula ukuthengiselana hlangana nokuhlukahluka kwe-gamma ye-metabolic nokukhethekile. Isiginali ye-JA idlala indima eqakathekileko kilokhu kuthengiselana. Ukwanda kokukhethekileko kwe-metabolome kuhambisana nokubikezela okukhulu kwe-OD begodu kuhlotjaniswa kuhle nesiginali ye-JA, ngesikhathi isiginali ye-JA ihlotjaniswa kumbi nokuhlukahluka kwe-gamma ye-metabolic. Amamodeli la atjengisa bona amandla we-OD weentjalo anqunywa khulu yiplastiki ye-JA, kungaba ngezinga lokuziphendukela kwemvelo okuncani nofana ngezinga elikhulu lokuziphendukela kwemvelo. Ukuhlolwa kokusetjenziswa kwe-JA kwangaphandle okudlula ukukhubazeka kwe-JA biosynthesis kuveza bona imihlobo yegwayi ehlobene khulu ingahlukaniswa ibe mihlobo ephendula isignali nengaphenduli isignali, njengendlela yayo ye-JA kanye ne-metabolome plasticity ebangelwa ziinlwana ezidla iintjalo. Imihlobo engaphenduli ngesiginali ayikwazi ukuphendula ngebanga lokungakghoni kwayo ukukhiqiza i-JA yangaphakathi begodu ngalokho-ke ingaphansi kwemikhawulo yomzimba. Lokhu kungabangelwa kutjhuguluka kwamanye amajini aqakathekileko endleleni yokubonisa i-JA (i-AOS ne-JAR4 ku-N. crescens) ye. Umphumela lo uveza bona amaphetheni wokuziphendukela kwemvelo hlangana nemihlobo emikhulu angaqhutshwa khulu matjhuguluko ekubonweni kwamahomoni wangaphakathi nokuphendula.
Ngaphezu kokuthintana hlangana kweentjalo neenlwana ezidla utjani, ukuhlolisisa ukuhlukahluka kwe-metabolic kuhlobana nayo yoke ituthuko eqakathekileko yethiyori ekurhubhululeni ukutjhuguluka kwezinto eziphilako emvelweni kanye nokuziphendukela kwemvelo kweempawu ezihlukahlukeneko. Ngokwanda kwenani ledatha etholwe ziinsetjenziswa ze-MS zesimanjemanje, ukuhlolwa kwemicabango ngokuhlukahluka kwe-metabolic manje kungadlula umehluko we-metabolite ngamunye/wesigaba begodu kwenziwe ukuhlaziywa kwephasi loke ukuveza amaphethini angakalindelwa. Esikhathini sokuhlaziywa okukhulu, isifaniso esiqakathekileko mqondo wokwenza ibalazwe elizwakalako elingasetjenziselwa ukuhlola idatha. Ngalokho-ke, umphumela oqakathekileko wokuhlanganiswa kwanje kwe-MS/MS metabolomics engathathi ihlangothi kanye nethiyori yolwazi kukuthi inikela ngemethrikhi elula engasetjenziselwa ukwakha amabalazwe ukuphequlula ukuhlukahluka kwe-metabolic emazingeni ahlukileko we-taxonomic. Yisidingo esiyisisekelo sale ndlela. Irhubhululo lokuziphendukela kwemvelo okuncani/okukhulu kanye nemvelo yomphakathi.
Ezingeni lokuziphendukela kwemvelo okukhulu, ummongo wethiyori yokuziphendukela kwemvelo ngokubambisana kweentjalo nezinambuzane zika-Ehrlich no-Raven (51) kukubikezela bona ukuhlukahluka kokuhlukahluka kwemihlobo yeentjalo kubangela ukuhlukahluka kwemihlobo yeentjalo. Kodwana, eminyakeni ematjhumi amahlanu solo kwagadangiswa umsebenzi lo oqakatheke khulu, umbono lo bewukavami ukuhlolwa (52). Lokhu kungenxa yobujamo be-phylogenetic bebujamo be-metabolic obufanako kiyo yoke imihlobo yeentjalo ezikude. Ukungavami kungasetjenziselwa ukubophelela iindlela zokuhlaziywa kwethagethi. Indlela yokusebenza ye-MS/MS yamanje esetjenziswe yithiyori yelwazi ilinganisa ukufana kwesakhiwo se-MS/MS sama-metabolites angaziwako (ngaphandle kokukhetha i-metabolite ngaphambilini) begodu itjhugulula ama-MS/MS la abe yisethi ye-MS/MS, ngalokho-ke ekusebenzeni kwe-metabolism yobungcweti Amamodeli we-macro-evolutionary amadaniswa ngesikali sokuhlukanisa. Iinkomba ezilula zezibalo. Ikambiso le iyafana nokuhlaziywa kwe-phylogenetic, okungasebenzisa ukulandelana kokulandelana ukulinganisa izinga lokuhlukahluka nofana ukutjhuguluka kwemilingiswa ngaphandle kokubikezela ngaphambilini.
Ezingeni le-biochemical, umbono wokuhlola ka-Firn no-Jones (53) utjengisa bona ukuhlukahluka kwe-metabolic kugcinwa emazingeni ahlukileko ukunikela ngezinto ezisetjenziswako zokusebenzisa imisebenzi ye-biological yama-metabolites ebegade angahlobene namkha athathelwe indawo. Iindlela zethiyori yolwazi zinikela ngohlaka lapho amatjhuguluko wokuziphendukela kwemvelo okwenzekako ngesikhathi sokukhetheka kwe-metabolite angalinganiswa njengengcenye yendlela yokuhlola yokuziphendukela kwemvelo ehlongozwayo: ukutjhuguluka okusebenzako kwezinto eziphilako ukusuka ekukhetheni okuphasi ukuya ekukhetheni okuphezulu Ama-metabolite avinjelwe wendawo enikelweko.
Sekukonke, emalangeni wokuthoma webhayoloji yamamolekhyuli, imibono eqakathekileko yokuzivikela kweentjalo yathuthukiswa, begodu iindlela eziqhutshwa yi-hypothesis zithathwa njengendlela yokuthuthuka kwezesayensi. Lokhu kungenxa yokulinganiselwa kobuchwepheshe bokulinganisa yonke i-metabolome. Nanyana iindlela eziqhutshwa yi-hypothesis ziwusizo khulu ekukhetheni ezinye iindlela ezibangelako, ikghono lazo lokuthuthukisa ukuzwisisa kwethu amanethiwekhi we-biochemical lilinganiselwe ukudlula iindlela zokubala ezitholakala njengamanje esayensini yedatha enengi. Ngalokho-ke, iimfundiso ezingakghoni ukubikezelwa zidlula khulu ububanzi bedatha etholakalako, ngalokho-ke ifomula yokucabanga/umjikelezo wokuhlolwa wentuthuko emkhakheni werhubhululo angeze waqedwa (4). Sibona ngaphambilini bona ukugeleza komsebenzi wekhomphyutha we-metabolomics okwethulwe lapha kungavuselela ikareko eendabeni zakamuva (njani) nezokugcina (kubayini) zokuhlukahluka kwe-metabolic, begodu kube nomthelela esikhathini esitjha sesayensi yedatha eqondiswa ngokwethiyori. Isikhathi sabuyekeza iimfundiso eziqakathekileko ezakhuthaza iinzukulwana zangaphambilini.
Ukudla okuqondileko kweenlwana ezidla iintjalo kwenziwa ngokukhulisa isibungu sesibili se-instar nofana isibungu se-Sl phezu kwekhasi elilodwa lesitjalo esinombala omnyama wesitjalo esisodwa esiqhakaza i-rose, esineentjalo ezili-10 eziphindaphindiweko ngesitjalo ngasinye. Iimvukuzane zezinambuzane zabotjhwa ngama-clamp, begodu amathitjhu wamakari aseleko aqoqwa ngemva kwama-awa ama-24 nama-72 ngemva kokutheleleka begodu afrizwa msinyana, begodu ama-metabolites akhishwa.
Lingisa ukwelatjhwa kokudla utjani ngendlela evumelana khulu. Indlela yokusebenzisa amasondo wephetheni yendwangu ukubhoboza imigqa emithathu yameva ngehlangothini ngalinye le-midrib yamakari amathathu anwebile ngokupheleleko wesitjalo ngesikhathi sesigaba sokukhula kwe-garland yendwangu, bese usebenzisa msinyana i-1:5 Diluted Ms. Nofana usebenzise iminwe enegilavu ​​ukufaka i-S1 OS esilondani sokubhoboza. Vuna begodu usebenze ikari njengombana kuchaziwe ngehla. Sebenzisa indlela echazwe ngaphambilini ukukhipha ama-metabolites aphambili namahomoni weentjalo (54).
Ukusetjenziswa kwe-JA yangaphandle, amakari amathathu we-petiole weentjalo ezisithandathu eziqhakazako zomhlobo ngamunye alatjhwa nge-20μl ye-lanolin paste ene-150μg ye-MeJA (Lan + MeJA), kanye ne-20μl ye-lanolin kanye nokwelatjhwa kwesilonda (i-Lan + W), nofana sebenzisa i-20μl ye-lanolin njengokulawula. Amakari avunwa ngemva kwama-awa ama-72 ngemva kokwelatjhwa, afakwa efrijini ngaphakathi kwe-nitrogen, begodu agcinwa ku--80°C bekwaba kulapho asetjenziswa khona.
Imigqa emine ye-JA ne-ET, okuyi-irAOC (36), irCOI1 (55), irACO kanye ne-sETR1 (48), sele ikhonjwe esiqhemeni sethu sokurhubhulula. I-irAOC itjengise khulu ukwehla kwamazinga we-JA ne-JA-Ile, lokha i-irCOI1 beyingakazweli kuma-JA. Nayiqathaniswa ne-EV, ukubuthelela kwe-JA-Ile kwanda. Ngokufanako, i-irACO izokwehlisa ukukhiqizwa kwe-ET, begodu nayiqathaniswa ne-EV, i-sETR1, engazweli i-ET, izokwandisa ukukhiqizwa kwe-ET.
I-photoacoustic laser spectrometer (Sensor Sense ETD-300 real-time ET sensor) isetjenziselwa ukwenza ukulinganisa kwe-ET ngaphandle kokuhlasela. Msinyana ngemva kokwelatjhwa, ingcenye yamakari yasikwa begodu yadluliselwa emgodini wengilazi evaliweko we-4-ml, begodu isikhala sehloko savunyelwa bona sibuthelele kungakapheli ama-awa ama-5. Ngesikhathi sokulinganisa, iviyo ngalinye lahlanzwa ngomsinga wamalitha amabili/i-awa womoya ohlanzekileko imizuzu ebunane, ebegade idlule nge-catalyst enikelwe yi-Sensor Sense ukususa i-CO2 namanzi.
Idatha ye-microarray yagadangiswa ekuthomeni ku-(35) begodu yagcinwa ku-National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (inomboro yokungena GSE30287). Idatha ehambelana namakari abangelwa kuphathwa kwe-W + OSMs kanye nokulawula okungalimalanga kwakhishwa kulolu cwaningo. Ukuqina okuhle ku-log2. Ngaphambi kokuhlaziywa kwezibalo, isisekelo satjhugululwa begodu senziwa sibe sephesentini yama-75 kusetjenziswa iphakheji yesofthiwe ye-R.
Idatha yokulandelana kwe-RNA (i-RNA-seq) yemihlobo ye-Nicotiana yatholwa ku-NCBI Short Reading Archives (SRA), inomboro yephrojekthi yi-PRJNA301787, eyabikwa ngu-Zhou et al. (39) begodu uragele phambili njengoba kuchaziwe ku (56). Idatha engakalungiswa esetjenziswe yi-W + W, W + OSM kanye ne-W + OSS1 ehambelana nemihlobo ye-Nicotiana yakhethwa bona ihlaziywe esifundweni lesi, begodu yasetjenzwa ngendlela elandelako: Kokuthoma, ukufundwa kwe-RNA-seq engakalungiswa kwatjhugululwa kwaba yifomethi ye-FASTQ. I-HISAT2 itjhugulula i-FASTQ ibe yi-SAM, begodu i-SAMtools itjhugulula amafayela we-SAM abe mafayela we-BAM ahlelweko. I-StringTie isetjenziselwa ukubala ukuvezwa kwezakhi zofuzo, begodu indlela yayo yokuvezwa kukuthi kuneengcezu ngayinye yeengcezu zesisekelo eziyi-1000 ngayinye yeengcezu zokutlolwa ezilandelanako eziyisigidi.
Ikholomu ye-Acclaim chromatographic (150 mm x 2.1 mm; ubukhulu bezinhlayiya 2.2μm) esetjenziswe ekuhlaziyweni kanye nekholomu yokuvikela engu-4 mm x 4 mm yenziwe ngezinto ezifanako. I-binary gradient elandelako isetjenziswa ku-Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC): imizuzu engu-0 ukuya ku-0.5, i-isocratic 90% A [amanzi ane-ionized, 0.1% (v/v) acetonitrile kanye ne-0.05% formic acid], 1% B, kanye ne-Acetonitrile 0.05% ye-formic acid); 0.5 ukuya ku-23.5 imizuzu, isigaba se-gradient siyi-10% A kanye ne-90% B, ngokulandelana; 23.5 ukuya ku-25 imizuzu, i-isocratic 10% A kanye no-90% B. Izinga lokugeleza lingu-400μl/min. Kikho koke ukuhlaziywa kwe-MS, faka i-eluent yekholomu ngaphakathi kwe-quadrupole kanye ne-time-of-flight (qTOF) analyzer efakwe umthombo we-electrospray osebenza ngendlela ye-ionization ehle (ivoltheji ye-capillary, 4500 V; ukuphuma kwe-capillary 130 V ; izinga lokutjhisa lokomisa 200°C; ukugeleza komoya okomisa 10/m/m).
Yenza ukuhlaziywa kwe-MS / MS (okuzokubizwa ngokuthi yi-MS / MS) okungakaqakatheki nofana okungahlukaniswa nedatha ukuthola ilwazi lesakhiwo mayelana nephrofayili ye-metabolic ebonakalako. Umqondo wendlela ye-MS/MS engakhethiko uthembele eqinisweni lokuthi i-quadrupole inefasitela elikhulu khulu lokuhlukanisa ubukhulu [ngalokho, cabanga ngawo woke amatshwayo we-mass-to-charge ratio (m/z) njengeengcezu]. Ngebanga lalesi sizathu, ngombana ithulusi le-Impact II belingakghoni ukwenza ukutjhinga kwe-CE, ukuhlaziywa okuzijameleko okunengi kwenziwa kusetjenziswa amanani akhuphukileko wamandla wokutjhayisana okubangelwa ukutjhayisana (CE). Ngamafuphi, kokuthoma hlaziya isampula nge-UHPLC-electrospray ionization/qTOF-MS ngokusebenzisa indlela eyodwa ye-mass spectrometry (ubujamo obuphasi bokuhlukana obukhiqizwa ku-in-source fragmentation), ukuskena ukusuka ku-m/z 50 ukuya ku-1500 nge-repetition frequency ye-5 Hz. Sebenzisa i-nitrogen njengegesi yokutjhayisana yokuhlaziywa kwe-MS/MS, begodu wenze iinlinganiso ezizijameleko kumavoltheji amane alandelako wokutjhayisana-induced dissociation voltage: 20, 30, 40, kanye ne-50 eV. Kiyo yoke ikambiso yokulinganisa, i-quadrupole inefasitela elikhulu lokuhlukanisa ubunzima, ukusuka ku-m/z 50 ukuya ku-1500. Lokha umzimba wangaphambili m/z nokuhlolwa kobubanzi bokuhlukanisa kusethwe ku-200, ububanzi bobunzima busebenza ngokuzenzakalelayo yisofthiwe yokusebenza yesisetjenziswa kanye no-0 Da. Skena iingcezu zesisindo njengemodi yesisindo esisodwa. Sebenzisa i-sodium formate (50 ml isopropanol, 200 μl formic acid kanye ne-1 ml 1M NaOH aqueous solution) ukulinganisa ubukhulu. Ngokusebenzisa i-algorithm yokulinganisa ephezulu ye-Bruker, ifayela ledatha liyalinganiswa ngemva kokusebenzisa i-avareji ye-spectrum ngesikhathi esithileko. Sebenzisa umsebenzi wokuthumela ngaphandle wesofthiwe ye-Data Analysis v4.0 (Brook Dalton, Bremen, Germany) ukutjhugulula amafayela wedatha engakalungiswa abe yifomethi ye-NetCDF. Isethi yedatha ye-MS/MS igcinwe ku-database ye-metabolomics evulekileko i-MetaboLights (www.ebi.ac.uk) nenombolo yokungena. MTBLS1471.
Ukuhlanganiswa kwe-MS/MS kungenziwa ngokuhlaziywa kokuhlobana hlangana kwe-MS1 kanye namasignali wekhwalithi ye-MS/MS yamandla aphasi naphezulu wokutjhayisana nemithetho esanda kusetjenziswa. Iskripthi se-R sisetjenziselwa ukufezekisa ukuhlaziywa kokuhlobana kokusabalala kwesandulela somkhiqizo, begodu iskripthi se-C# (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) sisetjenziselwa ukusebenzisa imithetho.
Ukuze sinciphise amaphutha angamanga abangelwa msindo wangemuva nokuhlobana okungamanga okubangelwa kutholakala kwezinto ezithileko ze-m/z kumasampula ambalwa kwaphela, sisebenzisa umsebenzi “we-filled peak” wephakheji le-R XCMS (yokulungisa umsindo wangemuva) Kufanele isetjenziselwe ukuthatha indawo ye-“NA” (i-peak engabonakaliko) ukuqina. Lokha nakusetjenziswa umsebenzi we-fill peak, kusese namagugu amanengi we-“0′′ wokuqina esethini yedatha azokuthinta ukubalwa kokuhlobaniswa. inani langemuva elibaliweko. Sibuye sacabangela kwaphela iimpawu ezinamandla wazo adlula inani lemuva kathathu begodu sazithatha njenge "ziqongo zamambala." Ekubalweni kwe-PCC, kucatshangelwa kwaphela amatshwayo we-m/z wesampula eyandulelako (MS1) kanye namasethi wedatha yeengcezu ezinama-peak abunane weqiniso okungenani.
Nangabe ukuqina kwesici sekhwalithi ye-precursor kiyo yoke isampula kuhlotjaniswa khulu nokuqina okunciphileko kwesici sekhwalithi efanako esibekelwe amandla aphasi nofana aphezulu wokutjhayisana, begodu isici singabhalwa njenge-isotope peak yi-CAMERA, singachazwa ngokuragela phambili. Bese, ngokubala woke ama-precursor-product pairs angaba khona ngaphakathi kwe-3 s (isikhathi esilinganiselwe sokugcinwa kokugcinwa okuphezulu), ukuhlaziywa kokuhlobana kwenziwa. Kuphela lokha inani le-m/z lingaphasi kwenani lesandulela begodu ukuhlukaniswa kwe-MS/MS kwenzeka endaweni efanako yesampula kusethi yedatha njengesandulela lapho ithathwe khona, lapho kuthathwa njengesiqephu.
Ngokuya ngale mithetho emibili elula, sikhipha iincezu ezitjhiwoko ezinamanani we-m/z amakhulu kune-m/z yesandulela esikhonjiweko, begodu ngokuya ngendawo yesampula lapho isandulela sivela khona nesiqetjhana esitjhiwoko. Kungenzeka nokuthi kukhethwe iimfanelo zekhwalithi ezenziwe ziingcezu ezinengi ezikhiqizwa nge-MS1 njengeenqalo ezikhethiweko, ngaleyo ndlela kukhiqizwe amakhompawundi we-MS/MS angatlhogekiko. Ukuze kwehlise ukungabi khona kwedatha, nangabe ukufana kwe-NDP kwama-spectra kudlula i-0.6, begodu kunge-chromatogram “pcgroup” ebhalwe yi-CAMERA, sizoyihlanganisa. Ekugcineni, sihlanganisa yoke imiphumela emine ye-CE ehlotjaniswa nesandulela kanye neengcezu zibe yi-spectrum yokugcina ehlanganisiweko ngokukhetha isiqongo esiphezulu khulu hlangana nazo zoke iinqophiso ezikhethiweko ezinenani elifanako le-m/z emandleni ahlukileko wokutjhayisana. Amagadango wokucubungula alandelako asekelwe emqondweni we-spectrum ehlangeneko begodu aqalele ubujamo obuhlukileko be-CE obutlhogekako ukukhulisa amathuba wokuhlukana, ngombana ezinye iingcezu zingatholakala ngaphasi kwamandla athileko wokutjhayisana.
I-RDPI (30) yasetjenziselwa ukubala ukuthonywa kwephrofayili yokugaya ukudla. Ukuhlukahluka kwe-metabolic spectrum (i-Hj index) kuthathwa ebunengini be-MS/MS precursors kusetjenziswa i-Shannon entropy ye-MS/MS frequency distribution kusetjenziswa i-equation elandelako echazwe ngu-Martínez et al. (8). Hj = −∑i = 1mPijlog2(Pij) lapho i-Pij ihambelana nefrekwensi ehlobene ye-i-th MS/MS ku-j-th isampula (j = 1, 2,…, m) (i = 1, 2, …, m) t).
Ukukhetheka kokugaya ukudla (inkomba ye-Si) kuchazwa njengobunjalo bokuvezwa kwe-MS/MS enikelweko malungana nokuvama hlangana namasampula aqakathekiswako. Ukukhethekileko kwe-MS/MS kubalwa njenge-Si = 1t (∑j = 1tPijPilog2PijPi)
Sebenzisa ifomula elandelako ukulinganisa i-δj index ye-metabolome yesampula ngayinye ye-j, kanye nesilinganiso se-MS/MS specificity δj = ∑i = 1mPijSi
I-MS/MS spectra ihlanganiswe ngababili, begodu ukufana kubalwa ngokuya ngamaphuzu amabili. Kokuthoma, ngokusebenzisa i-NDP ejwayelekileko (eyaziwa nangokuthi yindlela yokuhlobana ye-cosine), sebenzisa i-equation elandelako ukuthola ukufana kwesigaba hlangana nama-spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 lapho i-S1 ne-S2 Ngokuhambisana, nge-spectrum 1 ne-2, kanye ne-iWS, i2 imelela ubudisi obusekelwe ekuqineni kwesiqongo ukuthi umehluko wesiqongo esivamileko se-i phakathi kwama-spectra amabili ungaphasi kwe-0.01 Da. Ubunzima bubalwa ngalendlela: W = [i-peak intensity] m [ikhwalithi] n, m = 0.5, n = 2, njengombana kuphakanyiswe yi-MassBank.
Indlela yesibili yokufaka amaphuzu yasetjenziswa, eyayifaka hlangana ukuhlaziya i-NL eyabelwanako hlangana ne-MS/MS. Ukuze sifeze lokhu, sisebenzise amarhelo we-NL ama-52 avame ukuhlangana nawo ngesikhathi senqubo yokuhlukaniswa kwe-MS ngokulandelana, kanye ne-NL ekhethekileko (ifayela ledatha S1) ebegade ibhalwe ngaphambilini nge-MS/MS spectrum yama-metabolites wesibili wemihlobo ye-Nepenthes ebuthakathaka ( 9, 26). Yenza i-vector ye-binary ye-1 ne-0 ye-MS/MS ngayinye, ehambelana ne-NL yamanje nengakho ngokulandelana. Ngokuya ngokufana kwebanga le-Euclidean, isikolo sokufana se-NL sibalwa nge-pair ngayinye yama-vector we-NL kanambambili.
Ukwenza ukuhlanganiswa okubili, sisebenzise iphakheji ye-R i-DiffCoEx, esekelwe ekwandisweni kwe-Weighted Gene Co-expression Analysis (WGCNA). Ngokusebenzisa i-NDP kanye ne-NL scoring matrices ye-MS/MS spectra, sisebenzise i-DiffCoEx ukubala i-comparative correlation matrix. Ukuhlanganiswa kwe-binary kwenziwa ngokubeka ipharamitha ye-“cutreeDynamic” ku-method = “hybrid”, cutHeight = 0.9999, deepSplit = T, kanye ne-minClusterSize = 10. Ikhodi yomthombo ye-R ye-DiffCoEx yalandwa kufayela elengeziweko 1 ngu-Tesson et al. (57); Iphakheji yesofthiwe ye-R WGCNA etlhogekako ingatholakala ku-https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Ukuze senze ukuhlaziywa kwenethiwekhi yamamolekhyuli ye-MS/MS, sibale ukuxhumana kwe-spectral ngokuya ngemihlobo yokufana ye-NDP ne-NL, begodu sasebenzisa isofthiwe ye-Cytoscape ukubona ngeso lengqondo ithopoloji yenethiwekhi ngokusebenzisa ukuhleleka kwe-organic ku-CyFilescape yFiles layout algorithm extension application.
Sebenzisa i-R version 3.0.1 ukwenza ukuhlaziywa kwezibalo kudatha. Ukuqakatheka kwezibalo kwahlolwa kusetjenziswa ukuhlaziywa kweendlela ezimbili zokuhlukahluka (i-ANOVA), kulandelwe yi-Tukey's honestly significant difference (HSD) post-hoc test. Ukuze kuhlaziywe umehluko phakathi kokwelatjhwa kokudla utjani nokulawula, ukusabalalisa okunemisila emibili yamaqembu amabili wamasampula anokuhluka okufanako kwahlaziywa kusetjenziswa i-Student's t test.
Ukuthola izinto ezingezelelweko ze-atikili le, sibawa uqale i-http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
Le yi-atikili evulekileko esabalaliswa ngaphasi kwemibandela ye-Creative Commons Attribution-Non-Commercial License, evumela ukusetjenziswa, ukusabalalisa nokukhiqizwa kabutjha nganoma ngiyiphi indlela, inqobo nje nangabe ukusetjenziswa kokugcina kungasi kwezokurhweba begodu umqondo kukuthi umsebenzi wokuthoma ulungile. Ireferensi.
Tjheja: Sikubawa bona unikele ngekheli lakho le-imeyili ukwenzela bona umuntu omncomako ekhasini azi bona ufuna bona abone i-imeyili begodu akusiyo i-spam. Angeze sathatha amakheli we-imeyili.
Umbuzo lo usetjenziselwa ukuhlola bona usivakatjhi begodu uvimbele ukuthunyelwa kwe-spam okuzenzakalelayo.
Ithiyori yelwazi inikela ngemali ejayelekileko yokumadanisa ama-metabolome akhethekileko nokubikezela ithiyori yokuzivikela yokuhlolwa.
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