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Ngokungafaniyo nezilwanyana ezinethambo lomqolo, izinambuzane zikholelwa kakhulu ukuba azinazo iihomoni ze-steroid zesini ezikhethwe ngamadoda. Kwi-Anopheles gambiae, i-ecdysone steroid 20-hydroxyecdysone (20E) ibonakala ngathi iphuhlise ukulawula uphuhliso lweqanda xa yenziwe ngabasetyhini2 kwaye ibangele ixesha lokunganyamezelani xa idluliselwa ngokwesondo ngamadoda3. Ekubeni uphuhliso lweqanda kunye nokudibana ziimpawu ezibalulekileyo zokuzala, ukuqonda indlela iingcongconi ze-Anopheles ezingamabhinqa ezidibanisa ngayo le miqondiso yehomoni kunokunceda ukuyilwa kweenkqubo ezintsha zokulawula i-malaria. Apha, sityhila ukuba le misebenzi yokuzala ilawulwa zii-steroids zesini ezahlukeneyo ngenethiwekhi eyinkimbinkimbi yee-enzymes ezivuselela/ezivuselela i-ecdysteroid. Sichonge i-ecdysone e-oxidized ethile yendoda, i-3-dehydro-20E (3D20E), ekhusela ubuzali ngokuvala ukwamkelwa kwesondo kwabasetyhini emva kokudluliselwa ngokwesondo kunye nokusebenza nge-dephosphorylation. Okuphawulekayo kukuba, ukudluliselwa kwe-3D20E kukwabangele ukubonakaliswa kwezakhi zofuzo zokuzala ezigcina uphuhliso lweqanda ngexesha losulelo lwePlasmodium, ukuqinisekisa impilo yabasetyhini abanentsholongwane. I-20E evela kubasetyhini ayivezi impendulo yesondo, kodwa ivumela abantu abadibanayo ukuba babeke amaqanda emva kokuba i-20E-inhibiting kinases ithintelwe. Ukuchongwa kwale steroid hormone yesinambuzane ethile yamadoda kunye nendima yayo ekulawuleni ukwamkelwa kwesondo kwabasetyhini, ukuzala kunye nokusebenzisana nePlasmodium kubonisa ukuba kunokwenzeka ukunciphisa impumelelo yokuzala kweengcongconi ezisasaza imalariya.
Amatyala nokufa kwemalariya kuyanda kwakhona4 ngenxa yokuxhathisa izinambuzane kwiingcongconi zeAnopheles, ezikuphela kwento ebangela iintsholongwane zemalariya ebantwini.Ibhayoloji yokuzala kwezi ncongconi yeyona nto inomtsalane kakhulu kwiindlela ezintsha zokulawula imalariya kuba iimazi zizala kanye kuphela5; ukwenza esi siganeko sokuzala esinye singabi nazintsholongwane kuya kuba nethuba elikhulu lokunciphisa inani leengcongconi endle.
Abafazi bayakhubazeka ngokwesondo emva kokufumana ii-hormone ze-steroid eziphezulu ezivela kumadoda. Izifundo zibonise ukuba imbangela yobunzima ekuzalweni ngakumbi yi-20-hydroxyecdysone (20E), i-hormone ye-steroid eyaziwa ngcono njengomlawuli womjikelo wokubola kwinqanaba le-larval. Amandla amadoda okwenza nokudlulisa i-20E avele ngokukodwa kwiintlobo ze-Anopheles eziyinxalenye ye-subgenus Cellia7, esasazeke e-Afrika kwaye ibandakanya ii-vectors eziyingozi kakhulu ze-malaria, kubandakanya i-Anopheles gambiae. Oku kuyaphawuleka ngakumbi kuba kwezi ntlobo abafazi bavelisa i-20E emva kokutya kwegazi ngakunye, kwaye i-20E iqhuba umjikelo we-oogenesis (jonga ireferensi 8). Nangona kunjalo, kuncinci okwaziwayo malunga nendlela abafazi abadibanisa ngayo imiqondiso evela kwimithombo emibini eyahlukeneyo ye-ecdysone (ukudluliselwa kwamadoda kunye nokungeniswa kwegazi) ngaphandle kokubeka emngciphekweni amandla abo okutshata. Enyanisweni, ukuba i-20E eveliswa ngabafazi ibangela ukunganyamezelani ngokwesondo, oku kuya kukhokelela ekungazalini kubantu abancancisayo, into eqhelekileyo kwezi zinambuzane5.
Ingcaciso enokwenzeka kukuba iinkunzi ze-A. gambiae zidlulisela i-ecdysone eguquliweyo yendoda ethile, evuselela i-cascade yesignali kwindlela yokuzala yabasetyhini, nto leyo ebangela ukungazinzi kokudibana. Nangona kunjalo, nangona izilwanyana ezinethambo lomqolo zine-hormone ezininzi ze-steroid, ezifana ne-estrogen kunye ne-androgen (ehlaziyiweyo kwireferensi 9), ngokolwazi lwethu, ii-steroids ezichasene ne-androgen azikafunyanwa kwizinambuzane.
Sizimisele ukufumanisa uluhlu lwee-hormone ze-steroid kwi-male male accessory gland (MAG) ye-A. gambiae ekhulileyo ngokwesondo ekufuneni ii-steroids ezinokuthi ziguqulwe. Sisebenzisa i-chromatography yolwelo esebenza kakuhle edibene ne-tandem mass spectrometry (HPLC-MS/MS) endaweni yendlela engacacanga kangako esetyenzisiweyo ngaphambili, sifumanise i-ecdysone (E) kunye ne-20E kule tissue, siqinisekisa iziphumo zangaphambili. Nangona kunjalo, isampuli yayilawulwa yi-oxidized phosphorylated steroids, ehambelana nefomula ye-3-dehydro-20E-22-phosphate (3D20E22P)12 (Umfanekiso 1). Ezinye iintlobo ziquka i-3-dehydro-20E (3D20E) kunye ne-20E-22-phosphate (20E22P). Ubunzulu besignali ye-HPLC-MS/MS ye-3D20E22P yayizii-oda ezimbini zobukhulu obuphezulu kunefom yayo ye-dephosphorylated, i-3D20E, kunye ne-oda ezintathu zobukhulu obuphezulu kune-E kunye ne-20E (Umfanekiso 1). Nangona kwezinye iindawo zomzimba kunye nomzila wokuzala osezantsi (LRT; Extended Data Fig. 1a). Sikwahlalutye ii-ecdysteroids kumadoda nabafazi abasandula ukuvalwa (
Ukuze sihlolisise ukuba ingaba i-3D20E22P kunye ne-3D20E ziyadluliselwa na ngexesha lokudibana, sihlalutye ii-LRT zabasetyhini ngamaxesha ahlukeneyo emva kokudibana. Nangona i-ecdysone ingafumanekanga kwiintombi, sibonile ubungakanani obukhulu be-3D20E22P kwi-LRT emva kokudibana (iiyure eziyi-0.5 emva kokudibana, i-hpm), buyehla ngokuhamba kwexesha, ngelixa amanqanaba e-3D20E enyuka kakhulu (Umzobo 1). Sisebenzisa i-3D20E eyenziwe ngamakhemikhali njengomgangatho, sigqibe kwelokuba amanqanaba ale hormone ye-steroid kwi-LRTs zokudibana ayengaphezulu ngokuphindwe kalikhulu kune-20E (Itheyibhile yeDatha eYandisiweyo 1). Ke ngoko, i-3D20E22P yeyona ecdysone iphambili yamadoda edluliselwa kwi-LRT yabasetyhini ngexesha lokudibana, kwaye uhlobo lwayo olungenaphosphorylated, i-3D20E, luba lukhulu kakhulu emva kokudibana. Oku kubonisa indima ebalulekileyo kwi-ecdysone yokugqibela kwibhayoloji yabasetyhini emva kokudibana.
Emva kokuvelisa iseti entsha yedatha yokulandelelana kwe-RNA (i-RNA-seq) (Umzobo 2a), sisebenzisa umbhobho we-bioinformatics owakhelwe ngokwezifiso, sikhangele i-ecdysone kinase (EcK), i-ecdysone oxidase (EO), kunye ne-ecdysone encoding i-20E-modified phosphatase gene. I-EPP) ibonakaliswa kwizicubu zokuzala. Sichonge i-gene enye ye-EPP ekhethiweyo kunye ne-genes ezimbini ze-EcK ezinokubakho (i-EcK1 kunye ne-EcK2), kodwa asikwazanga ukufumana i-gene elungileyo ye-EO ekhethiweyo. Okuphawulekayo kukuba, ii-genes ze-EPP ngazinye zibonakaliswe kumanqanaba aphezulu (i-98.9th percentile) kwi-Gambian MAGs kodwa kungekhona kwi-LRTs zabasetyhini (Umzobo 2b), ngokuchaseneyo nolindelo lwethu kuba i-dephosphorylation ye-3D20E22P yenzekile kule tissue yabasetyhini. Ke ngoko, sikholelwa ukuba i-EPP yamadoda inokudluliselwa ngexesha lokudibana. Enyanisweni, sisebenzise ilebhile ye-isotope eqinileyo kwi-vivo ukufihla iprotheni yabasetyhini emva kokudibana, i-enzyme echongiwe yi-MS kwi-atrium yabasetyhini (Umzobo 2c kunye Itheyibhile eyoNgezelelweyo 1). Ubukho be-EPP kwi-MAG kunye ne-LRT yebhinqa edibeneyo (kodwa kungekhona emsulwa) nayo iqinisekisiwe kusetyenziswa ii-antibodies ezithile (Umzobo 2d).
a, Umbhobho we-bioinformatics owakhelwe ngokwezifiso ukukhangela izicubu zokuzala zesini ngasinye ukuze ufumane ii-genes ezifaka ii-EcKs, ii-EOs, kunye nee-EPPs. Amanani akufutshane neentolo abonisa inani labaviwa abangamadoda nabasetyhini kwinqanaba ngalinye. Olu hlalutyo luchonge i-gene enye ye-EPP (EPP) kunye ne-gene enye ye-EcK (EcK1) ebonakaliswa kumadoda, kunye ne-gene enye ye-EcK (EcK2) ebonakaliswa kuzo zombini izini kodwa ayivelisi i-gene ye-EO evunyiweyo.b, i-Heatmap ethelekisa ukubonakaliswa kwe-gene evunyiweyo kwi-virgin (V) kunye ne-mating (M) i-Anopheles gambiae kunye ne-Anopheles albicans tissues. I-Spca, ukuchumisa; ii-MAGs, ii-accessory glands kumadoda; amanye amalungu omzimba, kuquka amabele, amaphiko, imilenze, imizimba etyebileyo, kunye nezitho zangaphakathi kuzo zombini izini, kunye nama-ovari kuma-female. I-EcK2 ibonakaliswa kakhulu kwi-MAG nakwi-atria yaseGambia, ngelixa i-EPP ifumaneka kuphela kwi-MAG.c, uhlalutyo lwe-Proteomic lwe-mass ejaculate group translocation kwi-female atria kwi-3, 12 kunye ne-24 hpm, ebonisa iiproteni ezingama-67 ezininzi kakhulu. Ama-female akhuliswe ngokutya okuqulethe i-15N ukuze abhale iilebhile (kunye nemaski) zonke iiproteni. Amadoda angafakwanga i-tag adityaniswe nama-female tag, kwaye ama-LRT ama-female ahlulwe kwi-3, 12 kunye ne-24 hpm ukuze kuhlalutywe i-proteomic (jonga iTheyibhile eyoNgezelelweyo 1 ukuze ufumane uluhlu olupheleleyo lweeproteni zokukhupha i-ejaculatory). I-Inset, i-EPP, i-Eck1 kunye ne-EcK2 zifunyenwe kwi-MAG yama-female angoo-virgin ngohlalutyo lwe-proteomic lwala ma-tissue.d, i-EPP ifunyenwe yi-western blot kwi-MAG kunye ne-LRT yama-female angoo-virgin, kodwa kungekhona kuma-female angoo-virgin okanye amadoda okanye amanye ama-female. umzimba. Ii-membranes zihlolwe ngaxeshanye nge-anti-actin (ulawulo lokulayisha) kunye nee-anti-EPP antibodies. Onke amadoda angoontombi. Jonga uMfanekiso oNgezelelweyo 1 ukuze ufumane idatha yomthombo wejeli. Ii-Western blots zenziwe kabini ngeziphumo ezifanayo.
Umsebenzi we-ecdysteroid phosphophosphatase we-EPP uqinisekiswe emva kokufakwa kwi-incubation yi-HPLC-MS/MS nge-3D20E22P ehlukanisiweyo kwi-MAG (Umzobo weDatha oLandisiweyo 2a). Ngaphezu koko, xa sathulisa i-EPP ngokuphazamiseka kwe-RNA (RNAi), safumanisa ukuncipha okukhulu komsebenzi we-phosphatase kwizicubu zokuzala zala madoda (Umzobo 3a), kwaye amabhinqa adibene namadoda athuliswe yi-EPP abonise ukubaluleka Inani eliphantsi le-dephosphorylated 3D20E (Umzobo 3b) nangona i-gene ithulile kancinci (Umzobo weDatha oLandisiweyo 2b,c). Ngokwahlukileyo koko, asizange sifumane utshintsho olubalulekileyo kumlinganiselo we-20E22P/20E kwiingcongconi ezifanayo, nto leyo enokubonisa ukuba i-enzyme ikhethekileyo kwi-3D20E22P (Umzobo 3b).
a, Ukuncipha komsebenzi we-phosphatase kwi-MAG okubangelwa kukuthulisa i-EPP kusetyenziswa ulawulo lwe-EPP RNA (dsEPP) olunemicu emibini okanye ulawulo lwe-GFP RNA (dsGFP) olunemicu emibini. Kusetyenziswe amachibi e-MAG angamashumi amabini kwi-replica nganye (P = 0.0046, uvavanyo lwe-t oludibeneyo, olunemicu emibini), olumelwe ngamachaphaza ahlukeneyo.b, Abafazi abadibene namadoda anesithulisi se-EPP babenenani eliphantsi kakhulu le-3D20E e-dephosphorylated kwi-3 hpm (P = 0.0043, uvavanyo lwe-t olungadibaniyo, olunemicu emibini), ngelixa amanqanaba e-20E engachaphazelekanga (P = 0.063, olunemicu emibini). uvavanyo lwe-t, olunecala ezimbini). Idatha iboniswa njenge-avareji ± sem evela kumachibi amathathu eemazi ezili-13, 16 kunye ne-19 nganye. c, Iimazi ezidibene neemazi ezithuliswe yi-EPP zazinamazinga aphezulu kakhulu okuphinda zihlangane (P = 0.0002, uvavanyo oluchanekileyo lukaFisher, olunecala ezimbini). Iimazi zaqala ukunyanzelwa ukuba zihlangane ukuqinisekisa imeko yazo yokudibana; Emva kweentsuku ezi-2, badibana namanye amadoda aphethe isidoda esiguqulweyo ukuze kuvavanywe amazinga okuphinda ahlangane ngokufumanisa i-PCR yobuninzi be-transgene.d, iimazi ezondliwe ngegazi ezidibene namadoda athuliswe yi-EPP zazinciphise kakhulu ukuzala (P < 0.0001; uvavanyo lweMann-Whitney, olunecala elinye) kunye nenani lamaqanda elinciphileyo kancinci (P = 0.088, uvavanyo lweMann-Whitney, olunecala elinye), ngelixa izinga lokuzala lingachaphazelekanga (P = 0.94, uvavanyo oluchanekileyo lukaFisher, olunecala elinye). Kuzo zonke iiphaneli, u-n umele inani leesampuli zeengcongconi ezizimeleyo ngokwebhayoloji.NS, ayibalulekanga.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Okulandelayo, sivavanye ukuba i-ecdysone dephosphorylation ibalulekile na ekukhuthazeni ukumelana nokudibana kwabasetyhini. Okuphawulekayo kukuba, abafazi abadibene namadoda aphelelwe yi-EPP baphinda badibana rhoqo (44.9%) kunabasetyhini abalawulayo (10.4%) xa bevezwa kumadoda angaphezulu (aguqulweyo) (Umzobo 3c). Sikwabone ukwehla okukhulu kokuzala (Umzobo 3d, ekhohlo) kunye nokwehla okuncinci kwenani lamaqanda abekwe ngaba bafazi (Umzobo 3d, embindini), ngelixa ipesenti yamaqanda abekwe ngabasetyhini (enye impendulo evezwe kubafazi ngokudibana )) ayichaphazelekanga (Umzobo 3d, ekunene). Ngenxa yokuchazwa okubonakalayo kwe-EPP kwi-3D20E22P, ezi ziphumo zibonisa ukuba ukusebenza kwe-3D20E yi-EPP edluliselwe ngexesha lokudibana kunokuba nendima ebalulekileyo ekucimeni ukwamkelwa kwabasetyhini ekuhlanganeni ngakumbi, ukuziphatha okwabangelwa ngaphambili kukudluliselwa ngokwesondo kwe-20E. Ke ngoko, le hormone ethile yamadoda ikwachaphazela kakhulu ukuzala kwabasetyhini.
Okulandelayo, sithelekise imisebenzi ye-20E kunye ne-3D20E kwiimvavanyo zenaliti kwiintombi ezikhulileyo ngokwesondo sisebenzisa i-3D20E eyenziwe ngamakhemikhali (Umzobo 4a–c) kunye ne-20E ethengiswayo. Siqaphele ukuba i-3D20E yayisebenza ngakumbi kune-20E ekuvaleni uvakalelo lwabasetyhini ekutshateni kuzo zombini iindawo zoxinzelelo (Umzobo 4d). Okuphawulekayo kukuba, isiqingatha senqanaba le-physiological le-3D20E kwi-LRT (1,066 pg emva kokufakwa vs. 2,022 pg emva kokudibana) sabangela inani labasetyhini abachasayo elaliphezulu ngokuphindwe ka-20 kunenqanaba le-physiological le-20E (361 pg emva kokufakwa) kwiiyure ezingama-24 emva kokufakwa kwi-concentration ephezulu ye-18 pg emva kokudibana; Itheyibhile yeDatha eyongeziweyo 1). Esi siphumo sihambelana nombono wokuba ukudluliselwa ngokwesondo kwe-20E akubangeli amaxesha okudibana angaqhelekanga, kwaye siqhubeka sibonisa i-3D20E njengento ebalulekileyo ekuqinisekiseni ubudlelwane bomzali nomntwana. I-3D20E nayo yayisebenza kakhulu kune-20E kwiimvavanyo zokubeka amaqanda kwiintombi ezingatshatanga (Umzobo 4e), nto leyo ebonisa ukuba izinga eliqhelekileyo lokubeka amaqanda esilibonileyo emva kokuthulisa i-EPP kancinci kwakubangelwa bubukho bomsebenzi oseleyo we-3D20E osaveliswa zizinto ezibangelwa kukudibana kwabasetyhini.
(a,b) I-3D20E eyenziwe ngeekhemikhali ukusuka kwi-20E (a) ngokuguqulwa/ukusebenza kakuhle okuphezulu kakhulu (idatha evezwe njenge-avareji ± sem evela kwiimpendulo ezintathu ezizimeleyo zokwenziwa) (b).c, i-Mass spectrum (isiqingatha esisezantsi) ifana ncamashi ne-ecdysone efunyenwe kwi-LRT yabasetyhini edibeneyo (isiqingatha esiphezulu).d, Xa kuthelekiswa ne-20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Uvavanyo oluchanekileyo lukaFisher, olunecala ezimbini) kunye ne-10% ye-ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Uvavanyo oluchanekileyo lukaFisher, olunecala ezimbini), ngelixa i-20E yayiphezulu kakhulu kunolawulo kuphela kwiidosi eziphezulu (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Uvavanyo oluchanekileyo lukaFisher, 2-side).e, inaliti ye-3D20E ibangele amazinga aphezulu kakhulu okuzala kwiimazi eziziintombi kune-10% yolawulo lwe-ethanol (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; uvavanyo oluchanekileyo lukaFisher, olunecala ezimbini), ngelixa i-20E ithelekiswa nolawulo kuphela kwiidosi eziphezulu (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; uvavanyo oluchanekileyo lukaFisher, olunecala ezimbini).I-3D20E ibangele amazinga aphezulu kakhulu okuzala kune-20E kwiidosi eziphezulu (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; uvavanyo oluchanekileyo lukaFisher, olunecala ezimbini).Kuzo zonke iiphaneli, u-n umele inani leesampuli zeengcongconi ezizimeleyo ngokwebhayoloji.NS, ayibalulekanga.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. Idatha ivela kwiikopi ezintathu.
Kwizifundo zangaphambili, sifumanise ukuba ukudluliselwa ngokwesondo kwee-hormone ze-steroid kubangela ukuvezwa kwe-MISO (Mating-Induced Stimulator of Oogenesis 11), i-gene yokuzala yabasetyhini ekhusela iimazi ze-A. gambiae kwi-P. falciparum infection. Iindleko zempilo ezibangelwa yi-13, i-parasite ye-malaria yabantu ebulalayo. Ngenxa yokubaluleka kwe-MISO kwimpilo yokuzala ye-Anopheles kwiindawo ezixhaphakileyo kwi-malaria, sigqibe ekubeni simisele ukuba yeyiphi i-hormone i-3D20E okanye i-20E ebangela ukubonakaliswa kwale gene. Sifumanise ukuba ngelixa i-20E injection ngokukodwa okanye ngamandla ngakumbi ibangele ezinye ii-receptors ze-nuclear hormone (HR), ezifana ne-HR3 kunye ne-HR4, kunye neethagethi eziqhelekileyo ze-steroid, ezifana ne-yolkogenic genes Vg14, 15, 16, i-MISO yabangelwa ngamandla yi-3D20E (Umzobo weDatha oLwandisiweyo 3). Ke ngoko, ukudluliselwa ngokwesondo kwale hormone ye-androgenic steroid kubonakala ngathi kubangela iindlela ezikhusela amabhinqa kwiindleko ezibangelwa lusulelo lwe-parasitic. Ngaphezu koko, i-3D20E ichaphazela ngokwahlukileyo zombini ii-isoforms ze i-E receptor EcR, ebangela i-EcR-A kwaye icinezela i-EcR-B, kwaye ivuselela ngakumbi ezinye ii-genes ezibangela ukudibana, kuquka i-HPX15, echaphazela ukuzala kwabasetyhini. Oku kunokuchaza ukungazali okubalulekileyo okubonwa kubasetyhini abadibene namadoda athuliswe yi-EPP (Umzobo weDatha oLwandisiweyo 3). Ezi datha zibonisa ukuba kukho iindlela ezisezantsi ezivuselelwa ngokukhethekileyo zii-hormone ezimbini ze-ecdysone ezinokubangela umsebenzi othile wesini.
Okulandelayo, sivavanye umsebenzi wee-genes ezimbini ze-EcK ezichongiweyo kumbhobho wethu we-bioinformatics. Ukuthulisa i-EcK1 okanye i-EcK2 kubangele ukufa okukhulu kumadoda (Umzobo weDatha eNwetyisiweyo 4a), nto leyo ebonisa ukuba i-ecdysone phosphorylation, ngaloo ndlela ingasebenzi, kubalulekile ukuze kusinde. Ngenxa yokuba i-EcK2 ibonakaliswe kumanqanaba aphezulu kune-EcK1 kwaye yafunyanwa kwi-MAGs yi-proteomics (Umzobo 2b,c kunye neTheyibhile eyoNgezelelweyo 2), siqinisekisile umsebenzi wayo we-ecdysteroid kinase ngokuyifunxa nge-20E, nto leyo eyabangela i-phosphorylation 20E22P (Umzobo weDatha eNwetyisiweyo 2).4b). Xa sisebenzisa i-3D20E njenge-substrate, asikwazanga ukubona imveliso ye-phosphorylated 3D20E22P (Umzobo weDatha eNwetyisiweyo 4c), nto leyo ebonisa ukuba i-20E endaweni ye-3D20E isenokuba yeyona nto ikhethwayo ye-EcK2.
Ngokwe-RNA-seq analysis yethu, i-EcK2 ikwabonakala kakhulu kwi-LRT yeentombi eziziintombi, apho yayicinywa khona emva kokudibana (Umzobo 2b). Siqinisekisile le datha saza safumanisa ukuba ukubonakaliswa kwe-EcK2 akuchaphazelekanga kukondla ngegazi (Umzobo weDatha oNgezelelweyo. 5a). Sandisa uvavanyo lwethu lokuqala lwe-MS, safumanisa ukuba incopho ye-20E22P yayinxulumene kakhulu nencopho ye-20E (iiyure ezingama-22-26 emva kokutya igazi; Umzobo weDatha oNgezelelweyo. 5b). Ukuthuliswa kwe-EcK2 kwiintombi eziziintombi kubangele ukwanda okuphindwe kathathu kumlinganiselo ohambelanayo we-20E ukuya kwi-20E22P kwiiyure ezingama-26 emva kokutya igazi (IiDatha eziNgezelelweyo. 2c kunye ne-5c), kuqinisekisa ukuba i-EcK2 ikwaphosphorylates i-20E kwiintombi eziziintombi. Okuphawulekayo kukuba, iintombi eziziintombi eziphelelwe yi-EcK2 zigcine ukwamkelwa ngokupheleleyo ngokwesondo (Umzobo weDatha oNgezelelweyo. 5d,e), nto leyo ebonisa ukuba ukuveliswa kwe-20E yabasetyhini akukhuthazi ukuchasana kokudibana. amaxesha.Nangona kunjalo, la mabhinqa ayenyuke kakhulu amazinga okuzala amaqanda xa kuthelekiswa nalawo alawulwayo, kunye nama-30% eentombi ezizala amaqanda (Umzobo weDatha oNwetyisiweyo 5f).Ukuba iinaliti ze-Eck2 RNA (dsEcK2) ezinemicu emibini zenziwe emva kokondliwa ngegazi, ukuzala akuzange kwenzeke, apho incopho ye-20E ngenxa yokufunxwa kwegazi yehlile. Ngokubanzi, ezi ziphumo zixhasa imodeli eyenziwe yi-20E emva kokufunxwa kwegazi inokubangela ukuzala, kodwa kuphela xa ibhloko yokuzala (i-EcK2 kunye nezinye izinto) icinyiwe ngokufunxwa. Akukho naliti ze-20E okanye ze-3D20E ezithintele ukubonakaliswa kwe-EcK2 kwiintombi ezintombi (Umzobo weDatha oNwetyisiweyo 5g), nto leyo ebonisa ukuba ezinye izinto zilawula ukuthintelwa kwale kinase.Nangona kunjalo, amanqanaba e-20E emva kokondliwa ngegazi ayenganelanga ukubangela ukungakhululeki kokuzala, kodwa abangelwa ngempumelelo zii-titers eziphezulu ze-3D20E ezidluliselwa ngesondo.
Iziphumo zethu zibonelela ngengqiqo ebalulekileyo kwiindlela ezilawula impumelelo yokuzala ye-A. gambiae. Kuvele imodeli apho amadoda aphuhlileyo ukuze enze ii-titers eziphezulu ze-3D20E, i-ecdysone eguqulweyo ethile yendoda eqinisekisa ukuzala ngokususa iimvakalelo zamabhinqa ukuba aqhubeke nokuzala. Kwangaxeshanye, ezi vectors ze-malaria zikwaphuhlise inkqubo esebenzayo yokuqalisa i-3D20E kumabhinqa ukuphendula ekudlulisweni ngokwesondo kwe-EPP ethile yendoda. Ngokolwazi lwethu, lo ngumzekelo wokuqala wenkqubo ye-steroid hormone elawulwa ngamadoda nabafazi eyenza umsebenzi owahlukileyo nobalulekileyo kwizinambuzane. Umsebenzi we-ecdysone ethile yendoda uqikelelwe kodwa awuboniswanga ngokuqinisekileyo. Umzekelo, ingcamango engachanekanga kakhulu 18 kukuba le misebenzi inokwenziwa yi-20E precursor E1. Kuyaziwa ukuba kwi-Drosophila, i-monandry ibangelwa kukudluliselwa ngokwesondo kwee-peptides zesini ezincinci19,20 ezisebenzisana nee-neurons ezigcina indlela yokuzala yabasetyhini ngokusebenzisa ii-receptors ezithile ze-peptide zesini21,22. Umsebenzi owongezelelweyo uyafuneka ukumisela ukwehla ii-cascade zesignali ezilawulwa yi-3D20E kwiimazi ze-A. gambiae kunye nokufumanisa ukuba ezi cascade zinokugcinwa na phakathi kweengcongconi kunye ne-Drosophila.
Ngenxa yendima ebalulekileyo ye-3D20E ekuzaleni nasekuziphatheni kwabasetyhini echongiweyo kuphononongo lwethu, iindlela ezikhokelela ekuhlanganisweni nasekusebenzeni kwe-3D20E zibonelela ngamathuba amatsha kwizicwangciso zokulawula iingcongconi zexesha elizayo, ezifana nokuveliswa kweenkunzi ezikhuphisanayo ezingakwaziyo ukuzala kwiindlela zobuchwepheshe bezinambuzane ezingakwaziyo ukuzala. Sebenzisa ukukhululwa endle okanye ukulinganisa i-3D20E kumdlalo we-virgin. Umsebenzi we-3D20E okhethekileyo ngamadoda usenokuba wavela xa i-A. gambiae kunye nezinye iintlobo zeCellia zafumana amandla okuhlanganisa isidoda sazo kwiiplagi zokuzala, njengoko oku kuvumela ukudluliselwa okusebenzayo kwenani elikhulu leehomoni kunye nee-enzymes ezivuselela iihomoni. Kwelinye icala, i-3D20E evolution esebenzisa i-monandry ibonelela ngendlela yokuba amabhinqa (ngokubonakalisa i-MISO ephezulu) akhuthaze ukufaneleka kwawo kokuzala kwiindawo ezinobuninzi be-malaria, nto leyo enegalelo ngokungathanga ngqo ekudluliseleni kwePlasmodium. Ngenxa yokuba i-20E yabasetyhini ibonakalisiwe ukuba inefuthe elikhulu ekusindeni nasekukhuleni kwe-P. falciparum kwiingcongconi ze-Anopheles zabasetyhini, iindlela ze-steroid zamadoda kunye nabasetyhini ngoku ziyimiba ephambili yokusebenzisana kweengcongconi kunye neeparasite.
Iintlobo ze-A. gambiae ze-G3 zikhuliswe phantsi kweemeko eziqhelekileyo zezinambuzane (26-28 °C, 65-80% yomswakama, ixesha lokukhanya/elimnyama leeyure ezili-12:12). Iimbungu zondliwa ngokutya kweentlanzi okugutyiweyo (iiTetraMin Tropical Flakes, iiKoi Pellets kunye neeTetra Pond Sticks ngomlinganiselo we-7:7:2). Iincukuthu ezindala zondliwa ngesisombululo se-dextrose se-10% kunye negazi lomntu leveki (izinto zegazi ezifundwayo). Iincukuthu eziziintombi zifunyenwe ngokwahlula isini kwinqanaba le-pupal emva kokuhlola iziphelo nge-microscopy. Iinkunzi ezithwele i-DsRed transgene zichazwe ngaphambili.
Uvavanyo lokudibana ngenkani lwenziwa ngokwemigaqo echazwe ngaphambili. Kwindibano yendalo, iimazi eziziintombi ezineminyaka emi-4 ubudala zigcinwe kwi-1:3 ratio kunye neentombi eziziintombi ezikhulileyo ngokwesondo ubusuku obubini. Kwindibano apho amadoda afakwa i-dsEPP, ukudibana nge-co-caging kwahambelana neentsuku ezi-3-4 emva kokufakwa i-injection, xa umsebenzi we-phosphatase wawungacinywanga kakhulu (Umzobo weDatha oLwandisiweyo. 2b).
Izicubu zeengcongconi, izidumbu eziseleyo (umzimba wonke), okanye umzimba wonke zahlulwahlulwa zaba yi-100% methanol zaza zahlanganiswa nge-beader (iiglasi ze-2 mm, i-2,400 rpm, imizuzwana engama-90). Ubungakanani bezicubu kunye nomthamo we-methanol bezimi ngolu hlobo lulandelayo: ukuphumla komzimba, 50 kwi-1,000 µl; i-MAG, 50–100 80 µl; i-female LRT, 25–50 80 µl. I-precipitate yafakwa kwi-methanol yesibini enomthamo ofanayo we-methanol. Inkunkuma yeseli yasuswa nge-centrifugation. I-methanol evela kuzo zombini ezi ngcoliso yadityaniswa yaza yomiswa phantsi kokuhamba kwe-nitrogen, yaze yaphinda yaxhonywa kwi-80% methanol emanzini: ukuphumla komzimba, 50 µl; i-MAG kunye ne-female LRT, 30 µl.
Iisampulu zihlalutywe kwi-mass spectrometer (ID-X, Thermo Fisher) edityaniswe nesixhobo se-LC (Vanquish, Thermo Fisher). I-5 µl yesampulu ifakwe kwikholamu ye-3 µm, 100 × 4.6 mm (Inspire C8, Dikma) egcinwe kwi-25 °C. Izigaba ezihambayo ze-LC yayiyi-A (amanzi, i-0.1% formic acid) kunye ne-B (acetonitrile, i-0.1% formic acid). I-LC gradient yayingolu hlobo lulandelayo: 5% B umzuzu o-1, emva koko yandiswa yaya kwi-100% B kwimizuzu eli-11. Emva kwemizuzu esi-8 kwi-100%, phinda ulinganise ikholamu kwi-5% B imizuzu emi-4. Isantya sokuhamba yayiyi-0.3 ml min-1. I-Ionization kumthombo we-MS ifezekiswa yi-heated electrospray ionization kwiindlela ezilungileyo nezingalunganga.
I-mass spectrometer ilinganisa idatha kuluhlu lwe-m/z ukusuka kwi-350 ukuya kwi-680 kwisisombululo se-60,000 kwimodi epheleleyo ye-MS. Idatha ye-MS/MS ifunyenwe kwi-[M + H]+ (zonke iithagethi), [M - H2O + H]+ (zonke iithagethi), kunye ne-[M - H]- (iithagethi ze-phosphorylated). Idatha ye-MS/MS isetyenziselwe ukuqinisekisa iipropati ze-ecdysone zeethagethi apho kungekho mgangatho wawufumaneka khona. Ukuchonga ii-ecdysteroids ezingajoliswanga, idatha ye-MS/MS yazo zonke iincopho ze-HPLC ezinobuninzi obungaphezulu kwe-15% yahlalutywa. Ukulinganisa kusetyenziswa ii-standard curves ezenziwe kwimigangatho ecocekileyo (20E, 3D20E) ukubala izixa ezipheleleyo okanye ukuxutywa kwesampulu enye ethile (zonke ezinye iithagethi) ukubala ukulingana kwazo nezixa ezifunyenwe kwindoda enye. Kwi-3D20E, ukulinganisa kwenziwa kusetyenziswa isixa se-adducts ezilandelayo: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-.Idatha ikhutshiwe kwaye yalinganiswa kusetyenziswa iTracefinder (inguqulelo 4.1).Idatha ye-MS/MS ihlalutywe kusetyenziswa i-Xcalibur (inguqulelo 4.4).I-MS spectra ye-E, 20E kunye ne-3D20E ithelekiswa nemigangatho efanelekileyo.I-3D20E22P ihlalutywe ngokuthathwa kwe-reagent kaGirard.I-20E22P ihlalutywe ngomlinganiselo we-m/z.
I-3D20E22P ihlanjululwe kwi-MAG. Ukucoca kwenziwe kwisikali sohlalutyo kusetyenziswa i-chromatograph yolwelo esebenza kakhulu (i-Acquity, i-Waters) ene-quadrupole mass-based detector (i-QDa, i-Acquity, i-Waters) phantsi kweemeko ezifanayo ze-LC njengohlalutyo lwe-HPLC-MS/MS. Ukuqokelelwa kwamaqhezu kwaqaliswa xa i-m/z ehambelana ne-3D20E22P ifunyenwe ngexesha elifanayo lokugcinwa njengoko bekumiselwe ngaphambili. Ubumsulwa beekhompawundi ezikhutshiweyo bajongwa yi-HPLC-MS/MS njengoko kuchaziwe apha ngasentla.
I-RNA iyonke ikhutshwe kwizicubu zokuzala ezili-10-12 okanye kwezinye iindawo zomzimba (ezingenantloko) kusetyenziswa i-TRI reagent (Thermo Fisher) kulandela imiyalelo yomenzi. I-RNA iphathwe nge-TURBO DNase (Thermo Fisher). I-cDNA yenziwe kusetyenziswa i-Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) kulandela imiyalelo yomenzi. Ii-primers ze-reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) zapapashwa ngaphambili24 okanye zayilwa kusetyenziswa i-Primer-BLAST26, kukhethwe iimveliso ezinobukhulu obuyi-70-150 bp kwaye zithatha indawo yee-exon-exon junctions okanye ii-Primer pair primers ezahlukeneyo ii-exons. Iisampulu ze-cDNA ezivela kwii-replicas ezintathu ukuya kwezine zebhayoloji zaxutywa kane emanzini kwi-RT-qPCR. Ubungakanani benziwe kwii-replica reactions ezili-15 µl eziqulethe i-1 × PowerUp SYBR Green Master Mix (Thermo Fisher), ii-primers, kunye ne-5 µl ye-dilated cDNA. Iimpendulo zenziwa kwi-a Inkqubo ye-PCR ye-QuantStudio 6 Pro yexesha langempela (iThermo Fisher) kunye nedatha ziqokelelwe kwaye zahlalutywa kusetyenziswa uYilo kunye noHlalutyo (inguqulelo 2.4.3). Njengoko kubonisiwe kolu phononongo, izixa ezihambelanayo zilungisiwe kwi-ribosomal gene RpL19 (AGAP004422), apho ukubonakala kwayo kungazange kutshintshe kakhulu ngokutya igazi 27 okanye ukudibana 3.
Umgangatho we-RNA uhlolwe kusetyenziswa i-Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). Iilayibrari ze-Illumina paired-end zalungiswa zaza zaqhutywa kwi-Broad Institute of MIT naseHarvard. Ukulandelelana kokufundwa kwahambelana ne-A. gambiae genome (uhlobo lwe-PEST, uhlobo 4.12) kusetyenziswa i-HISAT2 (uhlobo 2.0.5) kunye neeparameters ezimiselweyo. Ukufundwa okune-mapping quality (MAPQ) scores <30 kususiwe kusetyenziswa i-Samtools (uhlobo 1.3.1). Inani lokufundwa okumaphiwe kwiigenes libalwe kusetyenziswa i-htseq-count (uhlobo 0.9.1) kunye neeparameters ezimiselweyo. Ukufundwa okuqhelekileyo kubalwe kwaye ukubonakaliswa kwe-gene okwahlukileyo kuhlalutywe kusetyenziswa iphakheji ye-DESeq2 (uhlobo 1.28.1) kwi-R (uhlobo 4.0.3).
Ii-gene eziguqula i-Ecdysone zichongiwe ngokuqala zikhangele i-genome ye-A. gambiae kusetyenziswa i-algorithm ye-PSI-BLAST (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), kusetyenziswa amaxabiso amiselweyo Iiparamitha ezinezi protocol zeprotheyini ezilandelayo: ukusuka kwi-Bombyx mori (Inombolo yokufikelela. NP_001038956.1), i-Musca domestica (Inombolo yokufikelela. XP_005182020.1, XP_005175332.1 kunye ne-XP_011294434.1) kunye ne-Microplitis demolitor (Inombolo yokufikelela. XP_008552646.1 kunye ne-XP_008552645.1) i-EcK evela kwi-B. mori (Inombolo yokufikelela. NP_001036900), i-Drosophila melanogaster (Inombolo yokufikelela. NP_651202), i-Apis i-mellifera (Inombolo yoQhagamshelo XP_394838) kunye ne-Acyrthosiphon pisum (Inombolo yoQhagamshelo XP_001947166); kunye ne-EPP evela kwi-B. mori (Inombolo yoQhagamshelo XP_001947166) NP_001177919.1 kunye ne-NP_001243996.1) kunye ne-EO ye-D. melanogaster (Inombolo yoQhagamshelo NP_572986.1) (inyathelo 1). Okulandelayo, isihluzi sichaphazela ngokusekwe kwi-mRNA expression ephezulu (>100 fragments/kilobase exons per million mapped reads (FPKM) okanye >85%) kwizicubu zokuzala (i-female LRT okanye i-MAG) eGambia (inyathelo 2). Ukuze siphucule ukucaciswa, sikhethe ii-enzymes ezikhethiweyo ezikwabonakaliswa kwizicubu zokuzala ze-A. albimanus, uhlobo lwe-anopheles olungavelisiyo okanye oludlulisela i-ecdysone ngexesha lokuzala. Iijini ezikhethiweyo zihluziwe ngokusekelwe ekubonakalisweni okuphantsi (<100 FPKM okanye <85th percentile) kwizicubu zokuzala ze-A. albimanus (inyathelo lesi-3). Njengesihluzo sokugqibela (inyathelo lesi-4), iijini ezikhethiweyo kufuneka zanelise ubuncinane enye yezi zilandelayo: (1) ziphuculwe kakhulu emva kokuzala (P < 0.05) ngokohlalutyo lweejini ezichazwe ngokwahlukileyo kunye (2) kwizicubu ezingezizo zokuzala (< 85% okanye <100 FPKM).
Sitshintshe iindlela ezichazwe ngaphambili ezingama-28,29,30 ukuze sifezekise ukulebhelwa kwe-isotopic ye-whole-organism. Ngamafutshane, uhlobo lwe-wild-type Saccharomyces cerevisiae uhlobo lwesibini (YSC2, Sigma) luvavanyiwe kwi-yeast nitrogen base (BD Difco, DF0335) equlethe (wt/vol) i-2% glucose (G7528, Sigma), i-1.7% amino acid-free kunye ne-ammonium sulfate. culture medium) kunye ne-5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) njengomthombo we-nitrogen kuphela. I-Yeast yafunyanwa nge-centrifugation kwaye iimbungu ze-mosquito zondliwa ngokukhawuleza de kube yi-pupation. Isongezelelo nge-fishmeal (0.5 mg kwi-300 yeembungu) ukuthintela ukufa kwe-instar yesine. Iimazi kuphela ezasetyenziswa kuvavanyo lokudibana neenkunzi ezingenalebula ukuhlalutya i-proteome yenkunzi edluliselwe ngexesha lokudibana.
Iimazi eziziintombi ezineminyaka emi-4 ukuya kwemithandathu ubudala ezine-tag ye-15N zanyanzelwa ukuba zitshate neenkunzi eziziintombi ezingalinganiyo ngeminyaka. Ukuzala okuphumelelayo kwaqinisekiswa ngokufumana iiplagi zokuzala phantsi kwe-epifluorescence microscopy. Kwi-3, 12, kunye ne-24 hpm, i-atria yeemazi eziziintombi ezingama-45-55 zahlulwahlulwa kwi-50 µl ye-ammonium bicarbonate buffer (pH 7.8) zaza zafakwa kwi-homogenate. I-homogenate yafakwa kwi-centrifuge kwaye i-supernatant yaxutywa ne-50 µl ye-0.1% RapiGest (186001860, Waters) kwi-50 mM ammonium bicarbonate. I-supernatant kunye ne-pellet evela kwisampuli nganye zaqhwatywa kwiqhwa elomileyo zaza zathunyelwa ngobusuku bonke kwi-MacCoss laboratory kwiYunivesithi yaseWashington, apho kwagqitywa khona ukulungiselela isampuli ye-LC-MS/MS. Phinda umise i-pellet kwi-50 µl ye-0.1% RapiGest kwi-50 mM. I-ammonium bicarbonate kunye ne-sonicate kwindawo yokuhlambela ngamanzi. Uxinzelelo lweproteni ye-pellet kunye ne-supernatant lulinganiswe ngovavanyo lwe-BCA, iisampulu zancitshiswa nge-5 mM dithiothreitol (DTT; Sigma), zaxutywa ne-15 mM iodoacetamide (Sigma) zaza zafakwa kwi-37 °C (1:0 50) iyure e-1 nge-trypsinization: trypsin: substrate ratio). I-RapiGest yahluzwa ngokongezwa kwe-200 mM HCl, kwalandela i-incubation kwi-37 °C imizuzu engama-45 kunye ne-centrifugation kwi-14,000 rpm imizuzu eli-10 kwi-4 °C ukususa ukungcola. Iisampulu zahlanjwa nge-dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) zaza zaphinda zaxhonywa kwi-0.1% formic acid ukuze kubekho uxinzelelo lokugqibela lweproteni lwe-0.33 µg µl-1. Iiproteome ze-MAG ezingenalebula nazo zahlalutywa ngokufanayo kwi-virgin. Amaduna. Kuhlalutywe iikopi ezimbini zohlalutyo kwisampulu nganye. Okulandelayo, i-1 µg nganye yahlalutywa kusetyenziswa ikholamu ye-25-cm fused silica 75-μm ene-4-cm fused silica Kasil1 (PQ) frit trap epakishwe nge-Jupiter C12 reversed-phase resin (Phenomenex) kunye ne-180-minute liquid chromatography. Iisampuli zokugaya – I-MS/MS iqhutywe kwi-Q-Exactive HF mass spectrometer (iThermo Fisher) ene-nanoACQUITY UPLC System (Waters). Idatha yokufunyanwa enxulumene nedatha eyenziweyo kwi-run nganye iguqulwe yaba yifomathi ye-mzML kusetyenziswa iProteowizard (inguqulelo 3.0.20287) kwaye kusetyenziswa iComet31 (inguqulelo 3.2) ngokuchasene nedathabheyisi yeFASTA equlethe ulandelelwano lweeproteni ezivela kwi-Anopheles gambiae (inguqulelo yeVectorBase 54), i-Anopheles coluzzi Uphendlo lwenziwe kwiMali-NIH (inguqulelo yeVectorBase 54), iSaccharomyces cerevisiae (Uniprot, Matshi 2021), i-A. gambiae RNA-seq, kunye neenguqulelo ezintathu zezakhelo zezinto ezingcolisayo zabantu ezaziwayo. Ii-FDR ezihambelana nemephu yePeptide zichongiwe kusetyenziswa iPercolator32 (inguqulelo 3.05) enomlinganiselo we-0.01, kwaye iipeptides zahlanganiswa zaba ziimpawu zeproteni kusetyenziswa i-protein parsimony kwiLimelight33 (uhlobo 2.2.0). Ubuninzi beeproteni ezihambelanayo buqikelelwe kusetyenziswa i-normalized spectral abundance factor (NSAF) ebalwe kwiproteni nganye kuluhlu ngalunye njengoko kuchaziwe ngaphambili. I-NSAF malunga neproteni nganye ilinganiswe kwiisampuli ezivela kwiikopi ezimbini ezahlukeneyo zebhayoloji. Ukufakwa kwelebheli ye-15N kugqume ngempumelelo iproteome yabasetyhini, nangona inani elincinci leproteni engabhalwanga linokufunyanwa kwiintombi ezibhalwe ilebheli. Sirekhode ukubonwa kokunciphisa iiproteni zamadoda (1-5 spectra) kwiisampuli eziluhlaza zabasetyhini kuphela kuluhlu lobuchwephesha, apho iisampuli eziluhlaza ziqhutywa emva kweesampuli zamadoda/zokudibana, ngenxa ye-HPLC "ethwala ngaphezulu". Iiproteni ngamanye amaxesha ezifunyenwe 'njengezingcolisi' ezivela kwiintombi ezibhalwe ilebheli zidweliswe kwiTheyibhile eyoNgezelelweyo 1.
Iipeptide ezimbini ze-antigenic, i-QTTDRVAPAPDQQQ (ngaphakathi kwe-isotype PA) kunye ne-MESGTTPSGDSEQ (ngaphakathi kwe-isotype PA kunye ne-PB) kwi-Genscript. Ezi peptide zimbini zadityaniswa, emva koko zadityaniswa kwiproteni yokuthwala i-KLH zaza zafakwa kwiimvundla zaseNew Zealand. Iimvundla zanikelwa emva kokufakwa kwesine, kwaye i-IgG iyonke yahlulwa ngokuhlanjululwa kwe-affinity. I-IgG evela kwimvundla ekhethekileyo ye-EPP yasetyenziselwa ukubhula kwentshona ngakumbi.
Kwi-western blots, i-MAG (n = 10, apho i-n imele inani leesampuli zeengcongconi ezizimeleyo ngokwebhayoloji) kunye ne-LRT yabasetyhini (n = 30) evela kwiinkunzi eziziintombi ezineminyaka emi-4 ubudala kunye neemazi eziziintombi okanye ezinyanzelekileyo (<10 emva kokudibana), i-Protein extraction buffer (50 mM Tris, pH 8.0; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 × protease inhibitor cocktail (Roche)) yongezwa ngokwahlukeneyo. Iisampuli zalungiswa ngoko nangoko emva kokususwa nge-beader (2 mm glass beads, 2,400 rpm, 90 sec). Iinkunkuma ezinganyibilikiyo zasuswa nge-centrifugation kwi-20,000 g kwi-4 °C. Iiproteni zalinganiswa nge-Bradford assay (Bio-Rad). Emva koko, i-20 µg yeproteni ye-MAG, i-40 µg yeproteni ye-LRT, kunye ne-20 µg yeproteni eseleyo. zahlulwe zaza zahlulwa yi-10% Bis-Tris NuPAGE kusetyenziswa i-MOPS buffer. Iiproteni zidluliselwe kwi-polyvinylidene fluoride membranes kusetyenziswa inkqubo yokudlulisa iBlot2 (Thermo Fisher). Ii-Membranes zahlanjwa kabini kwi-1× PBS-T (0.1% Tween-20 kwi-PBS) zaza zavalwa kwi-Odyssey blocking buffer (Li-Cor) kangangeyure e-1 kwi-22°C. Ii-Membranes zashukunyiswa ubusuku bonke kwi-4°C nge-custom rabbit anti-EPP polyclonal primary antibody (1:700 kwi-blocking buffer) kunye ne-rat anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000). Ii-Membranes zahlanjwa nge-PBS-T zaza zafakwa kwi-secondary antibodies (i-donkey anti-rabbit 800CW kunye ne-goat anti-rat 680LT (Li-Cor), zombini ziyi-1:20,000) kwi-blocking buffer equlethe i-0.01% SDS kunye ne-0.2% Tween. -20 iyure e-1 kwi-22 °C. Ii-membranes zihlanjwe nge-PBS-T zaza zathathwa imifanekiso nge-Odyssey CLx scanner. Imifanekiso iqokelelwe yaza yacutshungulwa kwi-Image Studio (inguqulelo 5.2). Ibhendi ethile ehambelana ne-EPP-RA isoform (82 kDa) ayifunyenwanga.
Iindawo zokubhala ikhowudi ze-EPP (njenge-isoform AGAP002463-RB equlethe i-histidine phosphatase domain, i-NCBI conserved domain search 34) kunye ne-EcK2 (AGAP002181) zadityaniswa kwi-pET-21a(+) plasmid (Novagen Millipore Sigma); Iiprimers zidweliswe kwiTheyibhile yeDatha eyoNgezisiweyo 2. Iilinki ezisibhozo zeGS4 (ezikwi-tandem) zifakwe ngaphambi kokuba i-C-terminal 6xHis tag ye-pET-21a(+)-EcK2 construct ifakelwe. Iiproteni eziphinda-phindayo zenziwe kusetyenziswa i-NEBEExpress cell-free E. coli protein synthesis reaction (New England BioLabs). Iiproteni eziphinda-phindayo zicocwe kusetyenziswa iikholamu ze-NEBEExpress Ni spin (New England BioLabs). Iproteni yokulawula i-Dihydrofolate reductase (DHFR) yenziwe kusetyenziswa itemplate ye-DNA evela kwi-NEBEExpress Cell-Free E. coli Protein Synthesis Kit. Iiproteni zigcinwe kwi-50% glycerol kwi-PBS kwi--20 °C ukuya kuthi ga kwiinyanga ezi-3.
Umsebenzi we-Phosphatase we-EPP kunye ne-tissue extracts ulinganiswe kusetyenziswa i-4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). I-reaction buffer yayine-25 mM Tris, i-50 mM acetic acid, i-25 mM Bis-Tris, i-150 mM NaCl, i-0.1 mM EDTA, kunye ne-1 mM DTT. I-tissue yahlanganiswa kwi-reaction buffer kwaye i-cell debris yasuswa nge-centrifugation. Qalisa i-reaction ngokongeza i-enzyme okanye i-tissue extract kwi-reaction buffer equlethe i-2.5 mg ml-1 pNPP. Umxube we-reaction wafakwa kwi-incubation kubushushu begumbi ebumnyameni, kwaye ubungakanani be-pNP obuguqulwe kwi-pNPP balinganiswa ngokulinganisa ukufunxwa kwi-405 nm ngamaxesha ahlukeneyo.
Kwi-in vitro EcK activity, iproteni ifakwe kwi-0.2 mg 20E okanye 3D20E kwi-200 µl buffer (pH 7.5) equlethe i-10 mM HEPES–NaOH, i-0.1% BSA, i-2 mM ATP kunye ne-10 mM MgCl2 iiyure ezi-2 kwi-27 °C. I-reaction yamiswa ngokongeza i-800 µl methanol, yaze yapholiswa kwi--20 °C iyure e-1, yaze yafakwa kwi-centrifuge kwi-20,000 g imizuzu eli-10 kwi-4 °C. I-supernatant yahlalutywa yi-HPLC-MS/MS. Ukuze kufudunyezwe iiproteni ezisetyenziswa kwiqela lolawulo, iiproteni zafakwa kwi-50% glycerol kwi-PBS imizuzu engama-20 kwi-95 °C.
Kwi-in vitro EPP activity, iprotheyini ifakwe kwi-3D20E22P (elingana nesixa esifunyenwe kwiipayi ezili-18 ze-MAG, ehlanjululwe yi-HPLC-MS/MS) kwi-100 µl buffer (pH 7.5) equlethe i-25 mM Tris, i-50 mM acetic acid, i-25 mM Bis-Tris, i-150 mM NaCl, i-0.1 mM EDTA, kunye ne-1 mM DTT iiyure ezi-3 kwi-27 °C. I-reaction yamiswa ngokongeza i-400 µl methanol yaza yapholiswa kwi--20 °C iyure e-1, yaze yafakwa kwi-centrifuge kwi-20,000 g imizuzu eli-10 kwi-4 °C. I-supernatant yahlalutywa yi-HPLC-MS/MS.
Iziqwenga ze-PCR ze-EPP (362 bp), i-EcK1 (AGAP004574, 365 bp) kunye ne-EcK2 (556 bp) zandiswa kwi-cDNA elungiselelwe kwizidumbu zeengcongconi ezingenazintloko zesini esixutyiweyo. Isiqwenga se-PCR solawulo lwe-eGFP (495 bp) sandiswa kwi-pCR2.1-eGFP echazwe ngaphambili; Iiprimer zePCR zidweliswe kwiTheyibhile yeDatha eyoNgezisiweyo 2. Iqhekeza lePCR lifakwe phakathi kweepromotha ze-T7 eziguqulweyo kwiplasmid ye-pL4440. Iiplasmid constructs zifunyenwe kwi-NEB 5-α competent E. coli (New England Biolabs) kwaye zaqinisekiswa yi-DNA sequencing ngaphambi kokusetyenziswa (jonga iDatha eyoNgezisiweyo 1 ukuze ufumane ulandelelwano lokufaka). Iiprimer ezihambelana nepromotha ye-T7 (iTheyibhile yeDatha eyoNgezisiweyo 2) zisetyenziselwe ukwandisa i-insert evela kwi-plasmid esekwe kwi-pL4440. Ubungakanani bemveliso yePCR buqinisekiswe yi-agarose gel electrophoresis.dsI-RNA ikhutshelwe kwiitemplate zePCR kusetyenziswa iMegascript T7 Transcription Kit (Thermo Fisher) kwaye yacocwa ngokwemiyalelo yomenzi kunye notshintsho oluchazwe ngaphambili.
Kwisitofu se-dsRNA, i-1,380 ng ye-dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) yafakwa kwi-concentration ye-10 ng nl-1 esifubeni samadoda okanye amabhinqa amadala (i-Nanoject III, iDrummond) kungekapheli usuku olu-1 emva kokuphuma kwe-eclosion. Amanqanaba okuthotywa kwe-gene amiselwe ubuncinane kwiikopi ezintathu zebhayoloji ngokukhupha i-RNA, ukwenziwa kwe-cDNA, kunye ne-RT-qPCR. Kwisitofu se-ecdysone, amabhinqa angoontombi okanye angoontombi aneentsuku ezi-4 ubudala atyalwe igazi afakwe i-0.13, 0.21, okanye i-0.63 µg ye-20E okanye i-3D20E (i-Nanoject III, iDrummond) kumanqanaba e-1.3, 2.1, ngokulandelanayo, kuxhomekeke kuyilo lovavanyo okanye i-6.3 ng nl-1. Faka i-100 nl ye-10% (vol/vol) ethanol emanzini; I-100 nl ye-3D20E22P kwi-10% ye-ethanol (elingana ne-75% yesixa esifunyenwe kwisibini see-MAG). Iingcongconi zabelwa ngokungacwangciswanga kwiqela elijojoweyo.
Kwiimvavanyo zokuzala, iimazi ezineentsuku ezi-3 ubudala zondliwa ngokukhawuleza egazini lomntu. Susa iingcongconi ezingondliyo okanye ezingandliwanga. Ngokuxhomekeke kunyango, iimazi zabekwa kwiikomityi zokuzala ezahlukeneyo ubusuku obune ubuncinane iiyure ezingama-48 emva kokutya igazi. Amaqanda abalwa phantsi kwe-stereoscope (Stemi 508, Zeiss); kwiimazi ezizalelweyo, amaqanda aqanduselwe abe yizibungu ayethathwa njenganobuvuvu.
Kwizilingo zokuzala, iimazi zavunyelwa ubuncinane iintsuku ezi-2 kuxhomekeke kunyango ukuze zikhule ukumelana nokuzala, kwaye iinkunzi ezilingana nobudala bezilwanyana zasendle zangeniswa kwikheyiji enye. Emva kweentsuku ezimbini, ii-vesicles zemazi ezichunyisiweyo zasikwa kwaye i-DNA ye-genomic yakhululwa ngokuqandisa kunye nokunika i-sonication kwi-buffer equlethe i-10 mM Tris-HCl, i-1 mM EDTA, kunye ne-25 mM NaCl (pH 8.2). Iisampuli zafakwa kwi-Proteinase K (0.86 µg µl-1) imizuzu eli-15 kwi-55 °C, yalandelwa yimizuzu eli-10 kwi-95 °C. Amalungiselelo e-DNA ye-genomic akrwada axutywa kalishumi kwaye afunyanwa kwi-qPCR ye-chromosome sequences; ii-primers zidweliswe kwiTheyibhile yeDatha eLandisiweyo 2. Ukungabikho kwe-chromosome sequence ye-Y kubonisa ukuba akukho kuzala.
Kwiimvavanyo zokuphinda utshate, iimazi ezidityaniswe ngenkani zahlolwa ukuba zikhona na iiplagi zokutshata ukuqinisekisa imeko yokutshata kwaye zavunyelwa iintsuku ezi-2 ukuba zikhule zingakwazi ukumelana nokutshata xa kungekho maduna, njengoko kuchaziwe ngaphambili. 36. Iinkunzi ezithwele isidoda se-DsRed transgenic zafakwa kwiikheyiji zamabhinqa. Emva kweentsuku ezimbini, ii-vesicles ezichumisa zahlulwa kwiimazi, kwaye i-DNA ye-genomic yalungiswa njengoko kuchaziwe apha ngasentla kwaye yafunyanwa yi-qPCR transgene ye-DsRed; ii-primers zidweliswe kwiTheyibhile yeDatha eyoNgezisiweyo 2. Ukungabikho kwe-DsRed transgene kwabonisa ukuba akukho kuphinda kutshate kwenzeke.
I-3D20E yenziwe njengoko kuchaziwe ngaphambili 37. Ngamafutshane, i-10 mg ye-20E (iSigma-Aldrich) yanyibilika kwi-10 ml yamanzi, kwalandela ukongezwa kwe-30 mg yeplatinum black (kwimo yomgubo, iSigma-Aldrich). Umjelo othambileyo we-O2 wawuqhubeka uvuthuzwa kumxube we-reaction, owavuthuzwa kubushushu begumbi. Emva kweeyure ezi-6, kongezwa i-30 mL ye-methanol ukuze kumiswe i-reaction. Umxube waxutywa nge-centrifuge ukususa amasuntswana e-catalyst. I-supernatant yatshintshwa yaba ngumphunga kwi-vacuo kubushushu begumbi. Imveliso ye-reaction eyomileyo yanyibilika kwi-10% ye-ethanol kunye ne-methanol ukuze ijojwe kuhlalutyo lwe-HPLC-MS/MS. Izinga lokuguqulwa (ukusuka kwi-20E ukuya kwi-3D20E) yayimalunga ne-97% (Umzobo 4b), kwaye i-MS spectrum ye-3D20E eyenziweyo yafana neyafunyanwa kwiimazi ezitshatileyo (Umzobo 4c).
Le ntsomi ineenkcukacha ezithile zovavanyo lwezibalo olwenziweyo. I-GraphPad (inguqulelo 9.0) isetyenzisiwe ukwenza uvavanyo oluchanekileyo lukaFisher, uvavanyo lweMantel-Cox, kunye novavanyo lwe-t lomfundi. Uvavanyo lweCochran-Mantel-Haenszel lwenziwe kusetyenziswa iskripthi se-R esenziwe ngokwezifiso (esifumaneka kwi-https://github.com/duopeng/mantelhaen.test). Ukusasazwa kwedatha kuhlolwe ukufaneleka kusetyenziswa uvavanyo lweShapiro-Wilk olunomda wokubaluleka we-0.05. Xa idatha ingaphumelelanga kuvavanyo lokufaneleka, kwenziwa uvavanyo lweMann-Whitney. Idatha yokusinda ihlalutywe kusetyenziswa uvavanyo lweMantel-Cox. Iphakheji ye-DESeq2 (inguqulelo 1.28.1) isetyenzisiwe ukwenza uhlalutyo lokubonisa umahluko we-RNA-seq gene. Ibha ethe tye kwigrafu imele i-median. Ixabiso lokubaluleka le-P = 0.05 lisetyenzisiwe njengomda wazo zonke iimvavanyo.
Ukuze ufumane ulwazi oluthe kratya malunga noyilo lophando, jonga isishwankathelo seNgxelo yoPhando lweNdalo esidityaniswe neli nqaku.
Idatha ye-MS proteomic ifakwe kwiProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) ngePRIDE Partner Repository (https://www.ebi.ac.uk/pride/) kunye nesazisi sedatha iPXD032157.
Idatha ye-RNA-seq ibekwe kwi-Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) phantsi kwerekhodi ye-serial GSE198665.
Iiseti zedatha ezongezelelweyo ezenziweyo kunye/okanye ezihlalutyiweyo ngexesha lophando lwangoku zinokufunyanwa kubabhali abafanelekileyo xa kuceliwe ngokufanelekileyo. Eli nqaku libonelela ngedatha yomthombo.
UDe Loof, A. Ecdysteroids: Iisteroids zesini sezinambuzane ezingahoywanga?Indoda: Ibhokisi emnyama.Isayensi yezinambuzane.13, 325–338 (2006).
I-Redfern, i-CPF 20-hydroxyecdysone kunye nophuhliso lwe-ovarian kwi-Anopheles stephens.J. I-Insect Physiology.28, 97–109 (1982).
Ixesha lokuthumela: Julayi-08-2022