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He rerekē ki ngā kararehe tuarā, e whakaarohia whānuitia ana kāore i te kitea he homoni steroid ira tāne e aro ana ki ngā pepeke. I roto i te Anopheles gambiae, te āhua nei kua whanake te steroid ecdysone 20-hydroxyecdysone (20E) hei whakahaere i te whanaketanga o te hua manu ina hangaia e ngā uwha2 me te whakaoho i te wā uaua ki te moe tahi ina whakawhitia ira tāne e ngā tāne3. Nā te mea he āhuatanga whakaputa uri nui te whanaketanga hua manu me te moe tahi, ko te mārama ki te whakauru o ngā waeroa uwha Anopheles i ēnei tohu homoni ka taea te āwhina i te hoahoa o ngā kaupapa whakahaere malaria hou. I konei, ka whakakitea e mātou ko ēnei mahi whakaputa uri ka whakahaerehia e ngā steroid ira motuhake mā roto i tētahi whatunga matatini o ngā whākōkī whakahohe/whakakore ecdysteroid. I tautuhia e mātou he ecdysone oxidized motuhake ki ngā tāne, 3-dehydro-20E (3D20E), e tiaki ana i te whānautanga mā te aukati i te urunga ira tāne wahine i muri i te whakawhiti ira tāne me te whakahohenga mā te dephosphorylation. He mea nui, ko te whakawhiti 3D20E i whakaoho anō i te whakaaturanga o ngā ira whakaputa uri e pupuri ana i te whanaketanga hua manu i te wā o te mate Plasmodium, e whakarite ana i te hauora o ngā uwha kua pangia. Kāore te 20E i ahu mai i te uwha e whakaoho i te ira tāne te urupare, engari ka taea e ngā tāngata e moe tahi ana te whakatakoto hua i muri i te aukati i ngā kinase aukati-20E. Ko te tautuhi i tēnei homoni steroid pepeke tāne-motuhake me tana mahi ki te whakahaere i te urunga ā-ira wahine, te momonatanga me te taunekeneke ki te Plasmodium e kī ana i te kaha ki te whakaiti i te angitu whakaputa uri o ngā waeroa e tuku ana i te malaria.
Kei te piki haere anō ngā keehi me ngā matenga o te malaria4 nā te ātete whānui o ngā waeroa Anopheles ki ngā patu pepeke, te kaikawe anake o ngā pirinoa malaria tangata. Ko te koiora moepuku o ēnei waeroa he tino whainga mō ngā wawaotanga whakahaere malaria hou nā te mea kotahi anake te moepuku a ngā uwha5; mā te kore e mate tēnei moepuku kotahi ka nui te kaha ki te whakaiti i ngā taupori waeroa i te parae.
Ka kore e taea e ngā wāhine te moe tāne i muri i te whiwhinga o ngā homoni steroid teitei mai i ngā tāne. Kua whakaatuhia e ngā rangahau ko te mea e uaua ai te moe tahi ko te 20-hydroxyecdysone (20E), he homoni steroid e mōhiotia whānuitia ana ko te kaiwhakahaere o te huringa whakawhānau i te wāhanga torongū. Kua whanake te kaha o ngā tāne ki te hanga me te whakawhiti i te 20E i roto i ngā momo Anopheles he wāhanga o te momo Cellia7, e horahia ana i Āwherika, ā, kei roto ko ngā kaikawe malaria tino kino, tae atu ki te Anopheles gambiae. He mea nui tēnei nā te mea i roto i ēnei momo ka whakaputa hoki ngā uwha i te 20E i muri i ia kai toto, ā, ko te 20E te kaiwhakahaere i te huringa oogenesis (tirohia te tohutoro 8). Heoi, he iti noa te mōhiotia mō te huarahi e whakauru ai ngā uwha i ngā tohu mai i ngā puna e rua o te ecdysone (te whakawhiti tāne me te whakaoho toto) me te kore e whakararu i tō rātou ake kaha ki te moe tāne. Inaa, ki te whakaoho te 20E i hangaia e ngā uwha i te kore manawanui moe tāne, ka arahi tēnei ki te kore tamariki i roto i ngā tāngata e whāngai ana i ngā wāhine, he whanonga tino noa i roto i ēnei waeroa5.
Ko tētahi whakamārama pea ko te whakawhiti a ngā tāne A. gambiae i tētahi ecdysone tāne-motuhake kua whakarerekētia, e whakahohe ana i tētahi rerenga tohu i roto i te ara whakaputa uri wahine, ka hua ake te kore pumau o te moepuku. Heoi, ahakoa he maha ngā homoni steroid a ngā vertebrates, pērā i te estrogen me te androgen (i arotakehia i te tohutoro 9), ki tā mātou e mōhio ana, kāore anō kia kitea ngā steroid androgenic-biased i roto i ngā pepeke.
I tīmata mātou ki te whakatau i te rārangi o ngā homoni steroid i roto i te repe tāpiri tāne tāne (MAG) o te A. gambiae kua pakeke i te ira tangata, hei rapu i ngā steroid whakarerekē pea. Mā te whakamahi i te chromatography wai mahi teitei me te tandem mass spectrometry (HPLC-MS/MS) kaua ki te tikanga iti ake te motuhake i whakamahia i mua, i kitea e mātou te ecdysone (E) me te 20E i roto i tēnei kiko, e whakaū ana i ngā hua o mua. Heoi, ko te tauira i tino nui ko ngā steroids phosphorylated oxidized, e rite ana ki te tātai 3-dehydro-20E-22-phosphate (3D20E22P)12 (Pikitia 1). Ko ētahi atu momo ko te 3-dehydro-20E (3D20E) me te 20E-22-phosphate (20E22P). Ko te kaha o te tohu HPLC-MS/MS o te 3D20E22P e rua ngā ota teitei ake i tōna ahua dephosphorylated, 3D20E, ā, e toru ngā ota teitei ake i tō E me te 20E (Pikitia 1). Ahakoa i ētahi atu wāhanga o te tinana me te ara whakaputa uri o raro (LRT; Raraunga Whānui Pikitia 1a). I tātarihia hoki e mātou ngā ecdysteroids i roto i ngā tāne me ngā uwha kua katihia (<1 rā te pakeke) ā, i kitea ko 3D20E me 3D20E22P anake i roto i te MAG; i kitea a E, 20E me 20E22P i roto i ngā ira e rua (Raraunga Whānui Pikitia 1b). E kī ana ēnei raraunga he nui ngā taumata o ngā homoni whakarerekē ka puta i ngā tāne pakeke A. gambiae i roto i ā rātou MAG kāore i hangaia e ngā uwha.
I tangohia te MAG me te LRT uwha (tae atu ki te atria, ngā vesicle seminal, me te parovarium) mai i ngā tāne wahine 4-rā te pakeke (4-rā te pakeke) me ngā uwha wahine me ngā uwha kua moea (0.5, 3, me te 12 hpm). I tātarihia te Ecdysone i roto i ēnei kiko mā te HPLC-MS/MS (toharite ± sem; whakamātautau-t kore takirua, taha-rua, reiti kitenga teka (FDR) i whakatikatikaina; NS, kāore i te nui; *P < 0.05, **P < 0.01 . 3D20E: 3 hāora vs. 0.5 hāora, P = 0.035; 12 hāora vs. 3 hāora, P = 0.0015; 12 hāora vs. 0.5 hāora, P = 0.030. 3D20E22P: 3 hāora vs. 0.5 hāora, P = 0.25; 12 hāora vs. 3 hāora, P = 0.0032; 12 hāora vs. 0.5 hāora, P = 0.015). Nō ngā tāruarua koiora e toru ngā raraunga. I tatauhia te horahanga tihi mō ia ecdysone e hiahiatia ana, ā, i whakatauritea mā te maha o ngā waeroa. Ko te tae e tohuhia ana mō Ecdysone penei: E, kakariki; 20E, karaka; 20E22P, papura; 3D20E, kikorangi; 3D20E22P, māwhero. Ka whakanuia e te whakaurunga te tauine i runga i te tuaka-y hei whakaatu i ngā taumata ecdysone iti iho.
Hei tirotiro mēnā ka whakawhitia te 3D20E22P me te 3D20E i te wā e moe ana, i wetewetehia e mātou ngā LRT uwha i ngā wā rerekē i muri i te moe. Ahakoa kāore i kitea te ecdysone i roto i ngā wahine tauhou, i kitea e mātou he nui te 3D20E22P i roto i te LRT i muri tonu i te moe (0.5 h i muri i te moe, hpm), ka heke haere i roto i te wā, i te mea ka piki ake ngā taumata 3D20E (Pikitia 1). Mā te whakamahi i te 3D20E i hangaia i te matū hei paerewa, i whakatauhia e mātou ko ngā taumata o tēnei homoni steroid i roto i ngā LRT moe he 100 ngā wā teitei ake i te 20E (Ripanga Raraunga Whānui 1). Nō reira, ko te 3D20E22P te ecdysone tāne matua e whakawhitia ana ki te LRT uwha i te wā e moe ana, ā, ko tōna āhua kua tangohia te phosphorylation, 3D20E, ka tino nui haere i muri tata i te moe. E whakaatu ana tēnei i tētahi tūranga nui mō te ecdysone whakamutunga i roto i te koiora i muri i te moe a te uwha.
I muri i te whakaputa i tētahi huinga raraunga RNA hou (RNA-seq) (Pikitia 2a), mā te whakamahi i tētahi paipa bioinformatics i hangaia kia rite ki te hiahia, i rapu mātou i te ecdysone kinase (EcK), te ecdysone oxidase (EO), me te ecdysone e whakakōrero ana i te ira phosphatase whakarerekētia 20E. Ko te EPP) e whakaatuhia ana i roto i ngā kiko whakaputa uri. I tautuhia e mātou tētahi ira EPP kaitono me ētahi ira EcK pea e rua (EcK1 me EcK2), engari kāore i kitea he ira EO kaitono pai. He mea nui, i whakaatuhia ngā ira EPP takitahi i ngā taumata teitei (98.9th percentile) i roto i ngā MAG Gambian engari kāore i roto i ngā LRT wahine (Pikitia 2b), he rerekē ki ā mātou tumanakohanga mai i te mea i puta te dephosphorylation o 3D20E22P i roto i tēnei kiko wahine. Nō reira, e whakapono ana mātou ka taea te whakawhiti i te EPP tāne i te wā e moe ana. Ina hoki, i whakamahia e mātou te tapanga isotope pumau in vivo hei huna i te pūmua wahine i muri i te moe, he whākōkī i tautuhia e te MS i roto i te atrium wahine (Pikitia 2c me te Ripanga Tāpiri 1). Ko te aroaro o I whakaūtia hoki te EPP i roto i te MAG me te LRT uwha kua whakaipoipohia (engari ehara i te wahine pūrotu) mā te whakamahi i ngā paturopi motuhake (Pikitia 2d).
a, He paipa hangarau koiora i hangaia kia rapua ai ngā kiko whakaputa uri o ia ira mō ngā ira e whakamārama ana i ngā EcK, EO, me ngā EPP. Ko ngā tau i te taha o ngā pere e tohu ana i te maha o ngā kaitono tāne me ngā kaitono wahine i ia taahiraa. I tautuhia e tēnei tātaritanga tētahi ira EPP (EPP) me tētahi ira EcK (EcK1) e whakaatuhia ana i roto i ngā tāne, me tētahi ira EcK (EcK2) e whakaatuhia ana i roto i ngā ira e rua engari kāore e puta he ira kaitono EO. b, Heatmap e whakataurite ana i te whakaaturanga ira kaitono i roto i ngā kiko wahine (V) me te taatai ​​(M) Anopheles gambiae me Anopheles albicans. Spca, whakatō; MAG, repe tāpiri i roto i ngā tāne; ētahi atu wāhanga o te tinana, tae atu ki ngā ū, ngā parirau, ngā waewae, ngā tinana momona, me ngā whekau o roto i ngā ira e rua, me ngā ovaries i ngā uwha. He nui te whakaaturanga o EcK2 i roto i te MAG me te atria o Gambia, engari ko te EPP ka kitea anake i roto i te MAG.c, Te tātaritanga proteomic o te nekehanga rōpū ejaculate tāne ki roto i te atria uwha i te 3, 12 me te 24 hpm, e whakaatu ana i ngā pūmua 67 tino nui. I whakatipuria ngā uwha ki runga i te kai kei roto te 15N hei tapanga (me te huna) i ngā pūmua katoa. I whakaipoipohia ngā tāne kāore i tohua ki ngā uwha kua tohua, ā, i wehea ngā LRT uwha i te 3, 12 me te 24 hpm mō te tātaritanga proteomic (tirohia te Ripanga Tāpiri 1 mō te rārangi katoa o ngā pūmua ejaculatory). I roto i te whakaurunga, i kitea a EPP, Eck1 me EcK2 i roto i te MAG o ngā tāne wahine mā te tātaritanga proteomic o ēnei kiko.d, I kitea a EPP mā te western blot i roto i te MAG me te LRT o ngā uwha kua whakaritea, engari kāore i roto i ngā uwha wahine, i ngā tāne rānei, i te toenga rānei o te tinana uwha. I te wā kotahi i i tirotirohia ki te anti-actin (whakahaere uta) me ngā paturopi anti-EPP. He wahine tāne katoa. Tirohia te Pikitia Tāpiri 1 mō ngā raraunga pūtake tiēre. E rua ngā wā i mahia ai ngā Western blots me ngā hua ōrite.
I manatokohia te mahi ecdysteroid phosphophosphatase o te EPP i muri i te whakatipuranga mā te HPLC-MS/MS me te 3D20E22P i wehea mai i te MAG (Raraunga Whānui Pikitia 2a). I tua atu, i te wā i whakamōkitohia ai e mātou te EPP mā te pokanoa RNA-mediated (RNAi), i kitea e mātou he whakahekenga kaha o te mahi phosphatase i roto i ngā kiko whakaputa uri o ēnei toa (Pikitia 3a), ā, ko ngā uwha i moe tahi me ngā toa i whakamōkitohia e te EPP i whakaatu i te ōwehenga A iti ake o te 3D20E i tangohia te phosphorylation (Pikitia 3b) ahakoa te whakamōkito ira wāhanga (Raraunga Whānui Pikitia 2b,c). He rerekē, kāore i kitea e mātou he huringa nui o te ōwehenga 20E22P/20E i roto i ngā waeroa kotahi, e kī ana pea he motuhake te whākōkī mō te 3D20E22P (Pikitia 3b).
a, Te hekenga o te mahi phosphatase i roto i te MAG i puta mai i te whakangāwari EPP mā te whakamahi i te EPP RNA aho-rua (dsEPP) me ngā mana whakahaere GFP RNA aho-rua (dsGFP). E rua tekau ngā puna MAG i whakamahia i roto i ia tāruarua (P = 0.0046, whakamātautau-t takirua, taha-rua), e tohuhia ana e ngā ira motuhake. b, Ko ngā uwha i moea ki ngā tāne kua whakangāwarihia e te EPP he iti ake te ōwehenga o te 3D20E kua tangohia te phosphorylation i te 3 hpm (P = 0.0043, whakamātautau-t kore takirua, taha-rua), engari kāore ngā taumata 20E i pāngia (P = 0.063, kore takirua). (whakamātautau-t, taha-rua). Ka whakaaturia ngā raraunga hei toharite ± uri mai i ngā puna e toru o ia uwha, 13, 16 me te 19. c, Ko ngā uwha i moea ki ngā tāne i whakawāteatia e te EPP, he nui ake te auau moea anō (P = 0.0002, whakamātautau tika a Fisher, taha-rua). I te tuatahi, i akiakihia ngā uwha ki te moea kia mau ai tō rātou tūnga moea; E rua ngā rā i muri mai, i whakapā atu rātou ki ētahi atu tāne e mau ana i te parāoa whakawhiti ira hei aromatawai i ngā reiti whakaipoipo anō mā te kitenga PCR tauanga o te ira whakawhiti. Ko ngā uwha whāngai toto i whakaipoipohia ki ngā tāne kua whakawāteahia e te EPP he tino heke te momonatanga (P < 0.0001; whakamātautau Mann-Whitney, taha-rua) me te paku heke te maha o ngā hua manu (P = 0.088, whakamātautau Mann-Whitney, taha-rua), engari kāore i pāngia te reiti whakaputa uri (P = 0.94, whakamātautau tika a Fisher, taha-rua). I roto i ngā panui katoa, ko te n te tohu o te maha o ngā tauira waeroa motuhake koiora. NS, kāore i te nui. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Muri iho, i arotakehia e mātou mēnā he mea nui te ecdysone dephosphorylation hei whakaoho i te ātete ki te moe tahi i roto i ngā uwha. He mea nui, ko ngā uwha i moe tahi me ngā tāne kua pau te EPP i moe tahi anō i te auau teitei ake (44.9%) i ngā uwha mana whakahaere (10.4%) i te wā i tūtaki ai ki ētahi atu tāne (whakawhitiwhiti) (Pikitia 3c). I kite anō mātou i te hekenga nui o te momonatanga (Pikitia 3d, maui) me te hekenga iti o te maha o ngā hua i whakatakotoria e ēnei uwha (Pikitia 3d, waenganui), engari kāore i pāngia te ōrau o ngā hua i whakatakotoria e ngā uwha (tētahi atu urupare i puta mai i roto i ngā uwha mā te moe tahi) (Pikitia 3d, matau). I runga i te motuhaketanga o te EPP mō te 3D20E22P, e kī ana ēnei hua ko te whakahohenga o te 3D20E e te EPP i whakawhitia i te wā moe tahi he mahi nui pea ki te whakakore i te aro o ngā uwha ki te moe tahi anō, he whanonga i kīia i mua ko te whakawhiti ira tangata o te 20E. Nō reira, he kaha te pānga o tēnei homoni motuhake ki te momonatanga o ngā wāhine.
Muri iho, i whakaritea e mātou ngā mahi a 20E me 3D20E i roto i ngā whakamātautau werohanga i roto i ngā wahine kua pakeke te ira tangata mā te whakamahi i te 3D20E i hangaia i roto i ngā matū (Pikitia 4a–c) me te 20E e wātea ana i te mākete. I kitea e mātou he nui ake te whai huatanga o te 3D20E i te 20E ki te aukati i te aro o ngā uwha ki te moepuku i ngā kukū e rua (Pikitia 4d). He mea nui, ko te haurua o te taumata ā-tinana o te 3D20E i roto i te LRT (1,066 pg i muri i te werohanga vs. 2,022 pg i muri i te moepuku) i puta ai he ōwehenga o ngā uwha uaua ki te werohanga he 20 ngā wā teitei ake i te taumata ā-tinana o te 20E (361 pg i muri i te werohanga) 24 hāora i muri i te werohanga i te kukū teitei rawa 18 pg i muri i te moepuku; Ripanga Raraunga Whānui 1). He rite tonu tēnei hua ki te whakaaro kāore te whakawhiti ira tangata o te 20E e puta ai ngā wā uaua ki te moe tahi, ā, e tohu ana ki te 3D20E hei take nui hei whakarite i te whanaungatanga mātua-tamaiti. He nui ake te kaha o te 3D20E i te 20E i roto i ngā whakamātautau whakaputa hua i roto i ngā uwha wahine (Pikitia 4e), e kī ana ko te tere whakaputa hua noa i kitea e mātou i muri i te whakangāwari hapa o te EPP i puta mai i te noho tonu o te mahi 3D20E toenga i hangaia e ngā take uwha i puta mai i te moe tahi.
(a,b) 3D20E i hangaia mā te matū mai i te 20E (a) me te tino teitei o te huringa/te whai huatanga (ngā raraunga i whakaaturia hei toharite ± hua mai i ngā tauhohenga hanganga motuhake e toru) (b).c, Ko te whānuitanga papatipu (te haurua o raro) e ōrite ana ki te ecdysone i kitea i roto i te LRT uwha kua whakaipoipohia (te haurua o runga).d, I whakaritea ki te 20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Whakamātautau tika a Fisher, taha-rua) me te 10% etanoru (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Whakamātautau tika a Fisher, taha-2), i te mea he nui ake te 20E i te mana whakahaere anake i ngā horopeta teitei ake (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Whakamātautau tika a Fisher, taha-2).e, I te werohanga 3D20E, i puta ake ngā reiti whakaputa uri teitei ake i roto i ngā uwha wahine i ngā mana whakahaere etanoru 10% (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; Whakamātautau tika a Fisher, taha-rua), i te mea ko te 20E i whakaritea ki ngā mana whakahaere i ngā horopeta teitei ake anake (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; Whakamātautau tika a Fisher, taha-rua). I puta ake ngā reiti whakaputa uri teitei ake i te 3D20E i ngā horopeta teitei ake (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; Whakamātautau tika a Fisher, taha-rua). I roto i ngā panera katoa, ko te n te tohu o te maha o ngā tauira waeroa motuhake koiora. NS, kāore i te hiranga. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. Nō ngā tāruarua e toru ngā raraunga.
I roto i ngā rangahau o mua, i whakatauhia e mātou ko te whakawhiti ira tangata o ngā homoni steroid ka whakaoho i te whakaaturanga o te MISO (Mating-Induced Stimulator of Oogenesis 11), he ira uri wahine e tiaki ana i ngā wahine A. gambiae mai i te mate P. falciparum. Ko ngā utu hauora i puta mai i te 13, te pirinoa malaria tangata tino whakamate. I runga i te hiranga o te MISO ki te hauora uri o Anopheles i ngā rohe e mate ana te malaria, i whakatau mātou ki te whakatau ko tēhea homoni 3D20E, 20E rānei ka whakaoho i te whakaaturanga o tēnei ira. I kitea e mātou ahakoa i whakaohohia e te werohanga 20E ētahi kaiwhakawhiwhi homoni karihi (HR), pērā i te HR3 me te HR4, me ngā whāinga steroid o raro, pērā i ngā ira yolkogenic Vg14, 15, 16, ko te MISO i whakaohohia e te 3D20E (Whakaahua Raraunga Whānui 3). Nō reira, ko te whakawhiti ira tangata o tēnei homoni steroid androgenic te āhua nei ka whakaoho i ngā tikanga e tiaki ana i ngā wahine mai i ngā utu i puta mai i te mate pirinoa. Hei tāpiri, he rerekē te pānga o te 3D20E ki ngā isoforms e rua o te Ka whakaoho te kaiwhakawhiwhi E i te EcR, ka akiaki i te EcR-A me te pehi i te EcR-B, ā, ka kaha ake te whakaoho i ētahi atu ira e whakaoho ana i te moepuku, tae atu ki te HPX15, e pā ana ki te momonatanga o te wahine. Tērā pea ka whakamārama tēnei i te kore tamariki nui i kitea i roto i ngā wahine i moepuku ki ngā tāne i whakawāteatia e te EPP (Raraunga Whānui Pikitia 3). E kī ana ēnei raraunga kei te noho ngā ara o raro e whakahohehia ana e ngā homoni ecdysone e rua e whai pānga ana ki te mahi motuhake o te ira tangata.
Muri iho, i whakamatauria e mātou te mahi a ngā ira EcK e rua i tautuhia i roto i tā mātou ara koiora-pūnaha. Ko te whakamōkito i a EcK1, i a EcK2 rānei i hua ake ai te mate nui o ngā tāne (Raraunga Whānui Pikitia 4a), e kī ana he mea nui te phosphorylation ecdysone, ā, nō reira, te whakakorenga, mō te oranga. Nā te mea i whakaatuhia a EcK2 i ngā taumata teitei ake i a EcK1, ā, i kitea i roto i ngā MAG e ngā proteomics (Pikitia 2b,c me te Ripanga Tāpiri 2), i whakamanahia e mātou tana mahi ecdysteroid kinase mā te whakatō ki te 20E, i hua ake ai te phosphorylation 20E22P (Raraunga Whānui Pikitia 2).4b). I te whakamahi i te 3D20E hei papa, kāore i taea e mātou te kite i te hua phosphorylated 3D20E22P (Raraunga Whānui Pikitia 4c), e kī ana ko te 20E, kaua ko te 3D20E, te mea pea e pai ake ana ki a EcK2.
E ai ki tā mātou tātaritanga RNA-seq, i tino kitea hoki te EcK2 i roto i te LRT o ngā uwha wahine, i reira i whakawetohia ai i muri i te moepuku (Pikitia 2b). I whakaūhia e mātou ēnei raraunga, ā, i whakatau mātou kāore te whāngai toto i pāngia e te whakaaturanga EcK2 (Raraunga Whānui Pikitia 5a). I te whakaroa i ā mātou whakamātautau MS tuatahi, i whakatau mātou ko te tihi o te 20E22P he hononga tata ki te tihi o te 20E (22-26 hāora i muri i te kai toto; Raraunga Whānui Pikitia 5b). Ko te whakamōkito i te EcK2 i roto i ngā uwha wahine i hua ake ai te pikinga 3-ngā wā i roto i te ōwehenga whanaunga o te 20E ki te 20E22P i te 26 hāora i muri i te kai toto (Ngā Pikitia Raraunga Whānui 2c me te 5c), e whakaū ana ka whakapūmau hoki a EcK2 i te 20E i roto i ngā uwha. He mea nui, i mau tonu te aro nui o ngā wahine kua pau te EcK2 ki te moepuku (Raraunga Whānui Pikitia 5d,e), e kī ana anō kāore te whakaputanga 20E a ngā uwha e whakaoho i ngā wā uaua ki te moepuku. Heoi, ko ēnei uwha he nui ake te pikinga o ngā reiti whakaputa hua manu ki te whakataurite ki ngā mana whakahaere, neke atu i te 30% o ngā wahine tauhou e whakaputa hua ana (Raraunga Whānui Pikitia 5f). Mena i werohia te Eck2 RNA aho-rua (dsEcK2) i muri i te whāngai toto, kāore i puta te whakaputa uri, i taua wā kua heke te tihi 20E nā te horomitanga toto. I te nuinga, e tautoko ana ēnei hua i tētahi tauira ka taea e te 20E i hangaia i muri i te ngote toto te whakaoho i te whakaputa uri, engari i te wā anake ka whakawetohia te poraka whakaputa uri (EcK2 me ētahi atu āhuatanga pea) e te moepuku. Kāore ngā werohanga 20E me te 3D20E i aukati i te whakaaturanga EcK2 i roto i ngā wahine tauhou (Raraunga Whānui Pikitia 5g), e kī ana ko ētahi atu āhuatanga hei arataki i te aukati i tēnei kinase. Heoi, kāore i ranea ngā taumata 20E i muri i te whāngai toto ki te whakaoho i te mamae o te moepuku, engari i whakaohohia e ngā taumata teitei o te 3D20E i whakawhitia ira tangata.
He māramatanga nui ā mātou hua ki ngā tikanga e whakahaere ana i te angitu o te whakaputa uri o A. gambiae. Kua puta he tauira i reira kua whanake ngā tāne ki te hanga i ngā titers teitei o te 3D20E, he ecdysone whakarerekētanga motuhake ki te tāne e whakapumau ana i te whānautanga mā te whakakore i te aro o ngā uwha ki te taatai ​​anō. I taua wā anō, kua whanakehia hoki e ēnei kawe malaria he pūnaha whai hua hei whakahohe i te 3D20E i roto i ngā uwha hei whakautu ki te whakawhiti ira tangata o te EPP motuhake ki te tāne. Ki tā mātou e mōhio ana, koinei te tauira tuatahi o te pūnaha homoni steroid e whakahaerehia ana e te tāne me te wahine e mahi ana i tētahi mahi ahurei me te nui i roto i ngā pepeke. Kua whakapaetia te mahi ecdysone motuhake ki te tāne engari kāore anō kia whakaaturia kia tino mārama. Hei tauira, ko tētahi whakapae kua whakahēngia nuitia 18 ko ēnei mahi ka mahia pea e te 20E precursor E1. E mōhiotia whānuitia ana i roto i te Drosophila, ka whakaohohia te monandry e te whakawhiti ira tangata o ngā peptides ira tangata iti19,20 e taunekeneke ana me ngā neurons e whakakorikori ana i te ara whakaputa uri wahine mā roto i ngā kaiwhakawhiwhi peptide ira tangata motuhake21,22. Me mahi anō kia whakatauhia te tohu i raro iho. ngā rerenga wai e whakahaerehia ana e 3D20E i roto i ngā uwha A. gambiae, me te whakatau mēnā ka taea te tiaki i ēnei rerenga wai i waenga i ngā waeroa me te Drosophila.
I runga i te tūranga nui o te 3D20E ki te momonatanga me te whanonga o ngā wāhine i kitea i roto i tā mātou rangahau, ko ngā ara e arahi ana ki te hanga me te whakahohenga o te 3D20E he whai wāhitanga hou mō ngā rautaki whakahaere waeroa ā muri ake nei, pērā i te whakaputanga o ngā tāne koretake whakataetae i roto i ngā rautaki hangarau pepeke koretake. Whakamahia hei tukunga mohoao, hei whakatauira rānei i te 3D20E i roto i te takaro wahine. Tērā pea i whanake te mahi motuhake a te tāne o te 3D20E i te wā i whiwhi ai a A. gambiae me ētahi atu momo Cellia i te kaha ki te whakakotahi i ō rātou purapura ki roto i ngā mono marena, nā te mea ka taea te whakawhiti pai o te maha o ngā homoni me ngā whākōkī whakahohe homoni. Hei utu, ko te whanaketanga 3D20E e whakatinana ana i te monandry ka whakarato i tētahi tikanga mō ngā wāhine (mā te whakaaturanga nui o te MISO) kia pai ake ai tō rātou hauora uri i ngā wāhi e nui ana te horapa o te malaria, e whai wāhi ā-kore ana ki te tuku Plasmodium. I te mea kua whakaatuhia he pānga nui o te 20E uwha ki te oranga me te tipu o te P. falciparum i roto i ngā waeroa uwha Anopheles,24 ko ngā ara homoni steroid tāne me te uwha he wāhanga matua o ngā taunekeneke waeroa-parata.
I whakatipuria ngā momo pepeke A. gambiae G3 i raro i ngā āhuatanga paerewa (26-28 °C, 65-80% haumākū whanaunga, 12:12 h te roa o te mārama/pōuri. I whāngaihia ngā torongū ki te paura kai ika (TetraMin Tropical Flakes, Koi Pellets me Tetra Pond Sticks i te ōwehenga 7:7:2). I whāngaihia ngā waeroa pakeke ki te otinga dextrose 10% me te toto tangata ia wiki (tēnā he rangahau i ngā wāhanga toto). I whiwhihia ngā waeroa wahine mā te wehewehe i ngā ira i te wāhanga pupal i muri i te tirotiro i ngā pito mā te karuārai. Kua whakaahuatia i mua ngā toa e mau ana i te ira whakawhiti DsRed.
I whakahaerehia ngā whakamātautau moe tāwhana ā-ture i runga anō i ngā tikanga kua whakamāramahia i mua. Mō te moe tūturu, i puritia ngā uwha wahine 4-rā te pakeke ki te ōwehenga 1:3 ki ngā tāne wahine kua pakeke mō ngā pō e rua. Mō ngā whakamātautau i werohia ai ngā tāne ki te dsEPP, i tū tahi te noho tahi me ngā rā 3-4 i muri i te werohanga, i te wā i tino whakamōkitohia ai te mahi phosphatase (Raraunga Whānui Pikitia 2b).
I wehea ngā kiko waeroa, ngā tinana mate e toe ana (te toenga o te tinana), te tinana katoa rānei ki roto i te 100% methanol, ā, i whakaranua ki te beader (ngā poi karāhe 2 mm, 2,400 rpm, 90 hēkona). Ko ngā rahinga kiko me ngā rahinga methanol penei: te toenga o te tinana, 50 i roto i te 1,000 µl; MAG, 50–100 80 µl; LRT uwha, 25–50 80 µl. I tukuna te matūriki ki te tangohanga methanol tuarua me te rahinga methanol ōrite. I tangohia ngā para pūtau mā te centrifugation. I whakakotahihia te methanol mai i ngā tangohanga e rua, ā, i whakamaroketia i raro i te rere o te hauota, kātahi ka whakatārewatia anō ki roto i ngā rahinga e whai ake nei o te 80% methanol i roto i te wai: te toenga o te tinana, 50 µl; MAG me te LRT uwha, 30 µl.
I tātarihia ngā tauira i runga i te ine ira papatipu (ID-X, Thermo Fisher) i honoa ki te taputapu LC (Vanquish, Thermo Fisher). I werohia he 5 µl o te tauira ki runga i te pou 3 µm, 100 × 4.6 mm (Inspire C8, Dikma) i puritia i te 25 °C. Ko ngā wāhanga neke mō te LC ko A (wai, 0.1% waikawa formic) me B (acetonitrile, 0.1% waikawa formic). Ko te pikinga LC penei: 5% B mō te 1 meneti, kātahi ka whakanuia ki te 100% B i roto i te 11 meneti. I muri i te 8 meneti i te 100%, whakatikatika anō te pou i te 5% B mō te 4 meneti. Ko te tere rere he 0.3 ml min-1. Ka tutuki te ionization i roto i te pūtake MS mā te ionization rehu hiko wera i roto i ngā aratau pai me te kino.
Ka ine te ine papatipu i ngā raraunga i te awhe m/z mai i te 350 ki te 680 i te taumira 60,000 i te aratau MS katoa. I kohia ngā raraunga MS/MS i runga i te [M + H]+ (ngā whāinga katoa), [M - H2O + H]+ (ngā whāinga katoa), me te [M - H]- (ngā whāinga phosphorylated). I whakamahia ngā raraunga MS/MS hei whakaū i ngā āhuatanga ecdysone o ngā whāinga kāore i te wātea he paerewa. Hei tautuhi i ngā ecdysteroids kāore i te whainga, i tātarihia ngā raraunga MS/MS mō ngā tihi HPLC katoa me te nui whanaunga >15%. Tātaihia mā te whakamahi i ngā ānau paerewa i hangaia mai i ngā paerewa parakore (20E, 3D20E) hei tatau i ngā rahinga tino, i ngā waimehatanga rānei o tētahi tauira motuhake (ngā whāinga katoa) hei tatau i tō rātou ōrite ki ngā rahinga i kitea i roto i te tāne kotahi. Mō te 3D20E, i mahia te tātaitanga mā te whakamahi i te tapeke o ngā tāpiringa e whai ake nei: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-. I tangohia ngā raraunga, ā, i tauangahia mā te whakamahi i te Tracefinder (putanga 4.1). I tātarihia ngā raraunga MS/MS mā te whakamahi i te Xcalibur (putanga 4.4). I whakaritea ngā whānuitanga MS o E, 20E me 3D20E ki ngā paerewa e pā ana. I tātarihia te 3D20E22P mā te tango i ngā matū me te matū a Girard. I tātarihia te 20E22P mā te ōwehenga m/z.
I purea te 3D20E22P mai i te MAG. I mahia te purenga i runga i te tauine tātari mā te whakamahi i tētahi chromatograph wai tino-mahi (Acquity, Waters) me tētahi pūoko papatipu-whā-pole (QDa, Acquity, Waters) i raro i ngā āhuatanga LC ōrite ki te tātari HPLC-MS/MS. I tīmata te kohikohinga wāhanga i te kitenga o te m/z e rite ana ki te 3D20E22P i te wā pupuri ōrite i whakatauhia i mua. Kātahi ka tirohia te parakore o ngā matū i tangohia mā te HPLC-MS/MS e ai ki te whakaahuatanga i runga ake nei.
I tangohia te RNA katoa mai i te 10-12 ngā kiko whakaputa uri, i ētahi atu wāhanga rānei o te tinana (kāore he pane) mā te whakamahi i te matū TRI (Thermo Fisher) e whai ana i ngā tohutohu a te kaihanga. I rongoatia te RNA ki te TURBO DNase (Thermo Fisher). I hangaia te cDNA mā te whakamahi i te Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) e whai ana i ngā tohutohu a te kaihanga. I whakaputaina i mua ngā primers mō te PCR taupū whakawhiti whakamuri (RT-qPCR; Ripanga Raraunga Whānui 2), i hangaia rānei mā te whakamahi i te Primer-BLAST26, me te hiahia ki ngā hua o te 70-150 bp te rahi me te horapa i ngā hononga exon-exon, i ngā primers takirua Primer rānei e wehe ana i ngā exon. I waimehahia ngā tauira cDNA mai i te toru ki te whā ngā tārua koiora kia whā ngā wā ki te wai mō te RT-qPCR. I mahia te ine i roto i ngā tauhohenga tārua 15 µl kei roto ko te 1× PowerUp SYBR Green Master Mix (Thermo Fisher), ngā primers, me te 5 µl o te cDNA waimeha. I whakahaerehia ngā tauhohenga ki runga i te QuantStudio 6 Pro. I kohia, i tātarihia hoki ngā raraunga me te pūnaha PCR wā-tūturu (Thermo Fisher) mā te whakamahi i te Hoahoa me te Tātari (putanga 2.4.3). E ai ki te whakaaturanga i roto i tēnei rangahau, i whakatauritea ngā rahinga whanaunga ki te ira ribosomal RpL19 (AGAP004422), kāore i tino rerekē te āhua o tōna whakaputanga me te whāngai toto 27, te whakaipoipo rānei 3.
I tirohia te kounga o te RNA mā te whakamahi i te Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). I whakaritea ngā whare pukapuka Illumina takirua-mutunga, ā, i whakahaerehia i te Broad Institute o MIT me Harvard. I whakaritea ngā pānui raupapatanga ki te ira A. gambiae (momo PEST, putanga 4.12) mā te whakamahi i te HISAT2 (putanga 2.0.5) me ngā tawhā taunoa. I tangohia ngā pānui me ngā kaute kounga mahere (MAPQ) <30 mā te whakamahi i te Samtools (putanga 1.3.1). I tatauhia te maha o ngā pānui i maheretia ki ngā ira mā te whakamahi i te htseq-count (putanga 0.9.1) me ngā tawhā taunoa. I tatauhia ngā tatau pānui kua whakatauritea, ā, i tātarihia te whakaaturanga ira rerekē mā te whakamahi i te mōkihi DESeq2 (putanga 1.28.1) i roto i te R (putanga 4.0.3).
I tautuhia ngā kaitono ira whakarerekē-Ecdysone mā te rapu tuatahi i te ira A. gambiae mā te whakamahi i te raupapa PSI-BLAST (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), mā te whakamahi i ngā uara taunoa Ngā tawhā me ngā raupapa pūmua patai e whai ake nei: mai i Bombyx mori (Tau Whakauru NP_001038956.1), Musca domestica (Tau Whakauru XP_005182020.1, XP_005175332.1 me XP_011294434.1) me Microplitis demolitor (Tau Whakauru XP_008552646.1 me XP_008552645.1) EcK mai i B. mori (Tau Whakauru NP_001036900), Drosophila melanogaster (Tau Whakauru NP_651202), Apis mellifera (Tau Whakauru XP_394838) me Acyrthosiphon pisum (Tau Whakauru XP_001947166); me EPP mai i B. mori (Tau Whakauru XP_001947166) NP_001177919.1 me NP_001243996.1) me EO o D. melanogaster (Tau Whakauru NP_572986.1) (taahiraa 1). Muri iho, ka tātarihia ngā pānga i runga i te whakaaturanga mRNA teitei (>100 ngā kongakonga/kirobase exons ia miriona pānui kua maheretia (FPKM) >85% rānei) i roto i te kiko whakaputa uri (LRT wahine, MAG rānei) i Gambia (taahiraa 2). Hei whakapai ake i te motuhaketanga, i tīpakohia e mātou ngā whākōkī kaitono e whakaatuhia ana hoki i roto i te kiko whakaputa uri o A. albimanus, he momo anopheles kāore e hanga, e whakawhiti rānei i te ecdysone i te wā e moe ana. I tātarihia ngā ira kaitono i runga i te iti o te whakaaturanga (<100 FPKM, <85 ōrau rānei) i roto i te kiko whakaputa uri o A. albimanus (taahiraa 3). Hei tātari whakamutunga (taahiraa 4), me tutuki i ngā ira kaitono te iti rawa i tētahi o ngā mea e whai ake nei: (1) he nui ake te pikinga i muri i te moe (P < 0.05) e ai ki te tātari o ngā ira rerekē te whakaaturanga me te (2) i roto i ngā kiko kore-whakaputa uri (<85%, <100 FPKM rānei).
I whakarerekētia e mātou ngā tikanga kua whakamāramahia i mua 28,29,30 kia tutuki ai te tapanga isotopic mō te rauropi katoa. I roto i te poto, i whakamatautauria te momo-mohoao Saccharomyces cerevisiae momo II (YSC2, Sigma) i roto i te turanga hauota rewena (BD Difco, DF0335) kei roto (wt/vol) 2% hukahuka (G7528, Sigma), 1.7% waikawa amino kore-waikawa me te haukini whanariki. reo ahurea) me te 5% 15N haukini whanariki (NLM-713, >99%, Cambridge Isotope Laboratories) hei puna hauota anake. I whakahokia mai te rewena mā te centrifugation, ā, i whāngaihia ngā torongū waeroa ad libitum tae noa ki te wā e puta ai te momo. Tāpirihia ki te paraoa ika (0.5 mg mō ia torongū 300) hei ārai i te mate o te wha o ngā tau. Ko ngā uwha anake i whakamahia i roto i ngā whakamātautau moepuku me ngā tāne kāore i tapaina hei tātari i te proteome tāne i whakawhitia i te wā moepuku.
I akiakihia ngā uwha wahine 4-6 rā te pakeke, he 15N-tohu, kia moe tahi me ngā tāne wahine kāore anō kia tohua, he rite te pakeke. I manatokohia te angitu o te moe tahi mā te kimi i ngā mono moe tahi i raro i te karuārai epifluorescence. I te 3, 12, me te 24 hpm, i wehea te atria o ngā uwha 45-55 kua moe tahi ki roto i te 50 µl o te ammonium bicarbonate buffer (pH 7.8) ā, i whakaranua ki te pistle. I whakatōngia te homogenate, ā, i konatunatua te wai whakapūkara ki te 50 µl o te 0.1% RapiGest (186001860, Waters) i roto i te 50 mM ammonium bicarbonate. I tiohia te wai whakapūkara me te pellet mai i ia tauira ki runga i te hukapapa maroke, ā, i tukuna mō te pō ki te taiwhanga MacCoss i te Whare Wānanga o Washington, i reira i oti ai te whakarite tauira mō te LC-MS/MS. Whakatārewatia anō te pellet ki roto i te 50 µl o te 0.1% RapiGest i roto i te 50 mM ammonium bicarbonate me I whakatōkia ki roto i te kaukau wai. I inehia te kukū pūmua o te pellet me te supernatant mā te whakamātautau BCA, i whakaitihia ngā tauira ki te 5 mM dithiothreitol (DTT; Sigma), i whakakōkīhia ki te 15 mM iodoacetamide (Sigma) ā, i whaowhia ki te 37 °C (1:0 50) mō te 1 hāora me te trypsinization: ōwehenga trypsin:substrate). I wehea a RapiGest mā te tāpiri i te 200 mM HCl, ā, i muri mai ka whaowhia ki te 37 °C mō te 45 meneti me te centrifugation i te 14,000 rpm mō te 10 meneti i te 4 °C hei tango i ngā paru. I horoia ngā tauira mā te tangohanga ā-wāhanga totoka aratau-rua (ngā kātene Oasis MCX, Waters) ā, i whakatārewatia anō ki roto i te 0.1% waikawa formic mō te kukū pūmua whakamutunga o te 0.33 µg µl-1. I tātarihia anō ngā proteome MAG kāore i tapaina mai i ngā tāne wahine. E rua ngā tārua tātari i tātarihia mō ia tauira. Muri iho, i tātarihia te 1 µg o ia tauira mā te whakamahi i tētahi pou silica 25-cm kua hanumi, he 75-μm te roa, me tētahi mahanga frit Kasil1 (PQ) silica 4-cm kua hanumi, kua whakakiia ki te kapia Jupiter C12 kua hurihia te āhua (Phenomenex) me te chromatography wai 180-meneti. Ngā Whakamātautau Tauira – I whakahaerehia te MS/MS i runga i te ine papatipu Q-Exactive HF (Thermo Fisher) me te Pūnaha nanoACQUITY UPLC (Waters). Ko ngā raraunga hopu raraunga i hangaia mō ia whakahaere i hurihia ki te hōputu mzML mā te whakamahi i a Proteowizard (putanga 3.0.20287) me te whakamahi i a Comet31 (putanga 3.2) ki te pātengi raraunga FASTA kei roto ngā raupapa pūmua mai i Anopheles gambiae (VectorBase putanga 54), Anopheles coluzzi. I rapua a Mali-NIH (VectorBase putanga 54), Saccharomyces cerevisiae (Uniprot, Maehe 2021), A. gambiae RNA-seq, me ngā whakamāoritanga anga-toru o ngā poke tangata e mōhiotia ana. I whakatauhia ngā FDR e rite ana ki te mahere peptide mā te whakamahi i a Percolator32 (putanga 3.05) me te paepae o te 0.01, ā, i huihuia ngā peptide ki roto i ngā tautuhi pūmua mā te whakamahi i te parsimony pūmua i roto i a Limelight33 (putanga 2.2.0). I whakatauhia te nui o te pūmua whanaunga mā te whakamahi i te tauwehenga nui o te whānuitanga kua whakaritea (NSAF) i tatauhia mō ia pūmua i ia oma e ai ki te whakaahuatanga o mua. I toharitehia te NSAF e pā ana ki ia pūmua puta noa i ngā tauira mai i ngā tauira koiora rerekē e rua. I angitu te huna e te tapanga 15N te proteome uwha, ahakoa i kitea he iti noa te pūmua kāore i tapaina mai i ngā wahine kua tapaina. I tuhia e mātou te kitenga o te whakahekenga pūmua tāne (1-5 whānuitanga) i roto i ngā tauira mata uwha i ngā oma hangarau anake, i reira i oma ai ngā tauira mata i muri i ngā tauira tāne/whakaipoipo, hei hua o te "kawe" HPLC. Ko ngā pūmua i kitea i ētahi wā he 'paru' mai i ngā wahine kua tapaina kua whakarārangihia ki te Ripanga Tāpiri 1.
E rua ngā peptide antigenic, ko QTTDRVAPAPDQQQ (i roto i te momo iso PA) me MESDGTTPSGDSEQ (i roto i te momo iso PA me te PB) i roto i te Genscript. I whakakotahihia ngā peptide e rua, kātahi ka honoa ki te pūmua kawe KLH, ā, ka werowerohia ki roto i ngā rāpeti o Aotearoa. I patua ngā rāpeti i muri i te weronga tuawhā, ā, i wehea te IgG katoa mā te purenga hononga. I whakamahia te IgG mai i te rāpeti tino motuhake ki te EPP mō te western blotting anō.
Mō ngā tohu uru, ko te MAG (n = 10, ko te n te tohu mō te maha o ngā tauira waeroa motuhake koiora) me te LRT uwha (n = 30) mai i ngā toa wahine 4-rā te pakeke me ngā uwha wahine, wahine rānei kua whakawhanaungatia (<10 i muri i te whakawhanaungatanga), i tāpirihia motuhaketia te pūhui tango pūmua (50 mM Tris, pH 8.0; 1% NP-40; 0.25% konutai deoxycholate; 150 mM NaCl; 1 mM EDTA; 1× protease inhibitor cocktail (Roche)). I whakaranua ngā tauira i muri tonu i te wetewete ki te beader (2 mm ngā pīrere karāhe, 2,400 rpm, 90 hēkona). I tangohia ngā paru kore-rewa mā te centrifugation i te 20,000 g i te 4 °C. I inehia ngā pūmua mā te Bradford assay (Bio-Rad). Kātahi ka tangohia te 20 µg o te pūmua MAG, te 40 µg o te pūmua LRT, me te 20 µg o te pūmua papatipu toenga. i whakakorengia te āhua, ā, i wehea mā te 10% Bis-Tris NuPAGE mā te whakamahi i te pūhui MOPS. I whakawhitia ngā pūmua ki ngā kiriuhi fluoride polyvinylidene mā te whakamahi i te pūnaha whakawhiti iBlot2 (Thermo Fisher). I horoia ngā kiriuhi e rua ngā wā ki roto i te 1× PBS-T (0.1% Tween-20 i roto i te PBS) kātahi ka āraia ki roto i te pūhui ārai Odyssey (Li-Cor) mō te 1 hāora i te 22°C. I rūrūtia ngā kiriuhi i te pō i te 4°C me te paturopi tuatahi polyclonal anti-EPP rāpeti ritenga (1:700 i roto i te pūhui ārai) me te paturopi tuatahi monoclonal anti-actin kiore MAC237 (Abeam; 1:4,000). I horoia ngā kiriuhi ki te PBS-T, kātahi ka whakatōkia ki ngā paturopi tuarua (anti-rāpeti kaihe 800CW me te anti-kiore koati 680LT (Li-Cor), e rua 1:20,000) i roto i te pūhui ārai kei roto ko te 0.01% SDS me te 0.2% Tween -20 mō te 1 hāora i te 22 °C. I horoia ngā kiriuhi ki te PBS-T, ā, i whakaahuahia ki te matawai Odyssey CLx. I kohia, ā, i tukatukahia ngā whakaahua i roto i te Image Studio (putanga 5.2). Kāore i kitea he roopu motuhake e rite ana ki te momo EPP-RA (82 kDa).
I tāruatia ngā rohe waehere o EPP (hei isoform AGAP002463-RB kei roto te rohe histidine phosphatase, te rapu rohe tiakina NCBI 34) me EcK2 (AGAP002181) ki roto i te plasmid pET-21a(+) (Novagen Millipore Sigma); Kua whakarārangihia ngā paraimere ki te Ripanga Raraunga Whānui 2. E waru ngā hononga GS4 (i te taha kotahi) i whakaurua i mua i te tohu C-terminal 6xHis o te hanganga pET-21a(+)-EcK2. I hangaia ngā pūmua recombinant mā te whakamahi i te tauhohenga hanganga pūmua E. coli kore-pūtau NEBExpress (New England BioLabs). I purea ngā pūmua recombinant mā te whakamahi i ngā pou takahuri NEBExpress Ni (New England BioLabs). I hangaia te pūmua whakahaere dihydrofolate reductase (DHFR) mā te whakamahi i te tauira DNA mai i te Kete Hanga Pūmua E. coli Kore-Pūtau NEBExpress. I rongoatia ngā pūmua ki roto i te 50% glycerol i roto i te PBS i te -20 °C tae atu ki te 3 marama.
I inehia te ngohe phosphatase o te EPP me ngā tango kiko mā te whakamahi i te 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). Kei roto i te pūhui tauhohenga he 25 mM Tris, 50 mM waikawa acetic, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, me te 1 mM DTT. I whakaranua te kiko ki roto i te pūhui tauhohenga, ā, i tangohia ngā para pūtau mā te centrifugation. Tīmatahia te tauhohenga mā te tāpiri i te whākōkī, te tango kiko rānei ki te pūhui tauhohenga kei roto he 2.5 mg ml-1 pNPP. I whaowhia te ranunga tauhohenga i te pāmahana rūma i te pōuri, ā, i inehia te nui o te pNP i tahurihia mai i te pNPP mā te ine i te mimiti i te 405 nm i ngā wā rerekē.
Mō te ngohe EcK in vitro, i whaowhia te pūmua ki te 0.2 mg 20E, 3D20E rānei ki roto i te 200 µl buffer (pH 7.5) kei roto ko te 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP me te 10 mM MgCl2 mō te 2 hāora i te 27 °C. I whakamutua te tauhohenga mā te tāpiri i te 800 µl methanol, kātahi ka whakamataohia ki te -20 °C mō te 1 hāora, kātahi ka whakatōngia ki te 20,000 g mō te 10 meneti i te 4 °C. Kātahi ka tātarihia te supernatant mā te HPLC-MS/MS. Hei whakakore wera i ngā pūmua i whakamahia i roto i te rōpū whakahaere, i whaowhia ngā pūmua ki roto i te 50% glycerol i roto i te PBS mō te 20 meneti i te 95°C.
Mō te ngohe EPP in vitro, i whaowhia te pūmua ki te 3D20E22P (he rite ki te nui i kitea i roto i ngā takirua 18 o te MAG, i purea e te HPLC-MS/MS) i roto i te 100 µl buffer (pH 7.5) kei roto ko te 25 mM Tris, 50 mM waikawa acetic, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, me te 1 mM DTT mō ngā hāora 3 i te 27 °C. I whakamutua te tauhohenga mā te tāpiri i te 400 µl methanol, ā, i whakamataohia ki te -20 °C mō te hāora 1, kātahi ka whakatōngia ki te 20,000 g mō ngā meneti 10 i te 4 °C. I tātarihia te supernatant e te HPLC-MS/MS.
I whakanuia ngā kongakonga PCR mō te EPP (362 bp), EcK1 (AGAP004574, 365 bp) me te EcK2 (556 bp) mai i te cDNA i whakaritea mai i ngā tūpāpaku waeroa kore-upoko ira-whakauru. I whakanuia te kongakonga PCR o te mana whakahaere eGFP (495 bp) mai i te pCR2.1-eGFP kua whakaahuatia i mua; Kei roto i te Ripanga Raraunga Whānui 2 ngā paraimere PCR. I whakaurua te kongakonga PCR ki waenganui i ngā kaiwhakatairanga T7 kua hurihia i runga i te plasmid pL4440. I tangohia ngā hanganga plasmid mai i te E. coli matatau NEB 5-α (New England Biolabs) ā, i manatokohia mā te raupapatanga DNA i mua i te whakamahinga (tirohia te Raraunga Tāpiri 1 mō te raupapatanga whakauru). I whakamahia ngā paraimere i taurite ki te kaiwhakatairanga T7 (Ripanga Raraunga Whānui 2) hei whakanui ake i te whakauru mai i te plasmid e hangai ana ki te pL4440. I whakaūhia te rahi o te hua PCR mā te electrophoresis agarose gel. I tuhia te dsRNA mai i ngā tauira PCR mā te whakamahi i te Kete Transcription Megascript T7 (Thermo Fisher) ā, i purea kia rite ki ngā tohutohu a te kaihanga me ngā whakarerekētanga kua whakaahuatia i mua.
Mō te werohanga dsRNA, i werohangahia he 1,380 ng o te dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) i te kukū o te 10 ng nl-1 ki roto i te uma o ngā tāne pakeke, o ngā uwha rānei (Nanoject III, Drummond) i roto i te 1 rā i muri i te eclosion. I whakatauhia ngā taumata knockdown ira i roto i te iti rawa e toru ngā tārua koiora mā te tango RNA, te hanga cDNA, me te RT-qPCR. Mō te werohanga ecdysone, i werohangahia ngā uwha wahine 4-rā te pakeke, ngā uwha wahine 6-rā rānei i whāngaihia ki te toto ki te 0.13, 0.21, 0.63 µg rānei o te 20E, 3D20E rānei (Nanoject III, Drummond) i ngā kukū o te 1.3, 2.1, ia, i runga i te hoahoa whakamātautau, i te 6.3 ng nl-1 rānei. Werohia he 100 nl o te 10% (vol/vol) etanoru ki roto i te wai; 100 nl o te 3D20E22P i roto i te 10% etanoru (he rite ki te 75% o te nui i kitea i roto i te takirua MAG). I tohaina matapōkeretia ngā waeroa ki te rōpū werohanga.
Mō ngā whakamātautau whakaputa uri, i whāngaihia ngā uwha e 3-rā te pakeke ki te toto tangata ad libitum. Tangohia ngā waeroa i whāngaihia hapa, kāore rānei i whāngaihia. I runga i te maimoatanga, i whakanohoia ngā uwha ki roto i ngā kapu whakaputa uri motuhake mō ngā pō e whā, i te iti rawa 48 hāora i muri i te kai toto. I tatauhia ngā hua i raro i te stereoscope (Stemi 508, Zeiss); mō ngā uwha kua moepuku, ko ngā hua i pao hei torongū i kiia he momona.
Mō ngā whakamātautau whakawhanaungatanga, i tukuna ngā uwha kia rua ngā rā neke atu rānei kia whanake ai te ātete ki te whakawhanaungatanga, ā, i runga i te maimoatanga, i whakaurua ngā tāne momo-mohoao kua rite te pakeke ki roto i te whare herehere kotahi. I ngā pō e rua i muri mai, i wehea ngā pūmua uwha kua whakatōkia, ā, i tukuna te DNA ira mā te whakarewa-tio me te sonication i roto i te pūhui kei roto ko te 10 mM Tris-HCl, 1 mM EDTA, me te 25 mM NaCl (pH 8.2). I whakatōngia ngā tauira ki te Proteinase K (0.86 µg µl-1) mō te 15 meneti i te 55 °C, ā, i muri mai ko te 10 meneti i te 95 °C. I whakawaiwaitia ngā whakaritenga DNA ira mata kia 10 ngā wā, ā, i tukuna ki te kitenga qPCR o ngā raupapa ira Y; kua whakarārangihia ngā paraimere ki te Ripanga Raraunga Whānui 2. Ko te ngaro o te raupapa ira Y e tohu ana kāore he whakawhanaungatanga.
Mō ngā whakamātautau whakawhanaungatanga anō, i tirotirohia ngā uwha kua whakawhanaungatia mō te aroaro o ngā mono whakawhanaungatanga hei whakaū i te tūnga whakawhanaungatanga, ā, i tukuna kia 2 ngā rā ki te whakawhanake i te ātete ki te whakawhanaungatanga i te ngaro o ngā tāne, e ai ki te whakaahuatanga o mua 36. Kātahi ka whakaurua ngā tāne e mau ana i te parāoa whakawhiti DsRed ki roto i ngā whare herehere uwha. I ngā pō e rua i muri mai, ka wehea ngā pūhui whakatō mai i ngā uwha, ā, ka whakaritea te DNA ira e ai ki te whakaahuatanga i runga ake nei, ā, ka tukuna ki te kitenga qPCR o te ira whakawhiti DsRed; kua whakarārangihia ngā paraimere ki te Ripanga Raraunga Whānui 2. Ko te ngaro o te ira whakawhiti DsRed i tohu kāore i puta he whakawhanaungatanga anō.
I hangaia te 3D20E e ai ki te whakaahuatanga o mua 37. I roto i te poto, i whakarewaina te 10 mg o te 20E (Sigma-Aldrich) ki roto i te 10 ml o te wai, ā, i muri mai ka tāpirihia te 30 mg o te pango konukawata (i roto i te ahua paura, Sigma-Aldrich). I pupuhi tonutia he awa ngawari o te O2 ki roto i te ranunga tauhohenga, i whakaohokia i te pāmahana rūma. I muri i ngā haora 6, i tāpirihia te 30 mL o te methanol hei whakamutu i te tauhohenga. I hurihia te ranunga hei tango i ngā matūriki whakakorikori. I mimitia te wai i roto i te korehau i te pāmahana rūma. I whakarewaina te hua tauhohenga maroke ki roto i te 10% etanoru me te methanol hei werohanga mō te tātari HPLC-MS/MS. Ko te tere tahuritanga (mai i te 20E ki te 3D20E) tata ki te 97% (Pikitia 4b), ā, ko te whānuitanga MS o te 3D20E i hangaia i rite ki te mea i kitea i roto i ngā uwha kua moe (Pikitia 4c).
Kei roto i te pūrākau ngā taipitopito motuhake o ngā whakamātautau tatauranga i mahia. I whakamahia te GraphPad (putanga 9.0) hei whakahaere i te whakamātautau tika a Fisher, te whakamātautau Mantel-Cox, me te whakamātautau-t a Student. I whakahaerehia ngā whakamātautau Cochran-Mantel-Haenszel mā te whakamahi i tētahi tuhinga R ritenga (e wātea ana i https://github.com/duopeng/mantelhaen.test). I whakamatautauria te tohatoha raraunga mō te ōritetanga mā te whakamahi i te whakamātautau Shapiro-Wilk me te paepae hiranga o te 0.05. I te korenga o ngā raraunga i te whakamātautau ōritetanga, i whakahaerehia te whakamātautau Mann-Whitney. I tātarihia ngā raraunga ora mā te whakamahi i te whakamātautau Mantel-Cox. I whakamahia te mōkihi DESeq2 (putanga 1.28.1) hei whakahaere i te tātaritanga rerekētanga taumata ira RNA-seq. Ko te pae whakapae i runga i te kauwhata e tohu ana i te tau waenga. I whakamahia te uara hiranga o P = 0.05 hei paepae mō ngā whakamātautau katoa.
Mō ētahi atu kōrero e pā ana ki te hoahoa o te rangahau, tirohia te whakarāpopototanga o te Pūrongo Rangahau Taiao e hono ana ki tēnei tuhinga.
I tukuna ngā raraunga proteomic MS ki roto i te ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) mā te PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) me te tohu raraunga PXD032157.
Kei roto i te Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) te huinga raraunga RNA-seq i raro i te rekoata raupapa GSE198665.
Tērā pea ka taea te tiki atu i ngā huinga raraunga tāpiri i hangaia, i tātarihia rānei i te wā o te rangahau o nāianei mai i ngā kaituhi e pā ana mēnā he tono tika. Kei roto i tēnei tuhinga ngā raraunga pūtake.
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