应用酵母双杂交系统筛选虾夷扇贝FOXL2的相互作用蛋白
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摘要
FOXL2是脊椎动物的雌性决定基因,在多种无脊椎动物中也被认为是雌性决定与分化的关键基因,但其在无脊椎动物中的作用机制目前尚不清晰。本文利用Clontech酵母双杂交系统筛查虾夷扇贝FOXL2的相互作用蛋白。研究首先构建诱饵质粒pGBKT7-FOXL2,并检测诱饵蛋白的毒性及自激活活性,结果显示诱饵蛋白对酵母菌株无毒,也无自激活活性。利用该诱饵质粒,本研究从虾夷扇贝酵母双杂交cDNA文库筛选得到17个阳性克隆,测序验证得到12个读码框正确的阳性克隆,它们来自4种蛋白:COP9信号复合体亚基5、钠/钾ATP酶β3、哺乳动物室管膜相关蛋白1和胰脂肪酶相关蛋白2。研究进一步将诱饵质粒与4种蛋白的猎物质粒进行共转化验证,结果均为阳性,表明这4种蛋白与虾夷扇贝FOXL2都可能存在相互作用。本文为研究FOXL2在无脊椎动物性别决定与分化中的作用提供了参考。
FOXL2,a female sex-determining gene in vertebrates,is also considered a key gene for female determination and differentiation in a variety of invertebrates.But how it works in invertebrates remains unclear.In this study,the interacting proteins of FOXL2 of scallop Patinopecten yessoensis were screened using Clontech yeast two-hybrid system.Bait plasmid pGBKT7-FOXL2 was constructed,and the toxicity and self-activation ability of the bait protein was detected.Results showed that bait protein was non-toxic to yeast strain and had no self-activating activity.A total of 17 positive clones were screened from yeast two-hybrid cDNA library of P.yessoensis.Among them,12 were confirmed to contain correct open reading frame.These proteins showed high sequence similarity with the following four proteins:COP9 signalosome complex subunit 5,sodium/potassium-transporting ATPase subunit beta-3,mammalian ependymin-related protein 1-like and pancreatic lipase-related protein 2-like.Co-transformation of the bait plasmid with prey plasmids of the 4 proteins confirmed that these 4 proteins could interact with scallop FOXL2.This study provides a basis for studying the role of FOXL2 in the sex determination and differentiation of invertebrates.
FOXL2,a female sex-determining gene in vertebrates,is also considered a key gene for female determination and differentiation in a variety of invertebrates.But how it works in invertebrates remains unclear.In this study,the interacting proteins of FOXL2 of scallop Patinopecten yessoensis were screened using Clontech yeast two-hybrid system.Bait plasmid pGBKT7-FOXL2 was constructed,and the toxicity and self-activation ability of the bait protein was detected.Results showed that bait protein was non-toxic to yeast strain and had no self-activating activity.A total of 17 positive clones were screened from yeast two-hybrid cDNA library of P.yessoensis.Among them,12 were confirmed to contain correct open reading frame.These proteins showed high sequence similarity with the following four proteins:COP9 signalosome complex subunit 5,sodium/potassium-transporting ATPase subunit beta-3,mammalian ependymin-related protein 1-like and pancreatic lipase-related protein 2-like.Co-transformation of the bait plasmid with prey plasmids of the 4 proteins confirmed that these 4 proteins could interact with scallop FOXL2.This study provides a basis for studying the role of FOXL2 in the sex determination and differentiation of invertebrates.
引文
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[15]Teaniniuraitemoana V,Huvet A,Levy P,et al.Molecular signatures discriminating the male and the female sexual pathways in the pearl oyster Pinctada margaritifera[J].Plos One,2015,10(3):e0122819.
[16]Si Y,Ding Y,He F,et al.DNA methylation level of cyp19a1a and Foxl2gene related to their expression patterns and reproduction traits during ovary development stages of Japanese flounder(Paralichthys olivaceus)[J].Gene,2016,575(2):321-330.
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[22]Doronkin S,Djagaeva I,Beckendorf S K.CSN5/Jab1mutations affect axis formation in the Drosophilaoocyte by activating a meiotic checkpoint[J].Development,2002,129(21):5053-5064.
[23]Smith P,Leungchiu W M,Montgomery R,et al.The GLH proteins,Caenorhabditis elegans P granule components,associate with CSN-5and KGB-1,proteins necessary for fertility,and with ZYX-1,apredicted cytoskeletal protein[J].Developmental Biology,2002,251(2):333-347.
[24]Kim K,Kim B,Kim H,et al.Pancreatic lipase-related protein(PY-PLRP)highly expressed in the vitellogenic ovary of the scallop,Patinopecten yessoensis[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology,2008,151(1):52-58.
[25]Dheilly N M,Lelong C,Huvet A,et al.Gametogenesis in the pacific oyster Crassostrea gigas:A microarrays-based analysis identifies sex and stage specific genes[J].Plos One,2012,7(5):e36353.
[26]Herrera L,Ottolenghi C,Garcia-Ortiz J E,et al.Mouse ovary developmental RNA and protein markers from gene expression profiling[J].Developmental Biology,2005,279(2):271-290.
[27]文玉杰,李晓玫.钠钾ATP酶的信号转导功能新进展[J].生理科学进展,2005,36(2):159-162.Wen Y J,Li X M.New advances in signal transduction of sodium/potassium-transporting ATPase[J].Progress in Physiological Sciences,2005,36(2):159-162.
[28]Castillo A R D,Battaner E,Guerra M,et al.Regional changes of brain Na+,K+-transporting adenosine triphosphatase related to ovarian function[J].Brain Research,1987,416(1):113-118.
[2]Nagahama Y.Molecular mechanisms of sex determination and gonadal sex differentiation in fish[J].Fish Physiology&Biochemistry,2005,31(2-3):105-109.
[3]Tu Q,Brown C T,Davidson E H,et al.Sea urchin Forkhead gene family:Phylogeny and embryonic expression[J].Developmental Biology,2006,300(1):49-62.
[4]Naimi A,Martinez A S,Specq M L,et al.Molecular cloning and gene expression of Cg-Foxl2during the development and the adult gametogenetic cycle in the oyster Crassostrea gigas[J].Comparative Biochemistry&Physiology Part B,2009,154(1):134-142.
[5]Adell T,Müller W E.Isolation and characterization of five Fox(Forkhead)genes from the sponge Suberites domuncula[J].Gene,2004,334(1):35-46.
[6]Loffler K A,Zarkower D,Koopman P.Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome:FOXL2is a conserved,early-acting gene in vertebrate ovarian development[J].Endocrinology,2003,144(7):3237-3243.
[7]Ottolenghi C,Omari S,Garcia-Ortiz J E,et al.FOXL2is required for commitment to ovary differentiation[J].Human Molecular Genetics,2005,14(14):2053-2062.
[8]Boulanger L,Pannetier M,Gall L,et al.FOXL2is a female sexadetermining gene in the goat[J].Current Biology Cb,2014,24(4):404-408.
[9]Oshima Y,Uno Y,Matsuda Y,et al.Molecular cloning and gene expression of Foxl2in the frog Rana rugosa[J].General&Comparative Endocrinology,2008,159(2-3):170-177.
[10]Wang D,Kobayashi T,Zhou L,et al.Molecular cloning and gene expression of Foxl2in the nile tilapia,Oreochromis niloticus[J].Biochemical&Biophysical Research Communications,2004,320(1):83-89.
[11]Zhang N,Xu F,Guo X.Genomic analysis of the Pacific oyster(Crassostrea gigas)reveals possible conservation of vertebrate sex determination in a mollusc[J].G3:Genes|Genomes|Genetics,2014,4(11):2207-2217.
[12]Liu X L,Zhang Z F,Shao M Y,et al.Sexually dimorphic expression of foxl2during gametogenesis in scallop Chlamys farreri,conserved with vertebrates[J].Development Genes&Evolution,2012,222(5):279-286.
[13]Liu X L,Li Y,Liu J G,et al.Gonadogenesis in scallop Chlamys farreri and Cf‐foxl2expression pattern during gonadal sex differentiation[J].Aquaculture Research,2016,47(5):1605-1611.
[14]Li Y,Zhang L,Sun Y,et al.Transcriptome sequencing and comparative analysis of ovary and testis identifies potential key sex-related genes and pathways in scallop Patinopecten yessoensis[J].Marine Biotechnology,2016,18(4):1-13.
[15]Teaniniuraitemoana V,Huvet A,Levy P,et al.Molecular signatures discriminating the male and the female sexual pathways in the pearl oyster Pinctada margaritifera[J].Plos One,2015,10(3):e0122819.
[16]Si Y,Ding Y,He F,et al.DNA methylation level of cyp19a1a and Foxl2gene related to their expression patterns and reproduction traits during ovary development stages of Japanese flounder(Paralichthys olivaceus)[J].Gene,2016,575(2):321-330.
[17]Hudson Q J,Smith C A,Sinclair A H.Aromatase inhibition reduces expression of FOXL2in the embryonic chicken ovary[J].Developmental Dynamics,2005,233(3):1052-1055.
[18]Markov G V,Tavares R,Dauphinvillemant C,et al.Independent elaboration of steroid hormone signaling pathways in Metazoans[J].Proceedings of the National Academy of Sciences of the U-nited States of America,2009,106(29):11913-11918.
[19]Wei N,Deng X W.The COP9signalosome[J].Annual Review of Cell&Developmental Biology,2003,19(1):261-286.
[20]Wei N,Serino G,Deng X W.The COP9signalosome:More than aprotease[J].Trends in Biochemical Sciences,2008,33(12):592-600.
[21]许红,姚斐,盛清,等.家蚕JAB1/CSN5蛋白的基因克隆表达及亚细胞定位[J].蚕业科学,2009,35(3):508-515.Xu H,Yao F,Sheng Q,et al.Gene cloning,expression and subcellular localization of JAB1/CSN5protein from silkworm,Bombyx mori[J].Science of Sericulture,2009,35(3):508-515.
[22]Doronkin S,Djagaeva I,Beckendorf S K.CSN5/Jab1mutations affect axis formation in the Drosophilaoocyte by activating a meiotic checkpoint[J].Development,2002,129(21):5053-5064.
[23]Smith P,Leungchiu W M,Montgomery R,et al.The GLH proteins,Caenorhabditis elegans P granule components,associate with CSN-5and KGB-1,proteins necessary for fertility,and with ZYX-1,apredicted cytoskeletal protein[J].Developmental Biology,2002,251(2):333-347.
[24]Kim K,Kim B,Kim H,et al.Pancreatic lipase-related protein(PY-PLRP)highly expressed in the vitellogenic ovary of the scallop,Patinopecten yessoensis[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology,2008,151(1):52-58.
[25]Dheilly N M,Lelong C,Huvet A,et al.Gametogenesis in the pacific oyster Crassostrea gigas:A microarrays-based analysis identifies sex and stage specific genes[J].Plos One,2012,7(5):e36353.
[26]Herrera L,Ottolenghi C,Garcia-Ortiz J E,et al.Mouse ovary developmental RNA and protein markers from gene expression profiling[J].Developmental Biology,2005,279(2):271-290.
[27]文玉杰,李晓玫.钠钾ATP酶的信号转导功能新进展[J].生理科学进展,2005,36(2):159-162.Wen Y J,Li X M.New advances in signal transduction of sodium/potassium-transporting ATPase[J].Progress in Physiological Sciences,2005,36(2):159-162.
[28]Castillo A R D,Battaner E,Guerra M,et al.Regional changes of brain Na+,K+-transporting adenosine triphosphatase related to ovarian function[J].Brain Research,1987,416(1):113-118.