青海湖裸鲤CYP17a2基因的克隆及表达分析
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摘要
CYP17a2基因是细胞色素CYP17(cytochrome P450,CYP)超家族成员之一,在卵巢发育过程中,可以与CYP17a1共同作用控制卵子的发育和成熟。研究和掌握青海湖裸鲤(Gymnocypris przewalskii)卵巢发育的基本规律,可为该鱼种的人工增殖放流及其资源保护和恢复提供技术支撑。本研究采用普通PCR和RACE方法克隆得到了青海湖裸鲤CYP17a2基因全长c DNA序列,并分析了其生物信息学意义,通过实时荧光定量PCR确定了CYP17a2基因在青海湖雌性裸鲤各组织中的表达分布特征。该基因的c DNA序列全长为1 871 bp,共编码390个氨基酸,所编码的氨基酸序列与斑马鱼和鲤鱼的同源性最高,分别为69%和64%。该基因在卵巢、卵巢膜、脑、肌肉、肝胰脏中均有表达,以在卵巢中表达量最高(p<0.01)。本研究结果将为进一步开展CYP17a2基因在青海湖裸鲤卵巢发育的分子调节机理的研究中提供基础数据。
CYP17a2 gene is one of the members in cytochrome CYP17(cytochrome P450,CYP) super family.CYP17a2 can interact with CYP17a1 to control the growth and maturation of ova during the process of ovarian development.To study and master the regulation of the ovary development of the naked carp(Gymnocypris przewalskii) in Qinghai Lake can provide technical support for the artificial reproduction and release,and the protection and recovery of resources of the species.In this study,we employed the ordinary PCR and RACE method to clone the full-length c DNA of CYP17a2 gene of Gymnocypris przewalskii and analysed its significance by bioinformatics.The distribution characteristics of CYP17a2 gene expression in different tissues of the naked carp in Qinghai Lake was determined by real-time fluorescence quantitative PCR.The full-length c DNA sequence was 1 871 bp,which encoded 390 amino acid residues.The coded amino acid sequences of CYP17a2 gene in Gymnocypris przewalskii was most homologous to Danio rerio and Cyprinus carpio,which reached 69% and 64%,respectively.The results showed that CYP17a2 existed in all the tested tissues including the ovary,ovarian membranes,brain,muscles and hepatopancreas,and the highest expression level was shown in ovary(p<0.01).In general,the results would provide theoretical basis for the further study on the molecular regulating mechanism of CYP17a2 gene in ovarian development of Gymnocypris przewalskii in Qinghai Lake.
CYP17a2 gene is one of the members in cytochrome CYP17(cytochrome P450,CYP) super family.CYP17a2 can interact with CYP17a1 to control the growth and maturation of ova during the process of ovarian development.To study and master the regulation of the ovary development of the naked carp(Gymnocypris przewalskii) in Qinghai Lake can provide technical support for the artificial reproduction and release,and the protection and recovery of resources of the species.In this study,we employed the ordinary PCR and RACE method to clone the full-length c DNA of CYP17a2 gene of Gymnocypris przewalskii and analysed its significance by bioinformatics.The distribution characteristics of CYP17a2 gene expression in different tissues of the naked carp in Qinghai Lake was determined by real-time fluorescence quantitative PCR.The full-length c DNA sequence was 1 871 bp,which encoded 390 amino acid residues.The coded amino acid sequences of CYP17a2 gene in Gymnocypris przewalskii was most homologous to Danio rerio and Cyprinus carpio,which reached 69% and 64%,respectively.The results showed that CYP17a2 existed in all the tested tissues including the ovary,ovarian membranes,brain,muscles and hepatopancreas,and the highest expression level was shown in ovary(p<0.01).In general,the results would provide theoretical basis for the further study on the molecular regulating mechanism of CYP17a2 gene in ovarian development of Gymnocypris przewalskii in Qinghai Lake.
引文
Ding Y.X.,He F.,Wen H.S.,Li J.F.,Ni M.,Chi M.L.,Qian K.,Bu Y.,Zhang D.Q.,Si Y.F.,and Zhao J.L.,2013,DNAmethylation status of cyp17-Ⅱgene correlated with its expression pattern and reproductive endocrinology during ovarian development stages of Japanese flounder(Paralichthys olivaceus),Gene,527(1):82-88
Goldstone J.V.,Mcarthur A.G.,Kubota A.,Zanette J.,Parente T.,Jonsson M.E.,Nelson D.R.,and Stegeman J.J.,2010,Identification and developmental expression of the full complement of cytochrome P450 genes in Zebrafish,BMC Genomics,11(1):643
Iwade R.,Maruo K.,Okada G.,and Nakamura M.,2008,Elevated expression of P450c17(CYP17)during testicular formation in the frog,General and Comparative Endocrinology,155(1):79-87
Jin G.X.,Wen H.S.,He F.,Li J.F.,Chen C.F.,Zhang J.R.,Chen X.Y.,Shi B.,Shi D.,Yang Y.P.,Qi B.X.,and Li N.,2012,Molecular cloning,characterization expression of P450c17-Ⅰand P450c17-Ⅱand their functions analysis during the reproductive cycle in males of barfin flounder(Verasper moseri),Fish Physiology and Biochemistry,38(3):807-817
Kazeto Y.,Ijiri S.,Todo T.,Adachi S.,and Yamauchi K.,2000,Molecular cloning and characterization of Japanese eel ovarian P450c17(CYP17)c DNA,General and Comparative Endocrinology,118(1):123-133
Lei L.,and Egli M.,2016,In situ proteolysis for crystallization of membrane bound cytochrome P450 17A1 and 17A2Proteins from Zebrafish,Current Protocols in Protein Science,29(16):1-19
Nematollahi M.A.,Van Pelt-Heerschap H.,Atsma W.,and Komen J.,2012,High levels of corticosterone,and gene expression of star,cyp17a2,hsd3b,cyp21,hsd11b2 during acute stress in common carp with interrenal hyperplasia,General and Comparative Endocrinology,176(2):252-258
Pallan P.S.,Nagy L.D.,Lei L.,Gonzalez E.,Kramlinger V.M.,Azumaya C.M.,Wawrzak Z.,Waterman M.R.,Guengerich F.P.,and Egli M.,2015,Structural and kinetic basis of steroid 17α,20-lyase activity in teleost fish cytochrome P45017A1 and its absence in cytochrome P450 17A2,Journal of Biological Chemistry,290(6):3248-3268
Su T.,Ijiri S.,Kanbara H.,Hagihara S.,Wang D.S.,and Adachi S.,2015,Characterization and expression of c DNAs encoding P450c17-Ⅱ(cyp17a2)in Japanese eel during induced ovarian development,General and Comparative Endocrinology,221:134-143
Wang F.F.,2016,Bioinformatics analysis of cyp17a2 in Nile Tilapia and differences of expression in gonads,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),44(7):103-106(王伟伟,2016,尼罗罗非鱼cyp17a2生物信息学和雌雄性腺表达差异分析,安徽农业科学,44(7):103-106)
Wang Y.,and Ge W.,2004,Cloning of zebrafish ovarian P450c17(CYP17,17alpha-hydroxylase/17,20-lyase)and characterization of its expression in gonadal and extra-gonadal tissues,General and Comparative Endocrinology,135(2):241-249
X iong F.,Chen D.,and Duan X.,2010,Threatened fishes of the world:Gymnocypris przewalskii(Kessler,1876)(Cyprinidae:Schizothoracinae),Environmental Biology of Fishes,87(4):351-352
Zhou L.Y.,Wang D.S.,Kobayashi T.,Yano A.,Paul-Prasanth B.,Suzuki A.,Sakai F.,and Nagahama Y.,2007a,A novel type of P450c17 lacking the lyase activity is responsible for C21-steroid biosynthesis in the fish ovary and head kidney,Endocrinology,148(9):4282-4291
Zhou L.Y.,Wang D.S.,Shibata Y.,Paul-Prasanth B.,Suzuki A.,and Nagahama Y.,2007b,Characterization,expression and transcriptional regulation of P450c17-Ⅰand-Ⅱin the medaka,Oryzias latipes,Biochemical and Biophysical Research Communications,362(3):619-625
Goldstone J.V.,Mcarthur A.G.,Kubota A.,Zanette J.,Parente T.,Jonsson M.E.,Nelson D.R.,and Stegeman J.J.,2010,Identification and developmental expression of the full complement of cytochrome P450 genes in Zebrafish,BMC Genomics,11(1):643
Iwade R.,Maruo K.,Okada G.,and Nakamura M.,2008,Elevated expression of P450c17(CYP17)during testicular formation in the frog,General and Comparative Endocrinology,155(1):79-87
Jin G.X.,Wen H.S.,He F.,Li J.F.,Chen C.F.,Zhang J.R.,Chen X.Y.,Shi B.,Shi D.,Yang Y.P.,Qi B.X.,and Li N.,2012,Molecular cloning,characterization expression of P450c17-Ⅰand P450c17-Ⅱand their functions analysis during the reproductive cycle in males of barfin flounder(Verasper moseri),Fish Physiology and Biochemistry,38(3):807-817
Kazeto Y.,Ijiri S.,Todo T.,Adachi S.,and Yamauchi K.,2000,Molecular cloning and characterization of Japanese eel ovarian P450c17(CYP17)c DNA,General and Comparative Endocrinology,118(1):123-133
Lei L.,and Egli M.,2016,In situ proteolysis for crystallization of membrane bound cytochrome P450 17A1 and 17A2Proteins from Zebrafish,Current Protocols in Protein Science,29(16):1-19
Nematollahi M.A.,Van Pelt-Heerschap H.,Atsma W.,and Komen J.,2012,High levels of corticosterone,and gene expression of star,cyp17a2,hsd3b,cyp21,hsd11b2 during acute stress in common carp with interrenal hyperplasia,General and Comparative Endocrinology,176(2):252-258
Pallan P.S.,Nagy L.D.,Lei L.,Gonzalez E.,Kramlinger V.M.,Azumaya C.M.,Wawrzak Z.,Waterman M.R.,Guengerich F.P.,and Egli M.,2015,Structural and kinetic basis of steroid 17α,20-lyase activity in teleost fish cytochrome P45017A1 and its absence in cytochrome P450 17A2,Journal of Biological Chemistry,290(6):3248-3268
Su T.,Ijiri S.,Kanbara H.,Hagihara S.,Wang D.S.,and Adachi S.,2015,Characterization and expression of c DNAs encoding P450c17-Ⅱ(cyp17a2)in Japanese eel during induced ovarian development,General and Comparative Endocrinology,221:134-143
Wang F.F.,2016,Bioinformatics analysis of cyp17a2 in Nile Tilapia and differences of expression in gonads,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),44(7):103-106(王伟伟,2016,尼罗罗非鱼cyp17a2生物信息学和雌雄性腺表达差异分析,安徽农业科学,44(7):103-106)
Wang Y.,and Ge W.,2004,Cloning of zebrafish ovarian P450c17(CYP17,17alpha-hydroxylase/17,20-lyase)and characterization of its expression in gonadal and extra-gonadal tissues,General and Comparative Endocrinology,135(2):241-249
X iong F.,Chen D.,and Duan X.,2010,Threatened fishes of the world:Gymnocypris przewalskii(Kessler,1876)(Cyprinidae:Schizothoracinae),Environmental Biology of Fishes,87(4):351-352
Zhou L.Y.,Wang D.S.,Kobayashi T.,Yano A.,Paul-Prasanth B.,Suzuki A.,Sakai F.,and Nagahama Y.,2007a,A novel type of P450c17 lacking the lyase activity is responsible for C21-steroid biosynthesis in the fish ovary and head kidney,Endocrinology,148(9):4282-4291
Zhou L.Y.,Wang D.S.,Shibata Y.,Paul-Prasanth B.,Suzuki A.,and Nagahama Y.,2007b,Characterization,expression and transcriptional regulation of P450c17-Ⅰand-Ⅱin the medaka,Oryzias latipes,Biochemical and Biophysical Research Communications,362(3):619-625