青虾Ran基因的克隆、表达及其在卵巢发育中的功能
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摘要
本研究应用RACE技术克隆了青虾(Macrobrachium nipponensis)Ran(Ras related nuclear protein,Ras相关核蛋白)基因全长cDNA序列,该基因cDNA全长1191 bp,包括218 bp的5'UTR,648 bp的开放阅读框(ORF),405 bp的3'UTR,编码215个氨基酸。青虾Ran基因属于P-loop-NTPase超级家族,拥有PTZ00132跨结构域,多肽分子量约为24.57 kDa,理论等电点7.13。系统进化树分析表明,在动物界进化中非常保守的青虾Ran多肽与罗氏沼虾(Macrobrachium rosenbergii)聚为一支,具有最近的亲缘关系。使用荧光定量PCR技术检测Ran基因在成体青虾不同组织和卵巢不同发育期的表达差异,结果显示,Ran基因在青虾不同组织中均有表达,其表达量在卵巢中最高,是精巢表达量的7~8倍;随着卵巢的发育,Ran基因的表达水平呈现上升趋势,在卵巢消退期又恢复到较低水平。RNA干扰后,实验组Ran基因表达量显著低于对照组(P<0.05),同时卵巢发育关键基因Vg(vitellogenin)在卵巢中的表达量也显著低于对照组(P<0.05),推测Ran基因参与雌性卵巢发育过程并对Vg基因的表达起到调控作用。
Ras-related nuclear protein(Ran) is a small GTPase with many functions, such as hydrolysis of GTP, control of cell development, replication of DNA, and RNA transcription. In this study, the cDNA-encoding Ran of the oriental river prawn(Macrobrachium nipponense) was cloned using expressed sequence tag(EST) analysis and a rapid amplification of cDNA ends(RACE) approach. The full-length cDNA of Ran was 1191 bp, comprising a 5' untranslated region of 218 bp, a 3' untranslated region of 405 bp, and an open reading frame of 648 bp. The deduced protein had 215 amino acid residues with a molecular mass of 24.6 kD, and 7.13 point of theoretical isoelectric. Ran belongs to the P-loop NTPase super family. Ran has a PTZ00132 model that crosses multiple domains. The members of P-loop NTPase super family have extremely conservative nucleotide sequences. Phylogenetic analyses indicate evolution of Ran proteins within the animal kingdom is very conservative, with that of M. nipponense most closely related to that of M. rosenbergii. Quantitative real-time RT-PCR analysis revealed the Ran gene was expressed in testis, ovary, brain, muscle, eyestalk, abdominal nerve, heart and gill tissues. The ovary has the highest level of expression and the eyestalk has the lowest level of expression(P<0.05). The Ran gene expression of ovary is seven-eight times higher than that of testis, and the expression level of Ran gene increased with the development of ovary. After ovulation in ovarian regression period, the expression level of Ran gene was at a low level. After RNA interference(RNAi), expression of Ran gene in an experimental group of adult females was significantly lower than in the control group(P<0.05). After RNA interference(RNAi) in the mature female prawns, the expression of Ran gene in experimental group(injected ds RNA solution into the shrimp's pericardial cavity) was significantly lower than in the control group(injected equal amount of DEPC water into the pranw's pericardial cavity)(P<0.05). The expression of Ran gene changes with the development of ovary. The expression of Ran gene increased from the early stage of ovarian development to the mature stage and decreased rapidly after ovulation in the ovary. Expression of Ran in ovarian tissues in the experimental group was also significantly lower than that of control group(P<0.05), indicating RNA interference was effective. Expression of vitellogenin was significantly affected by RNA interference, with expression in the experimental group significantly lower than in the control group(P<0.05). We speculate that the Ran gene plays a regulatory role in expression of the Vg gene, and that the Ran gene is involved in female ovary development.
Ras-related nuclear protein(Ran) is a small GTPase with many functions, such as hydrolysis of GTP, control of cell development, replication of DNA, and RNA transcription. In this study, the cDNA-encoding Ran of the oriental river prawn(Macrobrachium nipponense) was cloned using expressed sequence tag(EST) analysis and a rapid amplification of cDNA ends(RACE) approach. The full-length cDNA of Ran was 1191 bp, comprising a 5' untranslated region of 218 bp, a 3' untranslated region of 405 bp, and an open reading frame of 648 bp. The deduced protein had 215 amino acid residues with a molecular mass of 24.6 kD, and 7.13 point of theoretical isoelectric. Ran belongs to the P-loop NTPase super family. Ran has a PTZ00132 model that crosses multiple domains. The members of P-loop NTPase super family have extremely conservative nucleotide sequences. Phylogenetic analyses indicate evolution of Ran proteins within the animal kingdom is very conservative, with that of M. nipponense most closely related to that of M. rosenbergii. Quantitative real-time RT-PCR analysis revealed the Ran gene was expressed in testis, ovary, brain, muscle, eyestalk, abdominal nerve, heart and gill tissues. The ovary has the highest level of expression and the eyestalk has the lowest level of expression(P<0.05). The Ran gene expression of ovary is seven-eight times higher than that of testis, and the expression level of Ran gene increased with the development of ovary. After ovulation in ovarian regression period, the expression level of Ran gene was at a low level. After RNA interference(RNAi), expression of Ran gene in an experimental group of adult females was significantly lower than in the control group(P<0.05). After RNA interference(RNAi) in the mature female prawns, the expression of Ran gene in experimental group(injected ds RNA solution into the shrimp's pericardial cavity) was significantly lower than in the control group(injected equal amount of DEPC water into the pranw's pericardial cavity)(P<0.05). The expression of Ran gene changes with the development of ovary. The expression of Ran gene increased from the early stage of ovarian development to the mature stage and decreased rapidly after ovulation in the ovary. Expression of Ran in ovarian tissues in the experimental group was also significantly lower than that of control group(P<0.05), indicating RNA interference was effective. Expression of vitellogenin was significantly affected by RNA interference, with expression in the experimental group significantly lower than in the control group(P<0.05). We speculate that the Ran gene plays a regulatory role in expression of the Vg gene, and that the Ran gene is involved in female ovary development.
引文
[1]Liu P W,Qi M,Ren H Y.Functions of the small GTPase protein Ran in cell cycle regulation[J].Chinese Science Bulletin,2011,56(30):2472-2477.[刘佩伟,齐洺,任海云.小G蛋白Ran在细胞周期调控中的作用[J].科学通报,2011,56(30):2472-2477.]
[2]Vernoud V,Horton A C,Yang Z B,et al.Analysis of the small GTPase gene superfamily of arabidopsis[J].Plant Physiol,2003,131(3):1191-1208.
[3]Drivas G T,Shih A,Coutavas E,et al.Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line[J].Mol Cell Biol,1990,10(4):1793-1798.
[4]Bischoff F R,Ponstingl H.Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide[J].Proc Natl Acad Sci USA,1991,88(23):10830-10834.
[5]Koizumi K,Stivers C,Brody T,et al.A search for Drosophila neural precursor genes identifies ran[J].Dev Genes Evol,2001,211(2):67.
[6]Kimura T,Hashimoto I,Nishikawa M,et al.Nucleocytoplasmic transport of luciferase gene m RNA requires CRM1/Exportin1and Ran GTPase[J].Med Mol Morphol,2009,42(2):70-81.
[7]Ohba T,Nakamura M,Nishitani H,et al.Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran[J].Science,1999,284(5418):1356-1358.
[8]Clarke P R,Zhang C.Ran GTPase:a master regulator of nuclear structure and function during the eukaryotic cell division cycle[J].Trends Cell Biol,2001,11(9):366-371.
[9]Li C J,Liu J,Shi Y H,et al.Full length c DNA cloning and tissue expression specificity of Ran gene in color crucian carp[J].Zoological Research,2003,24(3):173-179.[李常健,刘军,石耀华,等.彩鲫Ran基因全长c DNA的克隆及其组织表达特异性分析[J].动物学研究,2003,24(3):173-179.]
[10]Li C J,Liu J,Gui J F.c DNA cloning of Ran gene and analyses on its expression characterization and role in sperm nuclei decondensation in vitro in gibel carp,Carassius auratus gibelio[J].Journal of Fisheries of China,2006,30(3):297-304.[李常健,刘军,桂建芳.银鲫Ran基因的c DNA克隆、表达特征及其在精核解凝中的作用[J].水产学报,2006,30(3):297-304.]
[11]Zhou F L,Zheng L,Yang Q B,et al.Molecular analysis of a ras-like nuclear(Ran)gene from Penaeus monodon,and its expression at the different ovarian stages of development[J].Mol Biol Rep,2012,39(4):3821-3827.
[12]Fu H T,Wan S Q,Fu C P,et al.Screening of microsatellite markers associated with growth traits in Macrobrachium nipponense[J].Acta Hydrobiologica Sinica,2010,34(5):1043-1048.[傅洪拓,万山青,付春鹏,等.青虾生长性状相关的微卫星标记筛选[J].水生生物学报,2010,34(5):1043-1048.]
[13]Qiao H,Xiong Y,Zhang W,et al.Characterization,expression,and function analysis of gonad-inhibiting hormone in Oriental River prawn,Macrobrachium nipponense,and its induced expression by temperature[J].Comp Biochem Physiol A:Mol Integr Physiol,2015,185:1-8.
[14]Du Y X,Ma K Y,Qiu G F.Discovery of the genes in putative Gn RH signaling pathway with focus on characterization of Gn RH-like receptor transcripts in the brain and ovary of the oriental river prawn Macrobrachium nipponense[J].Aquaculture,2015,442:1-11.
[15]Zhang F,Chen L,Ping W,et al.c DNA cloning and expres-sion of Ubc9 in the developing embryo and ovary of oriental river prawn,Macrobrachium nipponense[J].Comp Biochem Physiol B:Biochem Mol Biol,2010,155(3):288-293.
[16]Zhang F,Chen L,Qin J,et al.A novel gene with a v WD domain and three Kazal-type domains:Molecular cloning and expression in the ovary of the oriental river prawn,Macrobrachium nipponense[J].Genetika,2011,47(9):1190-1195.
[17]Liang G X,Fu H T,Qiao H.Molecular characterization and developmental expression of Gtsf1 in the oriental river prawn Macrobrachium nipponense[J].Acta Hydrobiologica Sinica,2016,40(2):261-267.[梁国霞,傅洪拓,乔慧,等.青虾精(卵)母细胞特有因子Gtsf1基因的克隆及其时空表达分析[J].水生生物学报,2016,40(2):261-267.]
[18]Qiao H,Xiong Y W,Jiang S F,et al.Gene expression profile analysis of testis and ovary of oriental river prawn,Macrobrachium nipponense,reveals candidate reproduction-related genes[J].Genet Mol Res Gmr,2015,14(1):2041-2054.
[19]Qiao H,Fu H,Jin S,et al.Constructing and random sequencing analysis of normalized c DNA library of testis tissue from oriental river prawn(Macrobrachium nipponense)[J].Comp Biochem Physiol D:Genom Proteom,2012,7(3):268-276.
[20]Huang P T.Condensed protocols from molecular cloning[M].Beijing:Chemical Industry Press,2008(6):707-707.[黄培堂.分子克隆实验指南精编版[M].北京:化学工业出版社,2008(6):707-707.]
[21]Jiang F W,Fu H T,Qiao H,et al.The RNA interference regularity of transformer-2 gene of oriental river prawn Macrobrachium nipponense[J].Chinese Agricultural Science Bulletin,2014(32):32-37.[江丰伟,傅洪拓,乔慧,等.青虾transformer-2基因RNA干扰规律的研究[J].中国农学通报,2014(32):32-37.]
[22]Zhai Z H,Chen X N,Wang J,et al.Primer design with Primer Primier 5.0[J].Northwest Medical Education,2008,16(4):695-698.[翟中会,陈希南,王娟.利用Primer Premier 5.0进行引物设计[J].西北医学教育,2008,16(4):695-698.]
[23]Zhang Y,Jiang S,Xiong Y,et al.Molecular cloning and expression analysis of extra sex combs gene during development in Macrobrachium nipponense[J].Turk J Fish Aquat Sci,2013,13(2):331-340.
[24]Ma K Y,Lin J Y,Guo S Z,et al.Molecular characterization and expression analysis of an insulin-like gene from the androgenic gland of the oriental river prawn,Macrobrachium nipponense[J].Gen Comp Endocrinol,2013,185(3):90-96.
[25]Wang Y T,Mao H,Hou C C,et al.Characterization and expression pattern of KIFC1-like kinesin gene in the testis of the Macrobrachium nipponense,with discussion of its relationship with structure lamellar complex(LCx)and acroframosome(AFS)[J].Mol Biol Rep,2012,39(7):7591-7598.
[26]Jin S B,Wang N,Qiao H,et al.Molecular cloning and expression of a full length c DNA encoding crustacean hyperglycemic hormone(CHH)in oriental river pawn(Macrobrachium nipponense)[J].Journal of Fishery Sciences of China,2013,37(1):82-92.[金舒博,王宁,乔慧,等.青虾高血糖激素基因全长c DNA序列的克隆及表达分析[J].中国水产科学,2013,37(1):82-92.]
[27]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[28]Zhang C,Hughes M,Clarke P R.Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts[J].J Cell Sci,1999,112(Pt 14):2453-2461.
[29]Moore J D.The Ran-GTPase and cell-cycle control[J].Bio Essays,2001,23(1):77–85.
[30]Ach R A,Gruissem W.A small nuclear GTP-binding protein from tomato suppresses a Schizosaccharomyces pombe cell-cycle mutant[J].Proc Natl Acad Sci USA,1994,91(13):5863-5867.
[31]Merkle T,Haizel T,Matsumoto T,et al.Phenotype of the fission yeast cell cycle regulatory mutant pim1-46,is suppressed by a tobacco c DNA encoding a small,Ran-like GTPbinding protein[J].Plant J Cell Mol Biol,1994,6(4):555-565.
[32]Sazer S.The search for the primary function of the Ran GTPase continues[J].Trends Cell Biol,1996,6(3):81-85.
[33]Seki T,Yamashita K,Nishitani H,et al.Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm[J].Mol Biol Cell,1993,3(12):1373-1388.
[34]Bai H K,Qiao H,Li F J,et al.Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense,and the effects of RNA interference and eyestalk ablation on ovarian maturation[J].Gene,2015,562(1):22-31.
[2]Vernoud V,Horton A C,Yang Z B,et al.Analysis of the small GTPase gene superfamily of arabidopsis[J].Plant Physiol,2003,131(3):1191-1208.
[3]Drivas G T,Shih A,Coutavas E,et al.Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line[J].Mol Cell Biol,1990,10(4):1793-1798.
[4]Bischoff F R,Ponstingl H.Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide[J].Proc Natl Acad Sci USA,1991,88(23):10830-10834.
[5]Koizumi K,Stivers C,Brody T,et al.A search for Drosophila neural precursor genes identifies ran[J].Dev Genes Evol,2001,211(2):67.
[6]Kimura T,Hashimoto I,Nishikawa M,et al.Nucleocytoplasmic transport of luciferase gene m RNA requires CRM1/Exportin1and Ran GTPase[J].Med Mol Morphol,2009,42(2):70-81.
[7]Ohba T,Nakamura M,Nishitani H,et al.Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran[J].Science,1999,284(5418):1356-1358.
[8]Clarke P R,Zhang C.Ran GTPase:a master regulator of nuclear structure and function during the eukaryotic cell division cycle[J].Trends Cell Biol,2001,11(9):366-371.
[9]Li C J,Liu J,Shi Y H,et al.Full length c DNA cloning and tissue expression specificity of Ran gene in color crucian carp[J].Zoological Research,2003,24(3):173-179.[李常健,刘军,石耀华,等.彩鲫Ran基因全长c DNA的克隆及其组织表达特异性分析[J].动物学研究,2003,24(3):173-179.]
[10]Li C J,Liu J,Gui J F.c DNA cloning of Ran gene and analyses on its expression characterization and role in sperm nuclei decondensation in vitro in gibel carp,Carassius auratus gibelio[J].Journal of Fisheries of China,2006,30(3):297-304.[李常健,刘军,桂建芳.银鲫Ran基因的c DNA克隆、表达特征及其在精核解凝中的作用[J].水产学报,2006,30(3):297-304.]
[11]Zhou F L,Zheng L,Yang Q B,et al.Molecular analysis of a ras-like nuclear(Ran)gene from Penaeus monodon,and its expression at the different ovarian stages of development[J].Mol Biol Rep,2012,39(4):3821-3827.
[12]Fu H T,Wan S Q,Fu C P,et al.Screening of microsatellite markers associated with growth traits in Macrobrachium nipponense[J].Acta Hydrobiologica Sinica,2010,34(5):1043-1048.[傅洪拓,万山青,付春鹏,等.青虾生长性状相关的微卫星标记筛选[J].水生生物学报,2010,34(5):1043-1048.]
[13]Qiao H,Xiong Y,Zhang W,et al.Characterization,expression,and function analysis of gonad-inhibiting hormone in Oriental River prawn,Macrobrachium nipponense,and its induced expression by temperature[J].Comp Biochem Physiol A:Mol Integr Physiol,2015,185:1-8.
[14]Du Y X,Ma K Y,Qiu G F.Discovery of the genes in putative Gn RH signaling pathway with focus on characterization of Gn RH-like receptor transcripts in the brain and ovary of the oriental river prawn Macrobrachium nipponense[J].Aquaculture,2015,442:1-11.
[15]Zhang F,Chen L,Ping W,et al.c DNA cloning and expres-sion of Ubc9 in the developing embryo and ovary of oriental river prawn,Macrobrachium nipponense[J].Comp Biochem Physiol B:Biochem Mol Biol,2010,155(3):288-293.
[16]Zhang F,Chen L,Qin J,et al.A novel gene with a v WD domain and three Kazal-type domains:Molecular cloning and expression in the ovary of the oriental river prawn,Macrobrachium nipponense[J].Genetika,2011,47(9):1190-1195.
[17]Liang G X,Fu H T,Qiao H.Molecular characterization and developmental expression of Gtsf1 in the oriental river prawn Macrobrachium nipponense[J].Acta Hydrobiologica Sinica,2016,40(2):261-267.[梁国霞,傅洪拓,乔慧,等.青虾精(卵)母细胞特有因子Gtsf1基因的克隆及其时空表达分析[J].水生生物学报,2016,40(2):261-267.]
[18]Qiao H,Xiong Y W,Jiang S F,et al.Gene expression profile analysis of testis and ovary of oriental river prawn,Macrobrachium nipponense,reveals candidate reproduction-related genes[J].Genet Mol Res Gmr,2015,14(1):2041-2054.
[19]Qiao H,Fu H,Jin S,et al.Constructing and random sequencing analysis of normalized c DNA library of testis tissue from oriental river prawn(Macrobrachium nipponense)[J].Comp Biochem Physiol D:Genom Proteom,2012,7(3):268-276.
[20]Huang P T.Condensed protocols from molecular cloning[M].Beijing:Chemical Industry Press,2008(6):707-707.[黄培堂.分子克隆实验指南精编版[M].北京:化学工业出版社,2008(6):707-707.]
[21]Jiang F W,Fu H T,Qiao H,et al.The RNA interference regularity of transformer-2 gene of oriental river prawn Macrobrachium nipponense[J].Chinese Agricultural Science Bulletin,2014(32):32-37.[江丰伟,傅洪拓,乔慧,等.青虾transformer-2基因RNA干扰规律的研究[J].中国农学通报,2014(32):32-37.]
[22]Zhai Z H,Chen X N,Wang J,et al.Primer design with Primer Primier 5.0[J].Northwest Medical Education,2008,16(4):695-698.[翟中会,陈希南,王娟.利用Primer Premier 5.0进行引物设计[J].西北医学教育,2008,16(4):695-698.]
[23]Zhang Y,Jiang S,Xiong Y,et al.Molecular cloning and expression analysis of extra sex combs gene during development in Macrobrachium nipponense[J].Turk J Fish Aquat Sci,2013,13(2):331-340.
[24]Ma K Y,Lin J Y,Guo S Z,et al.Molecular characterization and expression analysis of an insulin-like gene from the androgenic gland of the oriental river prawn,Macrobrachium nipponense[J].Gen Comp Endocrinol,2013,185(3):90-96.
[25]Wang Y T,Mao H,Hou C C,et al.Characterization and expression pattern of KIFC1-like kinesin gene in the testis of the Macrobrachium nipponense,with discussion of its relationship with structure lamellar complex(LCx)and acroframosome(AFS)[J].Mol Biol Rep,2012,39(7):7591-7598.
[26]Jin S B,Wang N,Qiao H,et al.Molecular cloning and expression of a full length c DNA encoding crustacean hyperglycemic hormone(CHH)in oriental river pawn(Macrobrachium nipponense)[J].Journal of Fishery Sciences of China,2013,37(1):82-92.[金舒博,王宁,乔慧,等.青虾高血糖激素基因全长c DNA序列的克隆及表达分析[J].中国水产科学,2013,37(1):82-92.]
[27]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[28]Zhang C,Hughes M,Clarke P R.Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts[J].J Cell Sci,1999,112(Pt 14):2453-2461.
[29]Moore J D.The Ran-GTPase and cell-cycle control[J].Bio Essays,2001,23(1):77–85.
[30]Ach R A,Gruissem W.A small nuclear GTP-binding protein from tomato suppresses a Schizosaccharomyces pombe cell-cycle mutant[J].Proc Natl Acad Sci USA,1994,91(13):5863-5867.
[31]Merkle T,Haizel T,Matsumoto T,et al.Phenotype of the fission yeast cell cycle regulatory mutant pim1-46,is suppressed by a tobacco c DNA encoding a small,Ran-like GTPbinding protein[J].Plant J Cell Mol Biol,1994,6(4):555-565.
[32]Sazer S.The search for the primary function of the Ran GTPase continues[J].Trends Cell Biol,1996,6(3):81-85.
[33]Seki T,Yamashita K,Nishitani H,et al.Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm[J].Mol Biol Cell,1993,3(12):1373-1388.
[34]Bai H K,Qiao H,Li F J,et al.Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense,and the effects of RNA interference and eyestalk ablation on ovarian maturation[J].Gene,2015,562(1):22-31.