牙鲆lin-29基因的全长cDNA克隆、鉴定及其在早期发育中的表达分析
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摘要
通过PCR技术,从牙鲆(Paralichtys olvaceus)总RNA中获得牙鲆lin-29基因c DNA序列,该序列全长1 769 bp,包括1 329 bp开放阅读框,编码442个氨基酸,预测其是一种多锌指结构蛋白;氨基酸序列对比分析显示,牙鲆LIN-29与大黄鱼(Larimichthys crocea)的同源性最高,达95.25%;系统进化树分析表明,牙鲆与其他鱼类聚集一支,并且与罗非鱼的进化关系最近。荧光定量PCR显示,在牙鲆胚胎和仔鱼发育阶段,以原肠胚期lin-29表达量最高;组织表达分析表明,脑中表达最高,肌肉中最低。外源甲状腺激素(TH)在牙鲆仔鱼发育初期(TH处理后2 d、5 d)下调lin-29表达,TH处理后21 d(36 dph)和26 d(41 dph)上调lin-29基因表达,同时,硫脲(TU)处理均显著上调lin-29表达。结果显示牙鲆lin-29基因可能在牙鲆早期发育过程中扮演着重要的角色,为进一步研究lin-29在牙鲆生长发育中的功能奠定基础。
Lin-29 gene from Japanese flounder( Paralichthys olivaceus) was cloned by PCR,and its gene sequence is 1 769 bp including a 1 329 bp open reading frame( ORF) which encodes a polypeptide of 442 amino acids. The lin-29 gene was predicted encoding a zinc finger protein and was highly similar to amino acid sequence in other fishes through phylogenetic analysis. BLAST analysis indicated that the lin-29 from P.olivaceus showed an average homology of 95. 25 % to Larimichthys crocea. Real-time quantitative PCR revealed that expression level of the lin-29 was the highest during gastrulation stage and in the tissue of brain.The expression of lin-29 was highly up or down-regulated by thyroid hormone( TH) at different stages while thiourea( TU) up-regulated significantly the lin-29 levels. The results showed that LIN-29 played an important role in development and metabolism of P. Olivaceus. These results provided the basic information for further study on the role of lin-29 gene in the development of P. olivaceus.
Lin-29 gene from Japanese flounder( Paralichthys olivaceus) was cloned by PCR,and its gene sequence is 1 769 bp including a 1 329 bp open reading frame( ORF) which encodes a polypeptide of 442 amino acids. The lin-29 gene was predicted encoding a zinc finger protein and was highly similar to amino acid sequence in other fishes through phylogenetic analysis. BLAST analysis indicated that the lin-29 from P.olivaceus showed an average homology of 95. 25 % to Larimichthys crocea. Real-time quantitative PCR revealed that expression level of the lin-29 was the highest during gastrulation stage and in the tissue of brain.The expression of lin-29 was highly up or down-regulated by thyroid hormone( TH) at different stages while thiourea( TU) up-regulated significantly the lin-29 levels. The results showed that LIN-29 played an important role in development and metabolism of P. Olivaceus. These results provided the basic information for further study on the role of lin-29 gene in the development of P. olivaceus.
引文
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[21]刘海金,王常安,朱晓琛,等.牙鲆单倍体、三倍体、雌核发育二倍体和普通二倍体胚胎发育的比较[J].大连水产学院学报,2008,23(3):161-167.LIU J H,WANG C A,ZHU X C,et al.Embryonic development of gynogenetic diploid and triploid Japanese flounder Paralichthys olivaceus[J].Journal of Dalian Fisheries University,2008,23(3):161-167.
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[23]FU Y S,SHI Z Y,WU M L,et al.Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder(Paralichthys olivaceus)[J].Plos One,2011,6(7):e22957.
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[25]INUI Y,MIWA S.Thyroid hormone inducesmetamorphosis of flounder larvae[J].General and Comparative Endocrinology,1985,60(3):450-454.
[26]MILLER J,MCLACHLAN A D,KLUG A.Repetitive zincbinding domains in the protein transcription factor IIIA from Xenopus oocytes[J].The Embo Journal,1985,4(6):1609-1614.
[27]LEE M S,GIPPERT G P,SOMAN K V,et al.Threedimensional solution structure of a single zinc finger DNAbinding domain[J].Science,1989,245(4918):635-637.
[28]CONRAD R,BARRIER M,FORD L P.Role of miRNA and miRNA processing factors in development and disease[J].Birth Defects Research Part C:Embryo Today:Reviews,2006,78(2):107-117.
[29]余刚.异时性基因在Caenorhabditis elegans寿命调控中的作用及在小鼠发育中的表达研究[D].厦门:厦门大学,2009.YU G.Heterochronic genes regulate the lifespan of Caenorhabditis elegans and the expression in the development of mouse[D].Xiamen:Xiamen University,2009.
[30]DE JESUS E G,HIRANO T,INUI Y.Changes in cortisol and thyroid hormone concentrations during early development and metamorphosis in the Japanese flounder,Paralichthys olivaceus[J].General and Comparative Endocrinology,1991,82(3):369-376.
[2]SMITH K K.Time's arrow:Heterochrony and the evolution of development[J].International Journal of Developmental Biology,2003,47(7-8):613-621.
[3]AMBROS V,HORVITZ H R.Heterochronic mutants of the nematode Caenorhabditis elegans[J].Science,1984,226(4673):409-416.
[4]AMBROS V,MOSS E G.Heterochronic genes and the temporal control of C.elegans development[J].Trends in Genetics,1994,10(4):123-127.
[5]LEE R C,FEINBAUM R L,AMBROS V.The C.elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75(5):843-854.
[6]MOSS E G,TANG L J.Conservation of the heterochronic regulator Lin-28,its developmental expression and microRNA complementary sites[J].Developmental Biology,2003,258(2):432-442.
[7]SLACK F J,BASSON M,LIU Z C,et al.The lin-41 RBCC gene acts in the C.elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor[J].Molecular Cell,2000,5(4):659-669.
[8]LIN S Y,JOHNSON S M,ABRAHAM M,et al.The C.elegans hunchback homolog,hbl-1,controls temporal patterning and is a probable microRNA target[J].Developmental Cell,2003,4(5):639-650.
[9]ABRAHANTE J E,DAUL A L,LI M,et al.The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1controls developmental time and is regulated by microRNAs[J].Developmental Cell,2003,4(5):625-637.
[10]JEON M,GARDNER H F,MILLER E A,et al.Similarity of the C.elegans developmental timing protein LIN-42 to circadian rhythm proteins[J].Science,1999,286(5442):1141-1146.
[11]PAPP A,ROUGVIE A E,AMBROS V.Molecular cloning of lin-29,a heterochronic gene required for the differentiation of hypodermal cells and the cessation of molting in C.elegans[J].Nucleic Acids Research,1991,19(3):623-630.
[12]MOSS E G,LEE R C,AMBROS V.The cold shock domain protein LIN-28 controls developmental timing in C.elegans and is regulated by the lin-4 RNA[J].Cell,1997,88(5):637-646.
[13]BAGGA S,BRACHT J,HUNTER S,et al.Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation[J].Cell,2005,122(4):553-563.
[14]PASQUINELLI A E,REINHART B J,SLACK F,et al.Conservation of the sequence and temporal expression of let-7heterochronic regulatory RNA[J].Nature,2000,408(6808):86-89.
[15]ROUGVIE A E,AMBROS V.The heterochronic gene lin-29encodes a zinc finger protein that controls a terminal differentiation event in Caenorhabditis elegans[J].Development,1995,121(8):2491-2500.
[16]EULING S,BETTINGER J C,ROUGVIE A E.The LIN-29transcription factor is required for proper morphogenesis of the Caenorhabditis elegans male tail[J].Developmental Biology,1999,206(2):142-156.
[17]HARRIS D T,HORVITZ H R.MAB-10/NAB acts with LIN-29/EGR to regulate terminal differentiation and the transition from larva to adult in C.elegans[J].Development,2011,138(18):4051-4062.
[18]BETTINGER J C,EULING S,ROUGVIE A E.The terminal differentiation factor LIN-29 is required for proper vulval morphogenesis and egg laying in Caenorhabditis elegans[J].Development,1997,124(21):4333-4342.
[19]田永胜,陈松林,徐田军,等.牙鲆不同家系生长性能比较及优良亲本选择[J].水产学报,2009,33(6):901-911.TIAN Y S,CHEN S L,XU T J,et al.The comparison of growth performances of Japanese flounder(Paralichthys olivaceus)families and selection of parents with good trait[J].Journal of Fisheries of China,2009,33(6):901-911.
[20]CHEN S L,ZHANG G J,SHAO C W,et al.Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle[J].Nature Genetics,2014,46(3):253-260.
[21]刘海金,王常安,朱晓琛,等.牙鲆单倍体、三倍体、雌核发育二倍体和普通二倍体胚胎发育的比较[J].大连水产学院学报,2008,23(3):161-167.LIU J H,WANG C A,ZHU X C,et al.Embryonic development of gynogenetic diploid and triploid Japanese flounder Paralichthys olivaceus[J].Journal of Dalian Fisheries University,2008,23(3):161-167.
[22]NAKAMURA A,SHIKANO L,MIZUTA A,et al.Melanophore development during metamorphosis in pseudoalbinic clone of Japanese flounder Paralichthys olivaceus[J].Nippon Suisan Gakkaishi,2005,71(5):729-735.
[23]FU Y S,SHI Z Y,WU M L,et al.Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder(Paralichthys olivaceus)[J].Plos One,2011,6(7):e22957.
[24]MINAMI T.The early life history of a flounder Paralichthys olivaceus[J].Bulletin of the Japanese Society of Scientific Fisheries,1982,48(11):1581-1588.
[25]INUI Y,MIWA S.Thyroid hormone inducesmetamorphosis of flounder larvae[J].General and Comparative Endocrinology,1985,60(3):450-454.
[26]MILLER J,MCLACHLAN A D,KLUG A.Repetitive zincbinding domains in the protein transcription factor IIIA from Xenopus oocytes[J].The Embo Journal,1985,4(6):1609-1614.
[27]LEE M S,GIPPERT G P,SOMAN K V,et al.Threedimensional solution structure of a single zinc finger DNAbinding domain[J].Science,1989,245(4918):635-637.
[28]CONRAD R,BARRIER M,FORD L P.Role of miRNA and miRNA processing factors in development and disease[J].Birth Defects Research Part C:Embryo Today:Reviews,2006,78(2):107-117.
[29]余刚.异时性基因在Caenorhabditis elegans寿命调控中的作用及在小鼠发育中的表达研究[D].厦门:厦门大学,2009.YU G.Heterochronic genes regulate the lifespan of Caenorhabditis elegans and the expression in the development of mouse[D].Xiamen:Xiamen University,2009.
[30]DE JESUS E G,HIRANO T,INUI Y.Changes in cortisol and thyroid hormone concentrations during early development and metamorphosis in the Japanese flounder,Paralichthys olivaceus[J].General and Comparative Endocrinology,1991,82(3):369-376.