虾夷扇贝beta-actin基因和G型溶菌酶基因的克隆与表达研究
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摘要
虾夷扇贝是20世纪80年代有日本引入我国,现已成为我国北方重要的贝类养殖品种,但近年来,虾夷扇贝陆续爆发大规模死亡,不但造成了巨大的经济损失,还严重影响了该产业的健康发展。病害的不断爆发以及病因的多样性迫切要求我们对此进行更加深入的研究。虾夷扇贝中抗病基因的克隆和表达无疑是研究养殖动物免疫防御机制,提高机体抗病力,实现遗传改良的基础和关键。
为进一步研究虾夷扇贝功能基因的表达调控。利用SMART cDNA文库构建试剂盒成功构建了健康虾夷扇贝外套膜和肾脏两种组织的cDNA文库。对随机选取的4009个克隆进行5’端测序,比对,从虾夷扇贝肾脏文库中筛选出1条β肌动蛋白同源序列,对此EST序列两端进行扩增、测序,得到肌动蛋白基因cDNA全长序列。该cDNA全长为1536bp,5’端非编码区84 bp, 3’端非编码区321 bp,编码区1131 bp,编码377个氨基酸;在基因组DNA中,该基因被一个内含子分为两段,内含子位于第41和第42个氨基酸之间,长度为1498bp。虾夷扇贝β-肌动蛋白基因可以被用于作为定量某种虾夷扇贝mRNA的标准,这为继续研究虾夷扇贝其他功能基因,及其分子生物的进一步研究、促进其他相关分子发育和系统进化研究奠定了基础。
从虾夷扇贝外套膜文库中筛选出G型溶菌酶同源序列,对此EST序列两端进行扩增、测序,得到G型溶菌酶基因cDNA全长序列。该基因序列全长为731 bp,5’端非编码区25 bp, 3’端非编码区100 bp,编码区606 bp,编码202个氨基酸。经过进一步分段扩增、测序,得到该基因的五个内含子,将该基因分成六个外显子。六个外显子长度分别为55 bp,60 bp,90 bp,113 bp,148 bp和140 bp;五个内含子的长度分别为7087 bp,5178 bp,3526 bp,710 bp和1400 bp;内含子的两侧都具有RNA正确剪接所必需的识别位点(GT/AG)。采用实时定量PCR方法,检测了虾夷扇贝G型溶菌酶基因在不同组织中的表达以及在菌注射后不同时段G型溶菌酶基因在血细胞中的表达状况。结果发现,该基因主要在外套膜和肝胰腺中表达,在鳃、肾脏、闭壳肌和血细胞中也有微量的表达,在鳗弧菌刺激后的3小时、6小时、9小时三个时段中,G型溶菌酶在血中的表达均显著高于对照组,第12小时、24小时低于对照组,第36小时高于对照组。另外,本研究随机克隆了虾夷扇贝5个不同个体的G型溶菌酶编码区并测序,发现有3个有义突变,由于测序容易产生误差,又对这3个有义突变进行了高通量SNP基因型分析,结果显示有1个有义突变是准确的。为进一步研究其物种的遗传多态性和基因表达差异奠定了基础。
Scallop aquaculture is a big industry and contributes enormously to the economic development of coastal provinces in China. In these years, large-scale mortality of cultured scallop has caused catastrophic losses to scallop aquaculture, which resulted in the production decreasing drastically. The durative outbreak of diseases has stimulated intensive efforts for the development of better health management strategies and characterization of original immune efforts for disease control. The identification and characterization of genes involved in scallop immune responses are now considered to be essential for the elucidation of immune defense mechanisms and disease control.
in this research, the cDNA library from mantle and kidney of mizuhopecten yessoensis was constructed using Creator SMART cDNA construction kit and large numbers of colonies were randomly picked and sequenced from the 5'. One EST with high homology withβ-actin gene of other species was found and then the complete express sequence ofβ-actin from mizuhopecten yessoensis was obtained by PCR. The cDNA of this gene was 1536bp with 1131 bp of ORF, which encodes 177 amino acids. The intron of this gene was 1498bp which cited between the 41st amino and the 42nd amino. The gene in this study is used for other genes’quantitative expression of mizuhopecten yessoensis as an intemal control.
The other EST is also found with high homology with G-type lysozyme genes of other species. and then the complete express sequence of G-type lysozyme genes from mizuhopecten yessoensis was obtained by PCR. The cDNA of this gene was 731bp with 606 bp of ORF, which encodes 202 amino acids. The gene has five introns which lengths are 7087 bp,5178 bp,3526 bp,710 bp and 1400 bp which cited the extron into 6 fragments which lengths are 55 bp,60 bp,90 bp,113 bp,148 bp and 140 bp. The expression of G-type lysozyme in various tissues was measured by using real time PCR analysis. The temporal expression of G-type lysozyme in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of G-type lysozyme in haemolymph was up-regulated evenly in the first 9 h, followed by a drastic increase. Meanwhile, we found several SNPs in the G-type lysozyme mRNA of mizuhopecten yessoensis.Through the method of High-throughput SNP genotyping by single-tube PCR with Tm-shift primers, it tune out that there is one Sense mutation in it. Which established the foundation for the study of genetic polymorphism and gene expression.
为进一步研究虾夷扇贝功能基因的表达调控。利用SMART cDNA文库构建试剂盒成功构建了健康虾夷扇贝外套膜和肾脏两种组织的cDNA文库。对随机选取的4009个克隆进行5’端测序,比对,从虾夷扇贝肾脏文库中筛选出1条β肌动蛋白同源序列,对此EST序列两端进行扩增、测序,得到肌动蛋白基因cDNA全长序列。该cDNA全长为1536bp,5’端非编码区84 bp, 3’端非编码区321 bp,编码区1131 bp,编码377个氨基酸;在基因组DNA中,该基因被一个内含子分为两段,内含子位于第41和第42个氨基酸之间,长度为1498bp。虾夷扇贝β-肌动蛋白基因可以被用于作为定量某种虾夷扇贝mRNA的标准,这为继续研究虾夷扇贝其他功能基因,及其分子生物的进一步研究、促进其他相关分子发育和系统进化研究奠定了基础。
从虾夷扇贝外套膜文库中筛选出G型溶菌酶同源序列,对此EST序列两端进行扩增、测序,得到G型溶菌酶基因cDNA全长序列。该基因序列全长为731 bp,5’端非编码区25 bp, 3’端非编码区100 bp,编码区606 bp,编码202个氨基酸。经过进一步分段扩增、测序,得到该基因的五个内含子,将该基因分成六个外显子。六个外显子长度分别为55 bp,60 bp,90 bp,113 bp,148 bp和140 bp;五个内含子的长度分别为7087 bp,5178 bp,3526 bp,710 bp和1400 bp;内含子的两侧都具有RNA正确剪接所必需的识别位点(GT/AG)。采用实时定量PCR方法,检测了虾夷扇贝G型溶菌酶基因在不同组织中的表达以及在菌注射后不同时段G型溶菌酶基因在血细胞中的表达状况。结果发现,该基因主要在外套膜和肝胰腺中表达,在鳃、肾脏、闭壳肌和血细胞中也有微量的表达,在鳗弧菌刺激后的3小时、6小时、9小时三个时段中,G型溶菌酶在血中的表达均显著高于对照组,第12小时、24小时低于对照组,第36小时高于对照组。另外,本研究随机克隆了虾夷扇贝5个不同个体的G型溶菌酶编码区并测序,发现有3个有义突变,由于测序容易产生误差,又对这3个有义突变进行了高通量SNP基因型分析,结果显示有1个有义突变是准确的。为进一步研究其物种的遗传多态性和基因表达差异奠定了基础。
Scallop aquaculture is a big industry and contributes enormously to the economic development of coastal provinces in China. In these years, large-scale mortality of cultured scallop has caused catastrophic losses to scallop aquaculture, which resulted in the production decreasing drastically. The durative outbreak of diseases has stimulated intensive efforts for the development of better health management strategies and characterization of original immune efforts for disease control. The identification and characterization of genes involved in scallop immune responses are now considered to be essential for the elucidation of immune defense mechanisms and disease control.
in this research, the cDNA library from mantle and kidney of mizuhopecten yessoensis was constructed using Creator SMART cDNA construction kit and large numbers of colonies were randomly picked and sequenced from the 5'. One EST with high homology withβ-actin gene of other species was found and then the complete express sequence ofβ-actin from mizuhopecten yessoensis was obtained by PCR. The cDNA of this gene was 1536bp with 1131 bp of ORF, which encodes 177 amino acids. The intron of this gene was 1498bp which cited between the 41st amino and the 42nd amino. The gene in this study is used for other genes’quantitative expression of mizuhopecten yessoensis as an intemal control.
The other EST is also found with high homology with G-type lysozyme genes of other species. and then the complete express sequence of G-type lysozyme genes from mizuhopecten yessoensis was obtained by PCR. The cDNA of this gene was 731bp with 606 bp of ORF, which encodes 202 amino acids. The gene has five introns which lengths are 7087 bp,5178 bp,3526 bp,710 bp and 1400 bp which cited the extron into 6 fragments which lengths are 55 bp,60 bp,90 bp,113 bp,148 bp and 140 bp. The expression of G-type lysozyme in various tissues was measured by using real time PCR analysis. The temporal expression of G-type lysozyme in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of G-type lysozyme in haemolymph was up-regulated evenly in the first 9 h, followed by a drastic increase. Meanwhile, we found several SNPs in the G-type lysozyme mRNA of mizuhopecten yessoensis.Through the method of High-throughput SNP genotyping by single-tube PCR with Tm-shift primers, it tune out that there is one Sense mutation in it. Which established the foundation for the study of genetic polymorphism and gene expression.
引文
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[2] Tuggle CK, Wang Y, Couture O. Advances in swine transcript omics. Int J Biol Sci, 2007, 3(3): 132?152.
[3] Jiang J, Jacob HJ. EbEST: an automated tool usingexpressed sequence tags to delineate gene structure.Genome Res, 1998, 8(3): 268?275.
[4] CHEN Wei, ZHANG Ge, ZHANG Si-Zhong. Discovery of candidate SNP by bioinformatic methods. Hereditas (Beijing), 2001, 23(2): 153?156.
[5] Kuehnlw M R, Peeken H, Troeder C et al. The Toroidal Drive. Mechanical Engineering, 1981, 103 (2):32-39.
[6] Li L, Cohen SN. Tsg101: A novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells. Cell, 1996, 85(3): 319?329.
[7] GUO Li-Li, CI Hong-Liang, SHAN Hong-Shuang, ZOUXing, ZHAI Yong-Gong, LI Yi-Ping. Molecular cloning and expression analysis of a novel human gene ZNF18. Hereditas (Beijing), 2005, 27(4): 523?530.
[8] Bernardi G D , Onofrio G, Caccio S. Molecular phylogeny of bony fishes , based on the amino acid sequence of the growth hormone [J ] . Journal of Molecular Evolution ,1993 , 37 : 6442649.
[9] Rubin D A , Dores R M. Obtaining a more resolute teleost growth hormone phylogeny by the introduction of gaps in sequence alignment [J ] . Molecular Phylogenetics and Evolution , 1995 , 4 : 1292138.
[10] Chen Y, Wang Y P , He S P , et al. Cloning and sequencing of the growth hormone gene of large yellow croaker and its phylogenetic significance [J ] . Biochemical Genetics , 2004 , 42 : 9210.
[11] Zang X N , Liu B , Liu S M , et al. Transformation and expression of Paralichthys olivaceus growth hormone cDNA in Synechocystis sp. PCC6803 [J ] . Aquaculture , 2007 , 266 : 63269.
[12] Tsai H J , Kuo J C , Lou S W, et al. Growth enhancement of juvenile striped mullet by feeding recombinant yeast containing fish growth hormone [J ] . Progress of Fish Culture , 1994 , 56 : 7212.
[13] Cavari B , Funkenstein B , Chen T T. Effect of growthhormone on the growth rate of the gilthead seabream( S parusaurata) and the use of different constructs for production of transgenic fish [ J ] . Aquacul2 ture , 1993 , 111 : 1892197.
[13] Jolles J, Jolles P. The losozyme from Asterias rubens. Eur J Biochem, 1975, 54(1): 19?23.
[14] Holtje JV. Bacterial lysozymes. EXS, 1996, 75: 65?74.
[15] Hultmark D. Insect lysozymes. EXS, 1996, 75: 87?102.
[16] Beintema JJ, Terwisscha van Scheltinga AC. Plant lysozymes. EXS, 1996, 75: 75?86.
[17] MEINKE D W,CHEN J ,BEACHY R N.Expression of storageprotein genes during soybean seed development[J].Planta 1981 (153): 130-139.
[18] Pet t D , Staron RS. Myosin isoforms , muscle fiber types and t ransitions. Microsc Res Tech , 2000 , 50 : 500—509
[19] Irving M , Piazzesi G. Motions of myosin heads t hat drive muscle cont raction. News Physio Sci , 1997 , 12 : 249—254
[20] Jowett T. Analysis of protein and gene expression. Met hods Cell Biol , 1999 , 59 : 63—85
[21] Frances E. Weaver K. Myosin heavy chain expression in t he red , white , and vent ricular muscle of juvenile stages of rainbow t rout . Zoology , 2001 , 290 : 751—758
[22] Greenberger PA. Immunotherpy update: mechanisms of action [ J ]. Allergy Asthma Proc, 2002, 23 (6) : 373 - 376.
[23] Norman PS. Immunotherapy: 199922004 [ J ]. J Allergy Clin Immu2 nol, 2004, 113 (6) : 1013 - 1023
[24] Ramirez NC, Ledford DK. Immunotherapy for allergic asthma [ J ]. Med Clin North Am, 2002, 86 (5) : 1091 - 1112.
[25] Ohtaki T , Shintani Y, Honda S , et al. Metastasis supp ressor gene KiSS - 1 encodes peptide ligand of a G - prot - ein - cou2 pled receptor [ J ]. Nature, 2001, 411 (6837) : 613～617.
[26] Song L, Zou H, Chang Y, Xu W, Wu L (2006) The cDNA cloning and mRNA expression of a potential seleniumbinding protein gene in the scallop Chlamys farreri. Dev Comp Immunol 30:265–273
[27] Gallois P, Makishima T, Hechtt V, Despres B, Laudie M, Nishimoto T, Cooke R (1997) An Arabidopsis thaliana cDNA complementing a hamster apoptosis suppressor mutant. Plant J 11:1325–1331
[28] Hoeberichts FA, Woltering EJ (2001) Cloning and analysis of a defender against apoptotic cell death (DAD1) homologue from tomato. J Plant Physiol 158:125–128
[29] Vaux DL, Weissman IL, Kim SK (1992) Prevention of programmed cell death in Caenorhabditis elegans by human bcl- 2. Science 258:1955–1957
[30] Young TE, Gallie DR, DeMason DA (1997) Ethylene mediated programmed cell death during maize endosperm development of Su and sh2 genotypes. Plant Physiol 115:737–751
[31] Kumar SK, Tamura K, Jakobsen IB, Nei M (2000) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245
[32] Kawai M, Pan L, Reed JC, Uchimiya H (1999) Evolutionally conserved plant homologue of the Bax inhibitor-1 (BI-1) gene capable of suppressing Bax-induced cell death in yeast. FEBS Lett 464:143–147
[33] Bennetzen, J.L., Hall, B.D., 1982. Codon selection in yeast. J. Biol. Chem. 257, 3026–3031.
[34] Mitta, G., Vandenbulcke, F., Roch, P., 2000a. Original involvement of antimicrobial peptides in mussel innate immunity. FEBS Lett. 486, 185–190. [25]
[35] Boman, H.G., 1998. Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scand. J. Immunol. 48, 15–25.
[36] Guo, X., Ford, S.E., Zhang, F., 1999. Molluscan aquaculture in China. J. Shellfish Res. 18, 19–31.
[37] Bulet, P., St¨ocklin, R., Menin, L., 2004. Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev. 198, 169–184.
[38] Hancock, R.E., Diamond, G., 2000. The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol. 8, 402–410.
[39] Roch, P., 1999. Defense mechanisms and disease prevention in farmed marine invertebrates. Aquaculture 172, 125–145.
[40] Patrzykat, A., Douglas, S.E., 2003. Gone gene fishing: howto catch novel marine antimicrobials. Trends Biotechnol. 121, 362–369.
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