墨吉明对虾肌肉生长抑制素基因（FmMstn）克隆及功能和多态性研究
发布时间：2019-06-14 07:39
【摘要】：肌肉生长抑制素(myostatin,Mstn)属于转化生长因子-beta(TGF-β)超家族中的一个重要成员,对动物肌肉的生长和发育具有重要的负调控作用。随着近年凡纳滨对虾养殖病害的频发,墨吉明对虾(Fenneropenaeus merguiensis)作为对虾养殖业的重要对虾养殖品种逐渐成为人们的研究热点。本实验克隆获得了墨吉明对虾FmMstn基因组DNA的全长序列和两个高度相似的FmMstn(短型和长型)cDNA序列,原因是第1个外显子和第一个内含子交界处有两个可选择的GT剪接位点。FmMstn基因结构与软体动物、鱼类和哺乳类动物的Mstn基因类似,含有3个外显子和2个内含子。在FmMstn基因上游的2160bp碱基启动子(promoter)序列,预测的转录因子结合位点包括CAAT box,TATA box,E-boxes和Mstn基因的标记性转录因子即肌增强因子(myocyte enhancing factor element,MEF)。短FmMstn开放阅读框(ORF)含1260bp碱基数,编码420氨基酸。长FmMstn cDNA比短FmMstn cDNA多出(GNNYTSQS)8个氨基酸序列,其他序列同源性一致。FmMstn基因与凡纳滨对虾(Litopenaeus vannamei)和斑节对虾(Penaeus monodon)的Mstn有较高的同源性(95%)。多重序列比对分析显示,FmMstn蛋白质结构与其他动物的Mstn结构类似,具有两个保守的切割位点(RXXR),高度保守的9个半胱氨酸残基和位于成熟肽区高保守TGF功能区。Real-time PCR结果显示,FmMstn在对虾心脏、肌肉、眼柄和胸节神经组织有高表达外,在其他组织微量表达;FmMstn在对虾蜕皮后期有较高的表达水平,随后蜕皮间期FmMstn的表达量呈现下降的趋势,而在蜕皮前期时呈上调趋势直到蜕皮;同一生长周期的对虾,较小个体的FmMstn表达水平较高。通过RNA干扰技术(RNAi),沉默FmMstn基因的表达,可显著延长对虾的蜕皮周期,甚至使对虾蜕皮失败。通过DNA测序结果的直接比对分析,结果显示FmMstn基因具有高度的多态性,且本研究识别了几个潜在的单核酸多肽(SNPs)位点。综上所述,本研究的结果显示墨吉明对虾FmMstn与对虾个体的蜕皮活动和肌肉生长有一定的相关性,而单核酸多态性(SNP)的存在说明FmMstn基因可作为墨吉明对虾遗传选育研究的候选基因。
[Abstract]:Myostatin (myostatin,Mstn), an important member of transforming growth factor-beta (TGF- 尾) superfamily, plays an important negative role in muscle growth and development in animals. With the frequent occurrence of diseases in Penaeus vannamei culture in recent years, (Fenneropenaeus merguiensis), as an important shrimp culture variety in shrimp culture industry, has gradually become a hot research topic. In this experiment, the full-length sequence of FmMstn genomic DNA and two highly similar FmMstn (short and long) cDNA sequences were obtained because there were two optional GT splicing sites at the junction of the first exon and the first intron. The structure of FmMstn gene was similar to that of molluscs, fish and mammals with three exons and two introns. In the (promoter) sequence of 2160bp base promoter upstream of FmMstn gene, the predicted transcription factor binding sites include the marker transcription factor of CAAT box,TATA box,E-boxes and Mstn gene, muscle enhancer (myocyte enhancing factor element,MEF). Short FmMstn open reading frame (ORF) contains 1260bp base number and encodes 420 amino acids. The (GNNYTSQS) 8 amino acid sequences of long FmMstn cDNA were more than those of short FmMstn cDNA, and the homology of other sequences was the same. FmMstn gene had high homology with (Litopenaeus vannamei) and (Penaeus monodon) Mstn of Penaeus vannamei (95%). Multiple sequence alignment analysis showed that the structure of FmMstn protein was similar to that of other animals, with two conserved (RXXR), highly conserved TGF residues and highly conserved TGF functional areas in mature peptide regions. Real-time PCR results showed that FmMstn was highly expressed in the heart, muscle, eye handle and thoracic ganglia nerve tissue of Penaeus chinensis, and was slightly expressed in other tissues. The expression level of FmMstn was higher in the late molting stage, and then decreased in the intermolting period, but up-regulated until molting in the early molting stage, and the FmMstn expression level in smaller individuals was higher in the same growth cycle. Silencing the expression of FmMstn gene by RNA interference technique can significantly prolong the molting cycle of Penaeus chinensis and even make the molting failure of Penaeus chinensis. Through the direct comparison and analysis of DNA sequencing results, the results showed that the FmMstn gene had a high degree of polymorphism, and several potential single nucleic acid polypeptide (SNPs) sites were identified in this study. To sum up, the results of this study showed that FmMstn of Penaeus Mojiming was related to molting activity and muscle growth of Penaeus vulgaris, and the existence of single nucleic acid polymorphism (SNP) suggested that FmMstn gene could be used as a candidate gene for genetic breeding of Penaeus Mojiming.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
本文编号：2499231
[Abstract]:Myostatin (myostatin,Mstn), an important member of transforming growth factor-beta (TGF- 尾) superfamily, plays an important negative role in muscle growth and development in animals. With the frequent occurrence of diseases in Penaeus vannamei culture in recent years, (Fenneropenaeus merguiensis), as an important shrimp culture variety in shrimp culture industry, has gradually become a hot research topic. In this experiment, the full-length sequence of FmMstn genomic DNA and two highly similar FmMstn (short and long) cDNA sequences were obtained because there were two optional GT splicing sites at the junction of the first exon and the first intron. The structure of FmMstn gene was similar to that of molluscs, fish and mammals with three exons and two introns. In the (promoter) sequence of 2160bp base promoter upstream of FmMstn gene, the predicted transcription factor binding sites include the marker transcription factor of CAAT box,TATA box,E-boxes and Mstn gene, muscle enhancer (myocyte enhancing factor element,MEF). Short FmMstn open reading frame (ORF) contains 1260bp base number and encodes 420 amino acids. The (GNNYTSQS) 8 amino acid sequences of long FmMstn cDNA were more than those of short FmMstn cDNA, and the homology of other sequences was the same. FmMstn gene had high homology with (Litopenaeus vannamei) and (Penaeus monodon) Mstn of Penaeus vannamei (95%). Multiple sequence alignment analysis showed that the structure of FmMstn protein was similar to that of other animals, with two conserved (RXXR), highly conserved TGF residues and highly conserved TGF functional areas in mature peptide regions. Real-time PCR results showed that FmMstn was highly expressed in the heart, muscle, eye handle and thoracic ganglia nerve tissue of Penaeus chinensis, and was slightly expressed in other tissues. The expression level of FmMstn was higher in the late molting stage, and then decreased in the intermolting period, but up-regulated until molting in the early molting stage, and the FmMstn expression level in smaller individuals was higher in the same growth cycle. Silencing the expression of FmMstn gene by RNA interference technique can significantly prolong the molting cycle of Penaeus chinensis and even make the molting failure of Penaeus chinensis. Through the direct comparison and analysis of DNA sequencing results, the results showed that the FmMstn gene had a high degree of polymorphism, and several potential single nucleic acid polypeptide (SNPs) sites were identified in this study. To sum up, the results of this study showed that FmMstn of Penaeus Mojiming was related to molting activity and muscle growth of Penaeus vulgaris, and the existence of single nucleic acid polymorphism (SNP) suggested that FmMstn gene could be used as a candidate gene for genetic breeding of Penaeus Mojiming.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
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