小尾寒羊TNNI2和TNNI3基因的克
发布时间：2018-11-02 08:46
【摘要】：肌钙蛋白I(Troponin I,Tn I)不仅对肌肉的正常收缩行使功能至关重要,还能从多个方面影响到肌肉的品质。肌钙蛋白I家族三个成员分别为肌钙蛋白I1(TNNI1)、肌钙蛋白I2(TNNI2)和肌钙蛋白I3(TNNI3)。本试验根据小尾寒羊和杜泊羊转录组测序数据,筛选到差异表达基因TNNI2和TNNI3。通过比对得到的相关基因保守序列,利用RT-PCR、3’RACE和5’RACE等技术方法克隆得到两个基因的cDNA全长;对两个基因的核苷酸序列和氨基酸序列进行了多种生物信息学比对分析;运用qRT-PCR在m RNA水平验证了两个基因在小尾寒羊和杜泊羊不同组织中的表达丰度。研究结果如下:(1)采用RACE等方法,以小尾寒羊股二头肌为试验材料,克隆得到TNNI2基因cDNA全长714bp,开放阅读框为549bp,编码182个氨基酸,3’端有88bp的非翻译区(不包括polyA尾),5’端有77bp的非翻译区。以小尾寒羊心肌提取mRNA为模板,克隆得到TNNI3基因cDNA全长847bp(不包括polyA尾),开放阅读框为639bp,编码212个氨基酸,3’端有65bp的非翻译区,5’端有143bp的非翻译区。提交GenBank,获得登录号分别为KX110054和KX110055。(2)使用多种软件和在线数据库分析,绵羊TNNI2蛋白和山羊同源性最高,分子量为21.4kDa,等电点为8.8,是一种亲水性较好的碱性蛋白;二级结构以α-螺旋为主,占到了70%以上,兼有少量的无规则卷曲;该蛋白是一种细胞内功能蛋白,并没有预测到信号肽位点;拥有1个O端糖基化位点和9个磷酸化位点。绵羊的TNNI3蛋白则与牛的该蛋白具有极高同源性,分子量为24.1kDa,等电点为9.8,也是一种亲水性较好的碱性蛋白;其二级结构与TNNI2具有较高的相似性,α-螺旋的比例为70.75%,其次是不规则卷曲;该蛋白未预测到信号肽位点;预测该蛋白具有多达14个磷酸化位点,但是没有任何糖基化位点。(3)小尾寒羊和杜泊羊不同组织中,两个基因在mRNA水平存在较大的表达差异。TNNI2基因的mRNA在肋间肌、背最长肌和股二头肌中高表达,在肺脏、胃、心脏和肝脏等组织中表达量很低,说明了TNNI2基因在表达上具有特异性。在不同的品种之间,TNNI2基因在杜泊羊肋间肌组织中的表达量极显著高于小尾寒羊(P0.01);在杜泊羊股二头肌和肺脏组织中表达量显著高于小尾寒羊(P0.05);但是在背最长肌、肝脏、胃和心脏组织中的表达量差异不显著。TNNI3基因在绵羊的心脏组织中高表达,而在股二头肌、肝脏、肺和胃中少量表达甚至不表达,说明了TNNI3基因的表达在生物体组织中有一定的空间特异性。TNNI3基因在绵羊心脏中的表达量极显著高于其他组织(P0.01),说明TNNI3基因具有心脏表达特异性。在不同的品种间,TNNI3基因在杜泊羊心脏组织中的表达量极显著高于小尾寒羊(P0.01),而在股二头肌、肝脏、肺和胃中两个品种羊的表达差异不显著。(4)TNNI2蛋白在小尾寒羊和杜泊羊背最长肌、血管、肋间肌、股二头肌、心肌、肝脏和肺脏六种组织中的表达存在一定差异,在肌肉组织中高表达,在其他组织中不表达或者很低表达。TNNI3蛋白特异在心肌中表达。综上所述,本研究通过对肌钙蛋白I家族的两个基因TNNI2和TNNI3的cDNA全长克隆,从不同的角度对两种蛋白的结构、功能和组织表达特异性进行了分析,为小尾寒羊的生产性状改良和分子标记辅助育种提供依据。
[Abstract]:Tropin I (Tn I) is not only essential for the normal contraction function of muscle, but also affects muscle quality from several aspects. Three members of the cardiac troponin I family are troponin I1 (TNNI1), troponin I2 (TNNI2), and troponin I3 (TNNI3), respectively. The differentially expressed genes TNNI2 and TNNI3 were screened according to the sequencing data of small tail Han sheep and Dupoyang transcription group. The full-length cDNA of the two genes was cloned by RT-PCR, 3 'RACE and 5' RACE techniques. The expression abundance of two genes in different tissues of small tail Han sheep and Dupoyang sheep was verified by qRT-PCR at m RNA level. The results are as follows: (1) The total length of TNNI12 gene cDNA is 714bp, the open reading frame is 549bp, 182 amino acids, 3 'end has 88bp non-translation region (excluding polyA tail), 5 The' end has a non-translated region of 77bp. The total length of TNNI3 gene cDNA was 847bp (excluding polyA tail), the open reading frame was 639bp, 212 amino acids, 3 'end 65bp non-translation region and 143bp non-translation region at 5' end. The GenBank accession number was KX110054 and KX110055, respectively. (2) Using a variety of software and on-line database analysis, sheep TNNI2 protein and goat have the highest homology, the molecular weight is 21. 4kDa, the isoelectric point is 8. 8, it is a hydrophilic relatively good basic protein; The protein is an intracellular functional protein that is not predicted to the signal peptide site; has 1 O-terminal glycosylation site and 9 phosphorylation sites. The TNNI3 protein of sheep has extremely high homology with the protein of bovine, the molecular weight is 24. 1kDa, the isoelectric point is 9. 8, and also is a hydrophilic relatively good basic protein; The protein does not predict the signal peptide site; it is predicted that the protein has up to 14 phosphorylation sites, but there is no glycosylation site. (3) In the different tissues of the small tail Han sheep and the Dupoyang sheep, the mRNA levels of the two genes were significantly different. The mRNA of TNNI2 gene is highly expressed in the intercostal muscles, the longest muscle and quadriceps, and the expression of TNNI2 gene is very low in lung, stomach, heart and liver. The expression of TNNI2 gene in the intercostal muscle of Dupoyang sheep was significantly higher than that of small tail Han sheep (P0.01). There was no significant difference in expression between stomach and heart tissue. TNNI3 gene is highly expressed in sheep's heart tissue, but expression of TNNI3 gene in the liver, lung and stomach is not even expressed, indicating that the expression of TNNI3 gene has certain spatial specificity in organism tissue. The expression of TNNI3 gene in sheep heart was significantly higher than that of other tissues (P0.01). The expression of TNNI3 gene in the heart of Dupoyang sheep was significantly higher than that of small tail Han sheep (P0.01), but there was no significant difference in the expression of two breeds of sheep in the liver, lung and stomach. (4) The expression of TNNI2 protein in the six tissues of the longest muscle, blood vessel, intercostal muscle, quadriceps femoris, myocardium, liver and lung of the small tail Han sheep and the Dupoyang sheep has a certain difference, which is highly expressed in muscle tissue and is not expressed or expressed very low in other tissues. TNNI3 protein is specifically expressed in the myocardium, and the structure, function and tissue expression specificity of the two proteins are analyzed from different angles through the full-length cloning of the two genes TNNI2 and TNNI3 of the troponin I family. It provides the basis for the improvement of production traits and molecular marker assisted breeding for small-tailed Han sheep.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S826;Q78
本文编号：2305526
[Abstract]:Tropin I (Tn I) is not only essential for the normal contraction function of muscle, but also affects muscle quality from several aspects. Three members of the cardiac troponin I family are troponin I1 (TNNI1), troponin I2 (TNNI2), and troponin I3 (TNNI3), respectively. The differentially expressed genes TNNI2 and TNNI3 were screened according to the sequencing data of small tail Han sheep and Dupoyang transcription group. The full-length cDNA of the two genes was cloned by RT-PCR, 3 'RACE and 5' RACE techniques. The expression abundance of two genes in different tissues of small tail Han sheep and Dupoyang sheep was verified by qRT-PCR at m RNA level. The results are as follows: (1) The total length of TNNI12 gene cDNA is 714bp, the open reading frame is 549bp, 182 amino acids, 3 'end has 88bp non-translation region (excluding polyA tail), 5 The' end has a non-translated region of 77bp. The total length of TNNI3 gene cDNA was 847bp (excluding polyA tail), the open reading frame was 639bp, 212 amino acids, 3 'end 65bp non-translation region and 143bp non-translation region at 5' end. The GenBank accession number was KX110054 and KX110055, respectively. (2) Using a variety of software and on-line database analysis, sheep TNNI2 protein and goat have the highest homology, the molecular weight is 21. 4kDa, the isoelectric point is 8. 8, it is a hydrophilic relatively good basic protein; The protein is an intracellular functional protein that is not predicted to the signal peptide site; has 1 O-terminal glycosylation site and 9 phosphorylation sites. The TNNI3 protein of sheep has extremely high homology with the protein of bovine, the molecular weight is 24. 1kDa, the isoelectric point is 9. 8, and also is a hydrophilic relatively good basic protein; The protein does not predict the signal peptide site; it is predicted that the protein has up to 14 phosphorylation sites, but there is no glycosylation site. (3) In the different tissues of the small tail Han sheep and the Dupoyang sheep, the mRNA levels of the two genes were significantly different. The mRNA of TNNI2 gene is highly expressed in the intercostal muscles, the longest muscle and quadriceps, and the expression of TNNI2 gene is very low in lung, stomach, heart and liver. The expression of TNNI2 gene in the intercostal muscle of Dupoyang sheep was significantly higher than that of small tail Han sheep (P0.01). There was no significant difference in expression between stomach and heart tissue. TNNI3 gene is highly expressed in sheep's heart tissue, but expression of TNNI3 gene in the liver, lung and stomach is not even expressed, indicating that the expression of TNNI3 gene has certain spatial specificity in organism tissue. The expression of TNNI3 gene in sheep heart was significantly higher than that of other tissues (P0.01). The expression of TNNI3 gene in the heart of Dupoyang sheep was significantly higher than that of small tail Han sheep (P0.01), but there was no significant difference in the expression of two breeds of sheep in the liver, lung and stomach. (4) The expression of TNNI2 protein in the six tissues of the longest muscle, blood vessel, intercostal muscle, quadriceps femoris, myocardium, liver and lung of the small tail Han sheep and the Dupoyang sheep has a certain difference, which is highly expressed in muscle tissue and is not expressed or expressed very low in other tissues. TNNI3 protein is specifically expressed in the myocardium, and the structure, function and tissue expression specificity of the two proteins are analyzed from different angles through the full-length cloning of the two genes TNNI2 and TNNI3 of the troponin I family. It provides the basis for the improvement of production traits and molecular marker assisted breeding for small-tailed Han sheep.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S826;Q78
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