猪GLP2R和MYO6基因cDNA克

发布时间：2018-06-13 01:02

本文选题：猪 + GLP2R　；参考：《湖南农业大学》2016年硕士论文

【摘要】：本研究克隆了猪GLP2R基因和MY06基因的cDNA并且分析其序列及其蛋白的生物学信息；以5个地方猪种和1个外来猪种为实验材料,研究了猪GLP2R基因和MY06基因的单核苷酸多态性,采用PCR-RFLP方法对该基因的遗传多态性进行了分析,并分别对这两个基因的SNP位点与猪达100 kg日龄以及校正背膘厚两个性状进行关联分析。结果如下：采用RACE技术成功克隆获得猪GLP2R基因的1,868 bp的cDNA序列,其中203bp的3'-UTR,1,665 bp的ORF,编码554个氨基酸,将该序列提交GenBank,序列收录号KF636493。猪GLP2R基因与其它物种构建的系统进化树表明,猪与虎鲸、家绵羊亲缘关系相对最近,与西部锦龟及游隼、斑胸草雀的亲缘关系相对最远。对猪GLP2R蛋白为水溶性蛋白质,有23个潜在蛋白激酶磷酸化位点和7个跨膜结构,其二级结构以无规则卷曲和a-螺旋为主,三级结构构成弯曲状螺旋结构。利用实时荧光定量PCR技术检测与分析不同猪种、不同组织中GLP2R mRNA的表达水平,结果表明,同一组织GLP2R基因在25日龄的大白猪和沙子岭猪的有不同程度的表达,且在背最长肌中高丰度表达,肾脏中几乎不表达。对于同一品种,背最长肌都与其他组织差异显著(P0.05),胰脏、腿肌、背肌在不同品种中差异显著(P0.05)。通过PCR-RFLP分析在745 bp扩增目的片段上244 bp处发现一个A/G突变位点,氨基酸由H突变为R,从多态信息含量上,大白猪、大围子猪和黔邵花猪都在该位点具有遗传多态性,并且具有较高的遗传变异。AA、AG和GG三种基因型在达100kg校正日龄和校正背膘厚差异均不显著(P0.05)。克隆得到猪MY06基因cDNA全长为4,186bp,包括ORF大小为3,765bp,编码1,254个氨基酸,5'-UTR 102 bp和3'-UTR 319 bp编码1,255个氨基酸。同源性分析发现,猪的MY06推测氨基酸序列与藏羚羊、家犬与猪氨基酸相似性最高,为97.13%；而斑马宫丽鱼与猪的氨基酸相似性最低,为80.46%。猪MY06基因与其它物种构建的系统进化树表明,猪与家牛、藏羚羊亲缘关系相对最近,与斑马宫丽鱼、玛丽鱼的亲缘关系相对最远。对猪MY06基因的生物信息学分析表明,MY06基因编码蛋白为水溶性蛋白质,含有47个潜在磷酸化位点,无跨膜结构,其二级结构以a-螺旋为主。通过PCR-RFLP分析在866 bp扩增目的片段上709 bp处发现一个A/G突变位点,导致氨基酸由K突变为R,从多态信息含量上看,大白猪、大围子猪、沙子岭猪和黔邵花猪具有遗传多态性,并且具有较高的遗传变异。AA、AG和GG三种基因型在达100kg校正日龄和校正背膘厚差异均不显著(P0.05)。本论文为进一步研究猪GLP2R和MY06基因的功能提供理论依据,为揭示猪GLP2R和MY06基因的分子遗传机制奠定基础,并为地方品种的保护提供有益的资料。
[Abstract]:In this study, the cDNA of porcine GLP2R gene and MY06 gene were cloned and the biological information of their sequences and proteins were analyzed, and the single nucleotide polymorphisms of porcine GLP2R gene and MY06 gene were studied with five local pig breeds and one foreign pig strain as experimental materials. The genetic polymorphism of the gene was analyzed by PCR-RFLP, and the SNP loci of the two genes were correlated with 100 kg days of pig age and corrected backfat thickness. The results were as follows: the 1868 BP cDNA sequence of porcine GLP2R gene was cloned successfully by race technique. The sequence encoding 554 amino acids was encoded by 203bp. The sequence was identified as KF636493. Phylogenetic tree constructed by porcine GLP2R gene and other species showed that the phylogenetic relationship between pig and orcas and domestic sheep was relatively close, and the phylogenetic relationship with western tortoise, peregrine falcon and sparrow was the furthest. The porcine GLP2R protein is a water-soluble protein with 23 potential protein kinase phosphorylation sites and 7 transmembrane structures. The secondary structure of GLP2R protein is mainly irregular crimp and a-helix. The expression of GLP2R mRNA in different pig breeds and tissues was detected and analyzed by real-time fluorescence quantitative PCR. The results showed that GLP2R gene was expressed in different degree in 25 day old white pig and Shailing pig. High abundance was expressed in the longissimus dorsi muscle and almost no expression in the kidney. For the same variety, the longissimus dorsi muscle was significantly different from other tissues (P0.05A), and the pancreas, leg muscle and dorsalis muscle were significantly different in different varieties. By PCR-RFLP analysis, an A- / G mutation site was found at 244 BP on the target fragment of 745 BP amplification, with amino acid mutation from H to R.The polymorphic information content in the polymorphic information showed that large white pig, big Weizi pig and Guizhou Shaohua pig all had genetic polymorphisms in this locus. There was no significant difference in the age of 100kg correction and the backfat thickness between the three genotypes, I. e. AA-AG and GG, with high genetic variation (P0.05). The full-length cDNA of porcine MY06 gene was 4186 BP, including ORF of 3765 BP, encoding 1254 amino acids (102bp) and 3H-UTR319bp (1255 amino acids). Homology analysis showed that the deduced amino acid sequence of swine was the highest with Tibetan antelope, the amino acid similarity between domestic dog and pig was 97.13, while the amino acid similarity between zebra gongyfish and pig was the lowest (80.46). Phylogenetic tree constructed by porcine MY06 gene and other species showed that the phylogenetic relationship between pig and domestic cattle and Tibetan antelope was relatively close, and the phylogenetic relationship with zebra gongyfish and Mary fish was the furthest. Bioinformatics analysis of porcine MY06 gene showed that the protein encoded by MY06 gene was water-soluble protein with 47 potential phosphorylation sites and no transmembrane structure. Its secondary structure was mainly a-helix. By PCR-RFLP analysis, an A / G mutation site was found at 709 BP on the target fragment of 866 BP amplification, which led to the mutation of amino acid from K to R. From the polymorphic information content, there were genetic polymorphisms in large white pig, big Weizi pig, Shailing pig and Guizhou Shaohua pig. There was no significant difference in the age of 100kg correction and the backfat thickness between the three genotypes, I. e. AA-AG and GG, with high genetic variation (P0.05). This paper provides a theoretical basis for further study on the function of porcine GLP2R and MY06 genes, lays a foundation for revealing the molecular genetic mechanism of porcine GLP2R and MY06 genes, and provides useful data for the protection of local breeds.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S828

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