从江香猪PHKG1和PHKG2基因克隆及其mRNA在组织中表达水平
发布时间：2018-07-22 21:02
【摘要】：磷酸化酶激酶γ1(phosphorylase kinase gamma1,PHKG1)和磷酸化酶激酶γ2(phosphorylase kinase gamma 2,PHKG2)基因是糖原代谢途径中的重要基因,具有分解糖原为机体肌肉收缩提供能量以及维持血糖平衡的功能。本研究为了对PHKG1和PHKG2基因的遗传机理进行探究,实验以贵州从江香猪(Sus scrofa)和大白猪为研究对象,利用T-A克隆的方法获取贵州从江香猪和大白猪PHKG1(Gen Bank:NM_001293144.1)、PHKG2(Gen Bank:NM_001166317)基因编码区序列,并比较2者CDS区序列差异,利用生物信息学软件预测2个基因的蛋白理化特性和功能等;同时采用实时荧光定量PCR技术分析PHKG1和PHKG2基因在贵州从江香猪和大白猪不同组织中表达差异。结果表明,从江香猪PHKG1基因CDS区全序列长1 167 bp,共编码388个氨基酸,PHKG2基因CDS区全序列长1 221 bp,共编码406个氨基酸,二者均构成具有S_TKc结构域的跨膜亲水性非分泌蛋白。对PHKG1和PHKG2蛋白结构预测以及进化树聚类分析表明这2个蛋白具有一定的同源性。并发现PHKG1基因存在4个突变位点,分别为A371G、A525G、T945C和C1050T,但是与大白猪相比存在T945C一处突变,均未引起氨基酸的改变,为同义突变;将从江香猪、大白猪、Gen Bank猪序列(NM_001166317)、巴马香猪(KJ 186785)PHKG2基因序列分析比较发现,G236A、C431T、A726G、A807G、A816G和A867G为大白猪、从江香猪、巴马香猪共有的突变,从江香猪存在A614G特殊的突变位点,并且引起了第205位谷氨酸变成了丙氨酸。q RT-PCR结果显示,PHKG1基因在2个猪品种中均能检测到表达,在背最长肌中表达量最高,在肺脏中大白猪的表达量显著高于从江香猪(P0.05);PHKG2基因在从江香猪和大白猪的脾、肺、大肠、小肠中的相对表达量都较高,而在心和背最长肌中的表达量较低。本研究为PHKG1和PHKG2基因后续真核表达的研究提供了理论依据。
[Abstract]:Phosphorylase kinase 纬 1 (phosphorylase kinase gamma1 (PHKG1) and phosphorylase kinase 纬 2 (phosphorylase kinase gamma 2 PHKG2) are important genes in glycogen metabolism pathway, which can decompose glycogen to provide energy for muscle contraction and maintain blood glucose balance. In order to study the genetic mechanism of PHKG1 and PHKG2 genes, the coding region of PHKG1 and PHKG2 (Gen BankVo NM00001166317) gene was obtained from Guizhou Congjiang Xiang pig (Sus scrofa) and large white pig (Sus scrofa) by T-A cloning. The sequence difference of CDS region was compared, and the protein physicochemical properties and function of the two genes were predicted by bioinformatics software. The expression of PHKG1 and PHKG2 genes in different tissues of Guizhou Congjiang Xiang pig and big white pig were analyzed by real-time fluorescent quantitative PCR. The results showed that the total length of CDS region of PHKG1 gene in Congjiang Xiang pig was 1 167 BP, the total length of CDS region of PHKG 2 gene encoding 388 amino acids was 1 221 BP, and the total number of amino acids was 406 amino acids. Both of them constituted transmembrane hydrophilic nonsecretory protein with STKc domain. The prediction of PHKG1 and PHKG2 protein structure and phylogenetic tree cluster analysis showed that the two proteins had some homology. It was also found that there were four mutation sites in the PHKG1 gene, namely A371GN A525GN T945C and C1050T, but there was a T945C mutation in comparison with the large white pig, none of which caused the amino acid change and was synonymous mutation. The gene sequence of GenBank (NM001166317) and PHKG2 of Bama Xiang pig (KJ 186785) were analyzed and compared. It was found that G236AfC431TUA726GFA816G and A867G were the big white pig, Congjiang Xiang pig and Bama Xiang pig had common mutation, Congjiang Xiang pig had a special mutation site of A614G, and Congjiang Xiang pig had a special mutation site of A614G, and A867G were the common mutants of big white pig, Congjiang Xiang pig and Bama Xiang pig. The results of RT-PCR showed that the PHKG1 gene could be detected in two pig breeds, and the highest expression level was found in the longissimus dorsi muscle. The relative expression of PHKG2 gene in spleen, lung, large intestine and small intestine of Congjiang Xiang pig and large white pig was higher than that in Congjiang Xiang pig (P0.05), but lower in heart and longissimus dorsi muscle. This study provides a theoretical basis for the subsequent eukaryotic expression of PHKG1 and PHKG2 genes.
【作者单位】： 贵州大学高原山地动物遗传育种与繁殖省部共建教育部重点实验室;贵州大学贵州省动物遗传育种与繁殖重点实验室;贵州大学动物科学学院;贵州大学生命科学学院;
【基金】：国家科技支撑计划(No.2015BAD03B02-3) 黔科合重大专项(黔科合NY字[2013]6008号)
【分类号】：Q78;S828
，
本文编号：2138564
[Abstract]:Phosphorylase kinase 纬 1 (phosphorylase kinase gamma1 (PHKG1) and phosphorylase kinase 纬 2 (phosphorylase kinase gamma 2 PHKG2) are important genes in glycogen metabolism pathway, which can decompose glycogen to provide energy for muscle contraction and maintain blood glucose balance. In order to study the genetic mechanism of PHKG1 and PHKG2 genes, the coding region of PHKG1 and PHKG2 (Gen BankVo NM00001166317) gene was obtained from Guizhou Congjiang Xiang pig (Sus scrofa) and large white pig (Sus scrofa) by T-A cloning. The sequence difference of CDS region was compared, and the protein physicochemical properties and function of the two genes were predicted by bioinformatics software. The expression of PHKG1 and PHKG2 genes in different tissues of Guizhou Congjiang Xiang pig and big white pig were analyzed by real-time fluorescent quantitative PCR. The results showed that the total length of CDS region of PHKG1 gene in Congjiang Xiang pig was 1 167 BP, the total length of CDS region of PHKG 2 gene encoding 388 amino acids was 1 221 BP, and the total number of amino acids was 406 amino acids. Both of them constituted transmembrane hydrophilic nonsecretory protein with STKc domain. The prediction of PHKG1 and PHKG2 protein structure and phylogenetic tree cluster analysis showed that the two proteins had some homology. It was also found that there were four mutation sites in the PHKG1 gene, namely A371GN A525GN T945C and C1050T, but there was a T945C mutation in comparison with the large white pig, none of which caused the amino acid change and was synonymous mutation. The gene sequence of GenBank (NM001166317) and PHKG2 of Bama Xiang pig (KJ 186785) were analyzed and compared. It was found that G236AfC431TUA726GFA816G and A867G were the big white pig, Congjiang Xiang pig and Bama Xiang pig had common mutation, Congjiang Xiang pig had a special mutation site of A614G, and Congjiang Xiang pig had a special mutation site of A614G, and A867G were the common mutants of big white pig, Congjiang Xiang pig and Bama Xiang pig. The results of RT-PCR showed that the PHKG1 gene could be detected in two pig breeds, and the highest expression level was found in the longissimus dorsi muscle. The relative expression of PHKG2 gene in spleen, lung, large intestine and small intestine of Congjiang Xiang pig and large white pig was higher than that in Congjiang Xiang pig (P0.05), but lower in heart and longissimus dorsi muscle. This study provides a theoretical basis for the subsequent eukaryotic expression of PHKG1 and PHKG2 genes.
【作者单位】： 贵州大学高原山地动物遗传育种与繁殖省部共建教育部重点实验室;贵州大学贵州省动物遗传育种与繁殖重点实验室;贵州大学动物科学学院;贵州大学生命科学学院;
【基金】：国家科技支撑计划(No.2015BAD03B02-3) 黔科合重大专项(黔科合NY字[2013]6008号)
【分类号】：Q78;S828
，
本文编号：2138564
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