【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)基因是糖原代謝途徑中的重要基因,具有分解糖原為機(jī)體肌肉收縮提供能量以及維持血糖平衡的功能。本研究為了對(duì)PHKG1和PHKG2基因的遺傳機(jī)理進(jìn)行探究,實(shí)驗(yàn)以貴州從江香豬(Sus scrofa)和大白豬為研究對(duì)象,利用T-A克隆的方法獲取貴州從江香豬和大白豬PHKG1(Gen Bank:NM_001293144.1)、PHKG2(Gen Bank:NM_001166317)基因編碼區(qū)序列,并比較2者CDS區(qū)序列差異,利用生物信息學(xué)軟件預(yù)測(cè)2個(gè)基因的蛋白理化特性和功能等;同時(shí)采用實(shí)時(shí)熒光定量PCR技術(shù)分析PHKG1和PHKG2基因在貴州從江香豬和大白豬不同組織中表達(dá)差異。結(jié)果表明,從江香豬PHKG1基因CDS區(qū)全序列長(zhǎng)1 167 bp,共編碼388個(gè)氨基酸,PHKG2基因CDS區(qū)全序列長(zhǎng)1 221 bp,共編碼406個(gè)氨基酸,二者均構(gòu)成具有S_TKc結(jié)構(gòu)域的跨膜親水性非分泌蛋白。對(duì)PHKG1和PHKG2蛋白結(jié)構(gòu)預(yù)測(cè)以及進(jìn)化樹(shù)聚類分析表明這2個(gè)蛋白具有一定的同源性。并發(fā)現(xiàn)PHKG1基因存在4個(gè)突變位點(diǎn),分別為A371G、A525G、T945C和C1050T,但是與大白豬相比存在T945C一處突變,均未引起氨基酸的改變,為同義突變;將從江香豬、大白豬、Gen Bank豬序列(NM_001166317)、巴馬香豬(KJ 186785)PHKG2基因序列分析比較發(fā)現(xiàn),G236A、C431T、A726G、A807G、A816G和A867G為大白豬、從江香豬、巴馬香豬共有的突變,從江香豬存在A614G特殊的突變位點(diǎn),并且引起了第205位谷氨酸變成了丙氨酸。q RT-PCR結(jié)果顯示,PHKG1基因在2個(gè)豬品種中均能檢測(cè)到表達(dá),在背最長(zhǎng)肌中表達(dá)量最高,在肺臟中大白豬的表達(dá)量顯著高于從江香豬(P0.05);PHKG2基因在從江香豬和大白豬的脾、肺、大腸、小腸中的相對(duì)表達(dá)量都較高,而在心和背最長(zhǎng)肌中的表達(dá)量較低。本研究為PHKG1和PHKG2基因后續(xù)真核表達(dá)的研究提供了理論依據(jù)。
[Abstract]:Phosphorylase kinase 緯 1 (phosphorylase kinase gamma1 (PHKG1) and phosphorylase kinase 緯 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.
【作者單位】： 貴州大學(xué)高原山地動(dòng)物遺傳育種與繁殖省部共建教育部重點(diǎn)實(shí)驗(yàn)室;貴州大學(xué)貴州省動(dòng)物遺傳育種與繁殖重點(diǎn)實(shí)驗(yàn)室;貴州大學(xué)動(dòng)物科學(xué)學(xué)院;貴州大學(xué)生命科學(xué)學(xué)院;
【基金】：國(guó)家科技支撐計(jì)劃(No.2015BAD03B02-3) 黔科合重大專項(xiàng)(黔科合NY字[2013]6008號(hào))
【分類號(hào)】：Q78;S828
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本文編號(hào)：2138564