陆川猪DGAT2基因克隆、序列分析及表达水平研究

Cloning,sequence analysis and expression of DGAT2 gene in Luchuan pigs

为了获得广西陆川猪二脂酰甘油酰基转移酶2(DGAT2)基因编码区(CDS)序列,并探讨该基因在陆川猪和杜长大猪肌肉中的表达水平差异,试验以陆川猪背最长肌组织总RNA为模板,克隆获得DGAT2基因CDS序列并利用生物软件进行序列分析,同时采用实时荧光定量PCR技术检测150日龄陆川猪和杜长大猪背最长肌中DGAT2基因的相对表达量。结果表明:陆川猪DGAT2基因CDS序列长度为1086 bp,与GenBank中的野猪、虎鲸、双峰骆驼、羊驼、佛罗里达海牛、野驴、马和家猫的同源性分别为99.8%、93.1%、93.0%、92.9%、92.1%、91.8%、91.7%和91.5%;陆川猪与野猪的遗传距离最近;陆川猪DGAT2蛋白由361个氨基酸组成,其中亮氨酸含量最高,半胱氨酸含量最少,DGAT2蛋白具有较弱的疏水性;在DGAT2蛋白高级结构中,α-螺旋占比39.34%,无规则卷曲占比43.21%,延伸链占比17.45%;150日龄陆川猪背最长肌中DGAT2基因相对表达量极显著高于同日龄的杜长大猪(P<0.01)。说明DGAT2基因在猪肌肉脂肪沉积过程中发挥了一定作用,但其作用于肌内脂肪的沉积机制尚不明确,可将DGAT2基因作为开展陆川猪肌内脂肪沉积分子机制研究的主要候选基因之一。

The objective of this study was to obtain the coding region sequence(CDS)of DGAT2 gene in Guangxi Luchuan pigs and to investigate the difference in the expression level of DGAT2 gene in the muscles of Luchuan pigs and Duroc-Landrace-Yorkshire(DLY)pigs.Total RNA from longissimus dorsi muscles of Luchuan pigs was used as the template.The CDS of DGAT2 gene was cloned and its sequence was analyzed by using biological software.Meanwhile,the relative expression of DGAT2 gene in longissimus dorsi muscles of Luchuan pigs and DLY pigs aged 150 days were detected by real-time fluorescence quantitative PCR.The results showed that the CDS region length of Luchuan pig DGAT2 gene was 1086 bp,and the homologies with Sus scrofa,Orcinus orca,Camelus ferus,Vicugna pacos,Trichechus manatus latirostris,Equus asinus,Equus caballus,and Felis catus that reported by GenBank were 99.8%,93.1%,93.0%,92.9%,92.1%,91.8%,91.7%,and 91.5%,respectively.Phylogenetic tree results showed that the genetic distance between Luchuan pigs and Sus scrofa was the closest.DGAT2 protein in Luchuan pigs was composed of 361 amino acids,of which leucine(Leu)content was the highest and Cysteine(Cys)content was the lowest,and DGAT2 protein possessed a low hydrophobicity.In the secondary structure of DGAT2 protein,the proportion of alpha-helix,random curl,and extended chains were 39.34%.43.21%,and 17.45%,respectively.The results of real-time fluorescence quantitative PCR showed that the relative expression of DGAT2 gene in the longissimus dorsi muscles of 150 days old Luchuan pigs was significantly higher than that in the same age of DLY pigs.Our results indicated that DGAT2 gene played a certain role in the process of muscle fat deposition in pigs,but the mechanism of intramuscular fat deposition was not yet clear.DGAT2 gene could be used as one of the main candidate genes for the molecular mechanism of intramuscular fat deposition in Luchuan pigs.