陆川猪脂蛋白脂酶基因的克隆与序列分析

Gene cloning and sequence analysis of lipoprotein lipase in Luchuan pig

【目的】对陆川猪脂蛋白脂酶(LPL)基因进行克隆与序列分析,为后期开展LPL基因表达与陆川猪肌内脂肪沉积的相关性研究奠定基础。【方法】根据GenBank中已公布的猪LPL基因序列设计引物,以陆川猪背最长肌组织总RNA为模板,利用RT-PCR扩增其LPL基因,测序结果用Lasergene 7.0软件进行序列分析,用MEGA 6.0软件进行同源性比较及构建系统进化树,同时利用在线蛋白质结构分析软件(PMP)预测陆川猪LPL蛋白二级结构。【结果】成功克隆获得陆川猪LPL基因编码区长1437 bp,与GenBank中已公布普通猪、大白猪、杜洛克猪、通城猪、贵州白香猪的基因同源性分别为99.6%、99.7%、99.7%、99.7%和99.4%。陆川猪LPL基因第760位点存在特有的碱基A,该突变导致缬氨酸突变为蛋氨酸。由基于LPL基因碱基同源性构建的物种系统进化树可知,广西陆川猪与大白猪的遗传距离最近,其次是山羊,最远的是小鼠。蛋白二级结构预测结果表明,陆川猪的LPL蛋白二级结构包含有N-端结构域和C-端结构域两个区域,且存在PLAT/LH2。【结论】LPL基因可作为研究陆川猪脂肪沉积的主要候选基因之一。

[Objective]Cloning and sequence analysis of lipoprotein lipase in Luchuan pig were conducted to provide references for further research on LPL gene expression association with intramuscular fat of Luchuan pig. [ Method ]According to the pig LPL gene sequence published in GenBank, a pair of primers was designed to amplify the LPL gene cDNA of Luehuan pig by RT-PCR. The sequence was analyzed with the use of Lasergene 7.0 software. Homology and phylogenetic tree were constructed using MEGA 6.0 software. The protein secondary structure of LPL was predicted and analyzed by PMP biological software. [ResultlThe length of the coding region for the cloned LPL gene in Luchuan pig was 1437 bp, and the sequence homologies of LPL gene compared with the sequence of ordinary, large white, Duroc, Tongcheng and Guizhou baixiang pig were 99.6%, 99.7%, 99.7%, 99.7% and 99.4%, respectively. It was found that Luehuan pig LPL gene existed in specific base point A at 760, which caused valine to methionine. The evolution of the species in the LPL gene phylogenetic tree showed that the genetic distance between Guangxi Luchuan pig and large white pig was the closest followed by the goat, and the furthest was mice. The prediction of LPL secondary structure was that Luchuan pig LPL contained an N-terminal region, a C-terminal domain, and PLAT/LH2. [Conelusion]LPL gene can be used as one of the main candidate genes related to intramuscular fat in Luchuan pig.