广西三黄鸡脂蛋白脂酶基因的克隆及蛋白构象分析

【目的】对广西三黄鸡和爱拔益加（AA）鸡的脂蛋白脂酶基因（LPL）进行克隆与蛋白质结构分析，为后期开展LPL基因表达与鸡肌内脂肪含量相关性研究及筛选出与优质肉质相关的分子遗传标记奠定基础。【方法】根据GenBank已公布的鸡“也基因序列设计引物，利用RT-PCR扩增广西三黄鸡和AA鸡的LPL基因cDNA序列，经双酶切鉴定和序列测定比对分析后，应用生物软件进行蛋白质二级结构预测分析。【结果】成功获得广西三黄鸡和AA鸡的LPL基因编码区序列（CDs），大小均为1473bp，两者的同源性为99．4％；将广西三黄鸡LPL基因序列提交至GenBank获得序列号JX090309。相对于AA鸡，广西三黄鸡LPL基因CDs存在9个位点的碱基突变，其中碱基^503T→C导致氨基酸^168Val→Ala，^606T→G导致^202Asp→Glu，^1066A→G导致^366Thr→Ala，^1277C→T导致^426Ser→Phe，^1420A→G导致^474Arp→Gly，^1432G→A导致^202Glu→Lys，这6个位点为错义突变；第166、372和1305位点的碱基突变为同义突变。蛋白质二级结构预测结果表明，广西三黄鸡与AA鸡LPL的C端结构域存在空间构象差异。【结论】LPL基因突变引起的氨基酸组成变化及蛋白质二级构象改变，可能影响鸡肌内脂肪沉积，进而决定肉质的优劣，即LPL基因可作为研究广西三黄鸡肌内脂肪代谢的主要候选基因。

Gene cloning and protein confirmation analysis of lipoprotein lipase in Guangxi Sanhuang chicken

Abstract： Objective ]To provide references for further research on LPL gene expression association with the intra- muscular fat （IMF） of Sanhuang chicken as well as selection of molecule genetic markers concerning quality meat, cloning and protein structure of lipoprotein lipase （LPL） in Guangxi Sanhuang chicken and Arbor Acre （AA） chicken were conducted. Method]A pair of primers was designed to amplify the LPL gene cDNA of these chicken using RT-PCR, accord- ing to the poultry LPL gene sequence published in GenBank. Following double digestion and sequence analysis, the protein secondary structure of LPL was predicted and analyzed by biological software. Result]Two LPL gene coding sequences （CDs） of Guangxi Sanhuang chicken and AA chicken were sequenced successfully, in which size was 1473 bp and homology was 99.4%. It was JX090309 that LPL gene in Guangxi Sanhuang chicken got from GenBank. Compared to AA chicken＇ s LPL gene CDs, CDs of Guangxi Sanhuang chicken had 9 loci base mutation, six of which were missense mutations. The base ^503T→C caused amino acid ^168Val→Ala,^606T→G led to ^202Asp→Glu,^1066A→G led to ^366Thr→Ala,^1277C→T led to ^426Ser→Phe,^1420A→G led to ^474Arp→Gly,^1432G→A led to^202Glu→Lys. The results of LPL secondary structure prediction showed that there were space conformational differences in the C-domain of LPL between Sanhuang chicken and AA chicken. Conclusion]The LPL gene mutations resulted in the changes of amino acids and protein secondary conformation, which might related to IMF of Sanhuang chicken. Consequently, LPL should be the dominant gene candidate for intramuscular fat research in Guangxi Sanhuang chicken.