家兔PPARγ基因的克隆表达及肌内脂肪含量的相关性分析

Cloning and expression of rabbit PPARγ gene and analysis on its correlation with intramuscular fat content

为了克隆家兔过氧化物酶体增殖物激活受体(peroxisome proliferative activated receptor gamma,PPARγ)基因蛋白质编码区(CDS)序列,并研究其生物学功能、组织表达规律及与肌内脂肪(IMF)含量的关系,试验克隆了家兔PPARγ基因CDS序列,并对其进行生物信息学分析;采集70日龄新西兰白兔心脏、肝脏、脾脏、肺脏、肾脏、子宫、胃、腿肌、腹肌、背最长肌,0,35日龄新西兰白兔背最长肌,70日龄豫丰黄兔腿肌、腹肌、背最长肌,通过qRT-PCR扩增检测了PPARγ基因在不同组织/器官中的相对表达量;测定了70日龄新西兰白兔和豫丰黄兔腿肌及背最长肌的IMF含量,并对PPARγ基因相对表达量与IMF含量进行相关性分析。结果表明:家兔PPARγ基因CDS序列长1428 bp,共编码475个氨基酸,其核苷酸序列与GenBank上公布的人、鼠、猪、绵羊、牛、马、犬及鸡等物种的氨基酸序列相似性分别为91.0%、90.5%、90.5%、90.2%、90.1%、90.0%、89.9%和80.5%,与小鼠的亲缘关系最近,与鸡的亲缘关系最远。PPARγ基因在70日龄新西兰白兔心脏、肝脏、脾脏、肺脏、肾脏、腿肌、子宫和胃中均有表达,在腿肌和胃中的相对表达量极显著高于其他器官(P<0.01),在腿肌中的相对表达量显著高于胃(P<0.05);在0,35,70日龄新西兰白兔背最长肌组织中的相对表达量差异不显著(P>0.05)。PPARγ基因在70日龄新西兰白兔和豫丰黄兔腹肌中的相对表达量极显著高于腿肌和背最长肌(P<0.01),豫丰黄兔腹肌、腿肌中的相对表达量显著高于新西兰白兔(P<0.05),在背最长肌中差异不显著(P>0.05)。PPARγ基因在腿肌和背最长肌中的相对表达量与IMF含量均呈正相关。说明PPARγ基因可能调控家兔IMF沉积过程,豫丰黄兔的肉品质可能优于新西兰白兔。

In order to clone the protein coding region(CDS) sequence of rabbit peroxisome proliferative activated receptor gamma(PPARγ) gene, and to study its biological function, tissue expression and its correlation with intramuscular fat(IMF). The CDS sequence of rabbit PPARγ gene was cloned and analyzed by bioinformatics. The test collected heart, liver, spleen, lung, kidney, muscle, uterus, stomach, leg muscle, abdominal muscle, longissimus dorsi muscle of 70-day-old New Zealand White rabbits;longissimus dorsi muscle of 0 and 35-day-old New Zealand White rabbits;leg muscles, abdominal muscles, longissimus dorsi muscle of 70-day-old Yufeng Yellow rabbits. The relative expression levels of PPARγ gene in different tissues and organs were detected by qRT-PCR amplification;the IMF contents in the leg muscles and the longissimus dorsi muscle of 70-day-old New Zealand White rabbits and Yufeng Yellow rabbits were determined, and the correlation between the relative expression of PPARγ gene and the IMF content was analyzed. The results showed that the CDS sequence of the rabbit PPARγ gene was 1 428 bp long and encoded a total of 475 amino acids;its nucleotide sequence was 91.0%, 90.5%, 90.5%, 90.2%, 90.1%, 90.0%, 89.9% and 80.5% similar to the amino acid sequence of human, mouse, pig, sheep, cattle, horse, dog and chicken published in GenBank, respectively;it was closest relative of the mouse, the most distant relative of the chicken. PPARγ gene was expressed in the heart, liver, spleen, lung, kidney, leg muscle, uterus and stomach of 70-day-old New Zealand White rabbits, and the relative expression in leg muscle and stomach were significantly higher than that in other organs(P<0.05). The relative expression in leg muscle was significantly higher than that in stomach(P<0.05). The relative expression levels in the longissimus dorsi of New Zealand White rabbits at 0, 35, and 70 days of age were not significantly different(P>0.05). The relative expression of PPARγ gene in abdominal muscles of 70-day-old New Zealand White rabbits and Yufeng Yellow rabbits was extremely significantly higher than in longissimus dorsi and leg muscles(P<0.01);the relative expression of Yufeng Yellow rabbits in abdominal muscle and leg muscle were significantly higher than that of New Zealand White rabbits(P<0.05) but not significantly different in longissimus dorsi muscle(P>0.05). The relative expression of PPARγ gene in leg muscle and longissimus dorsi was positively correlated with IMF content. The results suggested that PPARγ gene may regulate the deposition process of IMF in rabbits, and the meat quality of Yufeng Yellow rabbits may be better than that of New Zealand White rabbits.