摘要
FABPs属于脂结合蛋白超家族成员,是一类分子量较小而对脂肪酸有高亲和力的蛋白质,广泛存在于脊椎动物和非脊椎动物的细胞质中。FABPs担当细胞内脂肪酸的运输任务,它们与脂肪酸结合将其运输到脂肪酸氧化的位置、脂肪酸脂化成甘油三醋或磷脂的位置,或者进入细胞核内发挥其可能的调控功能。因此FABPs对脂类代谢具有重要的调控作用。本研究把L-FABP基因作为影响猪肌内脂肪含量的候选基因。为此,利用cDNA末端快速扩增(RACE)和PCR技术,克隆到猪肝脏型脂肪酸结合蛋白基因(L-FABP)的全长cDNA序列(GenBank登录号:AY960623)和部分基因组序列(GenBank登录号:DQ182323)。猪L-FABP基因的cDNA序列全长518bp,该序列包括起始密码子TGA和38bp的5′末端非编码区(5′URT),终止密码子TAG和99bp的3′末端非编码区(3′URT),在3′URT结构区域中包含polyA加尾信号序列AATAAA。猪L-FABP基因与其他FABPs基因一样,也由4个外显子(67bp、173bp、93bp和51bp)和3个内含子组成,内含子1和3的大小是1679bp和565bp,没有获得内含子2的序列,外显子和内含子剪接处符合GT/AG规律。应用ClustalW/X程序对猪L-FABP与其他物种的L-FABP进行多重序列比对,发现猪L-FABP与人、大鼠、鸡的L-FABP的相似性分别为89.8%、81.9%和72.4%。亲水性分析表明,猪L-FABP也是一个潜在的跨膜蛋白,在氨基酸残基57–65之间有一个明显的跨膜α螺旋。应用半定量RT-PCR分析发现,猪L-FABP在猪体组织中广泛存在,但在肝脏和小肠组织中表达量最为丰富。分析还发现,所克隆得到的编码区核苷酸序列与已知猪L-FABP基因的编码区核苷酸序列存在一定的变异,分别是外显子2中T→C(116位)、C→T(231位)、C→A(236位)和A→C(258位),演绎成氨基酸在Leu74Met存在差异。为进一步证实这些突变位点在猪群中真实存在,利用PCR-SSCP检测方法对4个猪种(藏猪、大河猪、雅南猪和约克夏)的157头个体的外显子2全序列进行SNP位点多态性片段的基因型分型,结果发现一个C→T的单核苷酸多态,等位基因频率在中国地方猪种(藏猪、大河猪、雅南猪)与国外约克夏猪种间存在极显著的差异(P<0.01)。连锁分析发现,基因型CC的肌内脂肪含量(4.86±0.22%)显著的高于基因型CT(4.16±0.23%)和TT(4.05±0.27%)的肌内脂肪含量(P<0.05)。因此,推测L-FABP基因可能是影响猪肌内脂肪含量的主效基因或与主效基因紧密连锁的标记基因,并且能够在分子标记辅助选择中用于对猪肌内脂肪含量的遗传改良。
In this study, the full-length cDNA of porcine liver-type fatty acid binding protein gene (L-FABP) was obtained by the rapid amplification of cDNA ends (RACE). The nucleotide sequence and the predicted protein sequence share a high sequence identity with their mammalian counterparts. Semi-quantitative RT-PCR revealed that porcine L-FABP gene is expressed in all twelve tissues studied, but a transcript is more abundant in liver and small intestine than in other tissues. The part genomic DNA of the porcine L-FABP gene was amplified by PCR. The coding region of the pig L-FABP gene is organized in four exons and spans an approximate 2.62 kb genomic region. Comparative sequencing of four pig breeds revealed a C→T single nucleotide polymorphism (SNP) within exon 2. The allele and genotype frequencies differed significantly between indigenous Chinese Zang, Dahe, and Yanan pigs with higher frequencies of allele C and genotype CC and Yorkshire pigs with higher frequencies of allele T and genotype TT (P 〈 0.01). The association analysis suggested that the C→T polymorphism was associated with intramuscular fat content, indicating that the SNP is a potential molecular marker for intramuscular fat content.
基金
This work was supported by the Ministry of Science and Technology of China (No. 2002BA514A-1-2).