藏猪与大约克夏猪GADD45A基因多态性与表达差异研究

Study on the polymorphism and expression difference of GADD45A gene between Tibetan pigs and Yorkshire pigs

为了探究GADD45A基因在猪肌肉生长中的作用,试验选择180日龄的藏猪和大约克夏猪各8头,采用实时荧光定量PCR法测定和分析两品种试验猪只背最长肌、腿肌和垂体组织中GADD45A基因的mRNA相对表达量;同时选择180日龄藏猪37头和大约克夏猪55头,PCR扩增两品种试验猪只GADD45A基因起始密码子上游3 000 bp左右的序列(5′侧翼区),通过混池测序分析序列的单核苷酸多态性(single nucleotide polymorphisms, SNPs)位点,同时分析SNPs对GADD45A基因转录的影响。结果表明:在背最长肌和腿肌组织中,藏猪GADD45A基因的mRNA表达量均极显著高于大约克夏猪(P<0.01);在垂体组织中,藏猪GADD45A基因的mRNA表达量显著高于大约克夏猪(P<0.05)。GADD45A基因5′侧翼区存在G1 952A、C1 689T、G1 614T 3个突变位点,并且这3个突变位点在藏猪和大约克夏猪中均差异极显著(P<0.01),且均符合哈迪-温伯格定律(P>0.05);经转录因子预测,在C1 689T位点中,当C碱基突变为T碱基后,出现了新的转录因子——叉头框转录因子D3(forkhead box D3, FOXD3);在G1 614T位点中,当G碱基突变为T碱基后,转录因子——同源框CUT样蛋白1(cut like homeobox 1,CUX1)消失。说明上述突变位点可能是导致180日龄藏猪和大约克夏猪GADD45A基因mRNA相对表达量差异极显著或显著的原因,并推测该位点是调控GADD45A基因表达的关键功能位点。

In order to explore the role of GADD45 A gene in pig muscle growth, eight 180-day-old Tibetan pigs and eight 180-day-old Yorkshire pigs were selected. The mRNA relative expression levels of GADD45 A gene in longissimus dorsi muscle, leg muscle and pituitary tissue of these two breeds of pigs were determined and analyzed by real-time fluorescence quantitative PCR. At the same time, 37 Tibetan pigs aged 180 days and 55 Yorkshire pigs aged 180 days were selected. The sequence of 3 000 bp upstream of the start codon(5′ flanks) of GADD45 A gene of these two breed pigs was amplified by PCR. The single nucleotide polymorphisms(SNPs) were analyzed by mixed pool sequencing, and the effect of SNPs on GADD45 A gene transcription was analyzed. The results showed that the mRNA expression of GADD45 A gene in the longissimus dorsi muscle and leg muscle of Tibetan pigs was significantly higher than that of Yorkshire pigs(P<0.01). Its expression level in pituitary tissue was significantly higher than that in Yorkshire pigs(P<0.05). Three mutation sites of G-1 952 A, C-1 689 T and G-1 614 T were found in flanking region 5 of GADD45 A gene, and the three mutation sites were significantly different between Tibetan pigs and Yorkshire pigs(P<0.01), and all conformed to Hardy Weinberg equilibrium(P>0.05). According to the prediction of transcription factors, a new transcription factor-forkhead box D3(FOXD3)appeared at the C-1 689 T site when C base was mutated to T base. At the G-1 614 T site, the transcription factor-cut like homeobox 1(CUX1) disappeared when G base was mutated to T base. These results indicated that the above mutation sites may be the reason for the extremely significant difference in the mRNA relative expression level of GADD45 A gene between Tibetan pigs and Yorkshire pigs at 180 days of age, and may be the key functional sites regulating the expression of GADD45 A gene.