皖南黑猪H-FABP基因5'-上游区和第二内含子的多态性分析及克隆测序

Sequence and Polymorphism Analysis of Wannan Black Pig H-FABP Gene 5'-Upstream Region and the Second Intron

对皖南黑猪心脏脂肪酸结合蛋白(H-FABP)基因5’-上游区和第二内含子的多态性进行分析及克隆测序,为开展皖南黑猪肉质性状分子育种奠定理论基础。采用PCR-RFLP(HinfⅠ、HaeⅢ、MspⅠ3种限制性内切酶)分子标记技术,分析了184头皖南黑猪H-FABP基因5’-上游区和第二内含子区的遗传变异情况,并对该基因的多态片段进行克隆和测序分析。结果显示:(1)在5’-上游区,HinfⅠ位点上存在多态性,等位基因H的基因频率为0.62,表现为中度多态(PIC=0.359 6);在第二内含子区,HaeⅢ位点上存在多态性,等位基因D的基因频率为0.92,表现为低度多态(PIC=0.130 7);而MspⅠ位点上尚未检测到多态,基因型均为AA型;HinfⅠ*位点上也存在多态性,等位基因B的基因频率为0.78,表现为中度多态(PIC=0.282 4);(2)χ2检验表明,除5’-上游区HinfⅠ多态位点外,其他3个位点均达到Hardy-Weinberg平衡状态(P>0.05);(3)对H-FABP基因3个多态片段进行克隆测序分析发现,HinfⅠ-RFLP是由于1 324 bp处存在T→C的突变引起,HaeⅢ-RFLP是由于1811bp处存在C→G的突变引起,HinfⅠ*-RFLP是由于1 970 bp处存在C→T的突变引起。本试验确证了皖南黑猪H-FABP基因5’-上游区的多态位点并在第二内含子区检测到了HaeⅢ和Hinf*Ⅰ多态位点,这可为进一步分析H-FABP基因不同基因型与IMF含量的关系,以及确定影响IMF沉积的主效基因提供一定的数据基础。

This paper analyzed,cloned and sequenced the polymorphism of H-FABP gene 5 ＇-upstream region and second intron to provide theoretical basis for the breeding of pork quality traits in Wannan Black Pig. The molecular marker techniques of PCR-RFLP（HinfⅠ,HaeⅢ,MspⅠ3 kinds of restriction enzymes）was used,and the genetic variation of H-FABP gene 5＇-upstream region and second intron in 184 Wannan Black Pigs was analyzed,moreover,the corresponding polymorphic fragments was cloned and sequenced. The results showed that：（1） there was polymorphism in loci of the Hinf in 5＇-upstream region,and the frequency of allele H was 0.62,showing moderate polymorphism（PIC = 0.359 6）; there was polymorphism in polymorphic loci Hae Ⅲ in the second intron allele the frequency of D was 0.92,showing low polymorphism（PIC = 0.130 7）. While there was no polymorphism detected in the loci of MspⅠ,which genotype was AA genotype. polymorphism of Loci of HinfⅠ＊ was also existed,the frequency of allele B was 0.78,showing moderate polymorphism（PIC = 0.282 4）;（2） Chi-square test showed that 3 polymorphic sites had reached the equilibrium of Hardy-Weinberg（P 0.05）except the polymorphic site of Hinf Ⅰ in the H-FABP gene 5＇-upstream region.（3） by cloning and sequence analysising three polymorphic fragments in H-FABP gene,we found that Hinf Ⅰ-RFLP was due to T →C mutation in 1 324 bp,HaeⅢ-RFLP was due to C→G mutation in 1 811 bp and HinfⅠ＊- RFLP was due to C→T mutation in 1 970 bp. The polymorphic loci of H-FABP gene in 5＇-upstream region of Wannan Black Pig was validated,and Hae Ⅲ and Hinf＊ Ⅰ polymorphic loci were detected in the second intron. The results provided certain data basis to determine the main effect gene that infect intramuscular fat（IMF） deposition for further investigating the relationship between different genotypes in H-FABP gene and variation in IMF content,whether H-FABP locus was the major gene for IMF deposition.