Isolation, Characterization, Genetic Effect Of H2A.Z And DAZL Gene Affecting Porcine Reproduction Traits

Litter size is one of the important factors which impact the benefit of porcine industry.In this study,the candidate gene approach has been emploied to discover new molecular markers associated with reproductive traits.According to physiological or biochemical functions of genes,QTL mapping results and pig-human comparative map, H2A.Z gene and DAZL gene were selected.The objective of this study was to isolate and character these candidate genes,to analyze the expression of these genes in different tissues,to identify mutations in these genes sequence and establish suitable method to detect polymorphisims,to determine associations between the polymorphisms and the reproductive performance in femal LargeWhite,Chinese lean type new line DIV and LargeWhite×Meishan F₂ resource populations.The main results are as following:1.The analysis results of genetic effect of H2A.Z gene(1) A 932bp cDNA sequence of porcine H2A.Z gene spanning complete coding sequences(CDS) was isolated by RT-PCR.A 1231bp genomic sequence encompassing complete intron 2,3 and 4 was amplified from total porcine genomic DNA.(2) A PCR-Bsu15I-RFLP assay was established to detect the 4bp deletion in intron 2.(3) Association analysis for the 4bp deletion with reproductive traits was preformed in nine independent populations.Statistical analysis demonstrated that Bsu15I polymorphism was associated with NBA in the first of DIV sows and Large White sows,an additive effect was detected(p＜0.05 or p＜0.01).2.The analysis results of genetic effect of DAZL gene(1) A 2805bp cDNA sequence of pig DAZL gene was isolated including 887bp complete coding sequences and 1917bp 3'UTR sequences.An genomic sequence of porcne DAZL which encompass the introns of 2,3,4,5,6,7,8,9 were amplified from porcine genomic DNA.(2) For DAZL gene:a PCR-MspI-RFLP assay was established to detect the A/G mutation in intron 7;and a PCR-TaqI-RFLP assay was estabilished to detect the A/C mutation in intron 9.(3) Association study for these SNPs with reproductive traits was preformed in two independent populations.Statistical analysis demonstrated that:TaqI polymorphism is associated with NBA in the first litters of both Large White sows(p＜0.05) and Line DIV(p=0.06),an additive effect was detected (p＜0.05)in Large White group.(4)An recombinated vector named DAZL-PGEX-kg was constructed and expressed in Escherichia coli expression system.Then the expressed protein was detected in PAGE gel,the result of which prove that the molecular weight of the expressed protein was in line with our prediction..The result shows that the Bsu15I-PCR-RFLP and TaqI-PCR-RFLP could be considered as the molecular markers of porcine litter size.The isolation and identification of these genes would give us a more profound.understanding of the functions of these genes,uncover the mechanism of pig litter size and give theoretics support for marker assistant selection(MAS).