DNA池快速筛查牛STAM1基因SNPs及估算等位基因频率

Rapidly Screening SNPs of Cattle STAM1 Gene and Estimating Allelic Frequencies by DNA Pooling

目的:旨在对不同牛种STAM1基因进行SNPs筛查,为地方牛种选种选育提供一定理论依据。方法:选取生长发育性状明显差异的务川黑牛和贵州荷斯坦奶牛2个牛种构建DNA池,设计1对引物分别扩增2个牛种STAM1基因第14外显子序列总长898bp。切胶回收后对PCR产物进行双向测序。结果:在牛STAM1基因中快速筛查到5个SNPs:A33C、C66G、C356T、T523A、T652C,其中A33C(Tyr→Ser)、C66G(Pro→Arg)、C356T(Glu→Lys)为错义突变,T523A为同义突变,T652C位于内含子区。贵州荷斯坦奶牛在T523A和T652C两个位点基因频率为1.000 0,而务川黑牛分别为0.673 0和0.810 6。生物信息学分析表明:突变前后STAM1的RNA二级结构和蛋白质二级、三级结构均有明显改变。结论:DNA池结合测序技术可快速筛选SNP位点,检测到STAM1基因第14外显子5个SNPs。

Objective ：This study was launched to screen the single nucleotide polymorphisms （SNPs）in STAM1 gene which could lay the experiment foundation for heredity selection of cattle. Method：Two cattle breeds （Wuchuan Black Cattle and Guizhou Holstein cow）with different growth and development traits were selected to construct DNA pools, the complete sequences of STAM1 exons 14 and part of 3＇ untranslated region（3＇ - UTR）were obtained by designing one pair specific primers. PCR production of 898bp conducted gel extraction to purify the production and sequenced subsequently. DNAStar and BLAST software were used to identify polymorphisms of cattle STAM1 genes. Resllit：Five SNPs were found in STAM1 gene which included three missense mutation（ A33C, Tyr→Ser, C66G, Pro→Arg, C356T, Glu→Lys） ,one synonymous mutation（T523A） and one SNP located in intron region respectively. The aUeIic frequencies of T523A and T652C was 1. 000 0 in Guizhou Holstein cow while was O. 673 0 and 0. 810 6 respectively in Wuchuan Black Cattle. Through bioinformatics analysis we found that the pelymorphisms leads to secondary structure change of RNA,secondary structure change and tertiary structure change of STAM1 protein. Condusion： DNA pooling could be used to screen SNPs of genes and Five SNPs were found in exon 14 of STAM1 gene by using this method.