山羊SKIP基因的分离、鉴定及与生长性状的关联分析

Isolation and identification of goat SKIP gene and its association with growth traits

为研究SKIP基因对山羊生长发育的影响,并寻找与山羊生长性状相关的分子标记,利用绵羊与牛SKIP基因的同源序列设计引物,RT-PCR扩增得到一段长1 350bp的山羊SKIP基因表达序列,包括其完整编码区序列,编码449个氨基酸的蛋白。通过序列Blast比较和系统发育树的构建,发现山羊SKIP与绵羊SKIP序列相似性最高(99.52%)。利用生物信息学分析SKIP蛋白的理化性质,预测SKIP蛋白分子质量为52ku,等电点为5.18,包含典型的疏水区域和磷脂酰肌醇5-磷酸磷酸酶结构域,定位在细胞质(60.9%)和细胞核(26.1%)中。间接免疫荧光试验验证了SKIP的细胞定位,并发现位于细胞核中的SKIP在成肌细胞分化过程中转移到细胞质。通过波尔山羊和麻城黑山羊的DNA混池测序,发现SKIP内含子2有一处G/A突变,利用PCRRFLP方法对波尔×麻城黑山羊F2代杂交后代进行基因分型,性状关联分析发现该单核苷酸多态性位点(SNP)与出生体质量、胸围、管围、身高等性状显著相关,等位基因G与生长呈正相关。

In order to explore the effect of the goat SKIPgene on growth and development,and find the molecular markers associated with growth trait in goats,primers were designed according to the homologous sequence of the bovine SKIP gene.A 1 350 bp expressed sequence of the goat SKIP gene was amplified by RT-PCR,including the complete coding sequence,encoding 449 amino acids.Through sequence comparison and phylogenetic analysis,it was found that the SKIP sequences between goat and sheep had the highest similarity（99.52%）.Physical and chemical characteristics of the SKIP protein were predicted by bioinformatic methods.The predicted SKIP protein has a calculated molecular weight of 52.0 ku and a pI of 5.18,and contains a typical phosphatidylinositol 5-phosphate phosphatase（COG5411）domain which is conserved among species.Most of the SKIP protein was located in the cytoplasm（60.9%）and nucleus（26.1%）,which was verified by indirect immunofluorescence assay.However,there was a dynamic transfer of SKIP from the nucleus to cytoplasm during myoblast differentiation.A single-nucleotide polymorphism（SNP）G/A was detected in the second intron of the SKIPin the Boer and Macheng Black goat breeds.PCR-RFLP was used to genotype this SNP in 318 F2 hybrids of Boer !Macheng Black goats.The SNP was significantly associated with four traits including birth weight,chest circumference,cannon circumference and body height.Allele G seemed to be associated with fast-growth trait.