水牛BCL2L10基因的克隆、序列分析及其在卵母细胞中的表达

Cloning and Sequence Analysis of Buffalo BCL2L10 Gene and Its Expression in Oocyte

本研究克隆了水牛BCL2-like 10(BCL2L10)基因序列,并用生物信息学技术分析了该序列的同源性、系统进化树、蛋白的理化性质以及高级结构。同时利用免疫荧光染色的方法检测了BCL2L10蛋白在水牛卵母细胞成熟前后的表达情况。结果表明,应用RT-PCR技术克隆得到的水牛BCL2L10基因序列全长600 bp,其中编码区长度为558 bp,共编码185个氨基酸。对该序列进行对比分析发现,水牛BCL2L10基因的核苷酸序列与野牛、牛、绵羊、小鼠、大鼠、野猪的同源性分别为99%、98%、96%、84%、81%、75%。生物进化树分析显示,水牛BCL2L10基因与牦牛、野牛的亲缘关系最近。生物信息学分析得到BCL2L10蛋白的分子量为21.6 k D,理论等电点为9.54。功能结构域预测显示BCL2L10蛋白属于BCL-2家族成员,参与细胞凋亡调控过程。免疫荧光染色结果发现,BCL2L10蛋白在水牛卵母细胞成熟过程中均有表达。

In this study, we cloned the CDS sequence of buffalo BCL2LIO gene, and used bioinformatics techniques to analyze the homology, phylogenetic tree of the sequence, and the physicochemical characteristics and secondary structures of BCL2L10 protein. At the same time, the expression pattern of BCL2L10 protein before and after the maturation of buffalo oocyte was investigated by immunofluorescence staining. The result showed that the buffalo BCL2LIO gene fragment cloned by RT-PCR was 600 bp in full length, including the whole CDS of 558 bp which encoded 185 amino acids. The result of multiple sequence alignment showed that the nucleotide sequence ofBCL2LIO gene of buffalo shared 99%, 98%, 96%, 84%, 81% and 75% homology with Bos mutus, Bos tauras, Ovis aries, Mus musculus, Rattus norvegicus and Sus scrofa respectively. The phylogenetic tree analysis showed that BCL2LIO gene of buffalo had the closest genetic relationship with Bos taurus and Bison. The bioinformatics analysis indicated that the molecuar weight and isoelectric point of BCL2L 10 protein were 21.6 kD and 9.54. The functional domain prediction showed that BCL2L10 protein belonged to BCL-2 family and was involved in the regulation process of cell apoptosis. The immunofluorescence staining showed that BCL2L10 protein expressed in the maturation process ofoocyte in buffalo.