豚鹿MHC-DOA基因克隆及其外显子2遗传多样性研究

Cloning of Axis porcinus MHC-DOA Gene and the Genetic Diversity of Its Exon 2 Region

主要组织相容性复合物(MHC)在脊椎动物的免疫系统中起着重要的作用,MHC-DOA基因是MHCⅡ类的非经典基因,其外显子2多态性丰富。豚鹿Axis porcinus是我国极度濒危的动物。本研究以成都动物园圈养的38头豚鹿为研究对象,提取豚鹿血液总RNA,反转录合成cDNA为模板,利用PCR方法克隆到豚鹿MHC-DOA序列;同时利用PCR方法扩增38头豚鹿的MHC-DOA基因外显子2基因并测序,再进行多态性分析。结果显示:豚鹿MHC-DOA基因开放阅读框长753 bp,编码250个氨基酸残基;蛋白质同源性分析表明,豚鹿MHC-DOA基因与东欧马鹿Cervus elaphus hippelaphus的同源性最高(98.4%);38头豚鹿的MHC-DOA外显子2共有10种单倍型,整体遗传多样性水平中等;MHC-DOA外显子2部分序列中发生了核苷酸的缺失和插入,进而引起DOA蛋白序列发生改变。豚鹿MHC-DOA多态性的研究对豚鹿种群遗传结构调查、遗传资源的保护具有重要意义。

The genes encoding the major histocompatibility complex(MHC) are important in the immune system of vertebrates. MHC-DOA gene is a non-classical gene of MHC class Ⅱ, of which exon 2 has rich polymorphisms. Hog deer(Axis porcinus) is an extremely endangered species in China. In this study, total RNA was extracted from the blood of A. porcinus and then the MHC-DOA gene was cloned by PCR from cDNA template. Meanwhile, the exon 2 of MHC-DOA genes in 38 A. porcinus from Chengdu Zoo were obtained by PCR and DNA-sequencing, followed by genetic polymorphism analysis. The results showed that the MHC-DOA gene of A. porcinus contained a 753 bp open reading frame, encoding a protein with a length of 250 amino acid residues. Sequence analysis predicted that DOA protein had high amino acids similarity(98.4%) with Cervus elaphus hippelaphus. A total of 10 haplotypes of DOA exon 2 were detected from the 38 individuals, indicating a medium level of genetic diversity in A. porcinus. Nucleotides insert and deletion were observed in MHC-DOA exon 2 gene, and these resulted in the change of MHC-DOA amino acid sequence. This study has significant importance in understanding the population genetic structure and the protection of A. porcinus in the future.