广西草龟GH基因全序列克隆及测序分析

Cloning and sequence analysis of growth hormone gene from Chinemys reevesii,collected in Guangxi

【目的】了解广西草龟生长激素(GH)基因全序列结构特点及其与其他物种间的关系,为进一步研究GH基因的结构、功能、遗传变异规律及其与生产性能的关系提供科学依据。【方法】根据GenBank上已公布的黄喉拟水龟GH基因序列设计合成9对特异性引物,以广西草龟基因组DNA为模板进行PCR扩增,所获得目的片段经纯化、克隆、鉴定和测序后,用Vector NTI Advance10软件将各扩增序列拼接,获得草龟生长激素基因全序列,并用DNASTAR软件MegAlign程序中的Alignment Report和NCBI的BLAST对草龟GH基因全序列进行同源性比对分析及构建基于GH基因编码序列的物种间亲缘进化树。【结果】广西草龟GH基因全长8538bp,与黄喉拟水龟GH基因全长8517bp相比,二者的同源性高达98%,其中有118个碱基发生突变,插入37个碱基,14个位点发生碱基缺失;与鸡、鸭、鹌鹑、鸵鸟、红树林蛇的同源性分别为88%、83%、87%、86%和84%。【结论】GH基因编码序列具有较高的保守性。

[Objective]The present experiment was conducted to analyze the sequence of growth hormone（GH） gene from Chinemys reevesii collected from Guangxi and its relationships with other species,in order to study the mechanism of its structure,functioning and genetic variations and its relationship with production performance.[Method]Based on the GH gene sequence in Mauremys mutica Cantor obtained from GenBank,nine pairs of primers were designed.PCR amplification was performed with the template DNA extracted from Chinemys reevesii.Each amplified product was purified,cloned,identified and sequenced,followed by linking of sequences using Vector NTI Advance 10 software to obtain whole gene sequence.Homology of gene sequences was compared（BLAST） and its evolutionary relationship with other species was analyzed by making phylogenetic dendrogram using MegAlign program of DNA STAR software.[Result]The full length of C.reevesii GH gene was 8538 bp and its homology with Mauremys mutica Cantor（8517 bp） was as high as 98%.There were 118 mutated bases in GH gene,37 bases were added and 14 bases were lost.Its homology with chicken,duck,quail,ostrich and mangrove snake was 88,83,87,86 and 84%,respectively.[Conclusion]Coding sequence of GH gene was relatively conservative.