瓜实蝇热激蛋白基因hsp70的克隆及序列分析

Cloning and sequence analysis of heat shock protein 70 gene (hsp70) from Bactrocera cucurbitae Coquillett

【目的】克隆分析瓜实蝇热激蛋白hsp70基因序列特征,为进一步研究瓜实蝇的热适应性机制及制定综合防治策略奠定基础。【方法】根据NCBI数据库中已报道的昆虫hsp70基因保守序列,设计合成扩增瓜实蝇hsp70基因部分片段的简并引物,并利用RACE-PCR扩增其全长序列。【结果】克隆获得瓜实蝇hsp70基因c DNA全长序列(获取号:KM112021)2271 bp,含1911 bp的开放阅读框,编码636个氨基酸,具有真核生物hsp70基因家族的3个明显基序标签,同时在C-末端具有EEVD基序,推测其属于胞质型热激蛋白。BLAST分析结果表明该序列与双翅目实蝇科昆虫hsp70基因高度相似,最高相似度达91%。氨基酸序列系统发育分析结果显示,瓜实蝇与双翅目实蝇科昆虫聚类为一个分支,证实hsp70基因的高度保守。【结论】瓜实蝇hsp70基因具有真核生物hsp70基因家族的特征,序列表现为高度的保守性,可作为今后研究瓜实蝇抗逆性机制的一个参数指标。

[Objective ]The present experiment was conducted to clone and analyze heat shock protein 70 gene （hsp70 gene） from Bactrocera cucurbitae Coquillett to lay the foundation for further study on heat adaptability mechanism of B.cu- curbitae and making comprehensive prevention and control strategy. [Methods]Based on the amino acid sequence of insect hsp70 from NCBI, the degenerate primers were designed to amplify full-length fragment of hspTO gene from B.cucurbitae by using RACE-PCR method. [Results]The result showed that, the obtained full-length fragment of hsp70 gene eNDA （accession number： KMl12021） was 2271 bp in length, which contained a open reading frame of 1911 bp encoding 636 amino acids with three sequence signatures of bsp70 gene family and EEVD motif in C-terminal region. Therefore, the hsp70 gene of B. cucurbitae was proven to belong to cytoplasmic heat shock protein. The BLAST analysis showed that the obtained hsp70 gene had high similarity with hspTO gene of insect from Diptera Tephritidae, which shared the highest similarity at 91%. The phylogenic tree based on the amino acid sequence showed B. cucurbitae and insects of Diptera Tephritidae could be classed into a branch, which certified that hsp70 gene was highly conserved sequence. [Conclusion] The hspTO gene is 2271 bp in length, has characteristic of eukaryote hspTO gene family, and shows highly conserved.