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采用RT-PCR、c DNA末端快速扩增法(RACE),克隆了象耳豆根结线虫Hsp70基因的全长c DNA(Me Hsp70)序列。Me Hsp70 c DNA全长2 203 bp,含有1 959 bp的开放阅读框,编码653个氨基酸,相对分子量为71.09 k Da,具有3段Hsp70家族的签名序列,Gen Bank登录号KF739434。同源性分析表明,氨基酸序列与其他真核生物的Hsp70序列具有很高的相似性。该Hsp70与其他物种中的Hsp70进行系统进化分析,结果显示,Hsp70的系统发育树不能体现物种间的亲缘关系,推测其反映的是不同物种间Hsp70生物学功能的相似性程度。构建了一个原核表达载体p EASY-E1-Me Hsp70,当IPTG终浓度为0.4~1.0 mmol/L时,能诱导表达融合蛋白。Me Hsp70基因的克隆和表达,将为象耳豆根结线虫的生态适应性机理研究提供依据。
The full-length c DNA (Me Hsp70) sequence of Hsp70 gene of Meloidogyne incognita was cloned by RT-PCR and rapid cDNA amplification (RACE). The full length of Me Hsp70 c DNA was 2 203 bp with an open reading frame of 1 959 bp encoding a protein of 653 amino acids with a relative molecular mass of 71.09 kDa and a signature sequence of 3 Hsp70 families and Gen Bank accession number KF739434. Homology analysis shows that the amino acid sequence has high similarity with Hsp70 sequences of other eukaryotes. The phylogenetic analysis of Hsp70 and Hsp70 in other species showed that the phylogenetic tree of Hsp70 can not reflect the genetic relationship among species, suggesting that Hsp70 reflects the similarity of the biological functions of Hsp70 among different species. A prokaryotic expression vector pEASY-E1-Me Hsp70 was constructed. When the final concentration of IPTG was 0.4-1.0 mmol / L, the fusion protein was expressed. The cloning and expression of Me Hsp70 gene will provide the basis for the study of the ecological adaptability mechanism of Meloidogyne incognita.