大豆胞囊线虫热激蛋白基因Hsp70的克隆与原核表达

采用RT-PCR技术结合基因组步移技术,从大豆胞囊线虫(Heterodera glycines)中克隆了热激蛋白70基因(Hsp70)的全长cDNA序列(Hg-Hsp-70),全长1 953 bp,GenBank登录号为FJ816100.1。碱基序列分析结果表明,该序列含有9个外显子与8个内含子,与其它线虫具有较高的同源性。氨基酸序列分析表明,该基因编码650个氨基酸,相对分子量为70.7 kD,具有2个Hsp70基因家族的签名序列,与其它线虫的该基因氨基酸序列具有很高的同源性。构建了原核表达载体Hsp70pEASY-E1,采用IPTG诱导蛋白表达,SDS-PAGE电泳结果表明,当IPTG终浓度为0.2～1.2 mmol.L-1时均能显著诱导表达;以终浓度0.8 mmol.L-1的IPTG诱导5 h蛋白表达量达到最大值。

Cloning and Prokaryotic Expression of Hsp70 from Soybean Cyst Nematode ( Heterodera glycines )

Heat shock protein 70(Hsp70) family is one of the most important heat shock proteins and an evolutionarily conserved protein.It plays an important role in environmental adaptability and stress tolerance of organisms.In this paper,Hsp70 complete cDNA sequence(Hg-Hsp-70)was cloned from Heterodera glycines by RT-PCR and genome walking technology,the full length is 1953 bp,and the Genbank accession number is FJ816100.1.The sequence structure analysis showed that the genomic sequences contained 9 exons and 8 introns,and the sequence homology was higher than other nematodes.This gene encoded 650 amino acids,and the calculated molecular weight was 70 kD,carried two important intact Hsp70 signature sequences.The result of amino acids sequences analysis revealed that translated molecules showed high homology with other nematodes.The prokaryotic expression vector Hsp70pEASY-E1 was constructed for Hg-Hsp-70.Hg-Hsp-70 protein was obviously induced to express by 0.2-1.2 mmol·L-1 IPTG and reached the maximum expression,after induced 5 h by 0.8 mmol·L-1 IPTG.