家蝇Hsp20.6基因原核表达及其在高温胁迫下的表达分析

Prokaryotic expression of Hsp20.6 gene in Musca domestica L. under heat stress and its expression characteristics

目的对家蝇小热休克蛋白Hsp20.6基因进行原核表达,并研究高温胁迫下该基因在家蝇各生长发育阶段及成虫不同部位的表达变化情况,为最终阐明其生物学功能奠定基础。方法利用p ET28a(+)原核表达载体构建家蝇Hsp20.6基因的重组表达载体,酶切、测序鉴定后,诱导蛋白的表达。采用半定量RT-PCR和实时荧光定量PCR研究Hsp20.6在家蝇卵、3日龄幼虫、蛹、成虫4个生长发育阶段及成虫头部、胸部、腹部的表达情况。结果成功构建p ET28a(+)-Hsp20.6重组表达载体,IPTG诱导表达产物大小约为Mr21 000,与生物信息学预测结果相符。RT-PCR与实时荧光定量PCR表明Hsp20.6基因在家蝇各个发育时期和成虫各部位有不同程度的表达,热激能上调该基因的表达(P<0.05),以幼虫和成虫头部组织上调量最高。结论 Hsp20.6基因属于热诱导型基因,参与了家蝇适应热胁迫的调控,可能在其中具有某种重要功能。

To construct a recombinant prokaryotic expression vector of Hsp20.6 gene of Musca domestica and analyze its expression characteristics, c DNA of Hsp20.6 was amplified by RT-PCR and then connected to p ET-28a（＋）vector. After identification with PCR, restriction enzyme digestion and nucleotide sequencing, the recombinant plasmid was transformed into Escherichia coli BL21（DE3） and then induced with IPTG for expression. SDS-PAGE result showed that the molecular weight of recombinant protein was about 21 KD, consistent with the deduced size.To determine the expression patterns of Hsp20.6 in response to heat shock, RT-PCR and real-time quantitative PCR were employed to detect m RNA accumulations at different developmental stages and in different tissues of the adult. The result showed that Hsp20.6 was expressed at all tested developmental stages and tissues of the adult.Furthermore, the protein expression level of Hsp20.6 was significantly increased by heat shock（P 0.05）, especially in larvae stage and in the head of adult. This study suggests that Hsp20.6 may play an important role in high temperature tolerance of Musca domestica, which would provide a foundation for further researches on the biological functions of Hsp20.6 gene.