Cloning And Expression Analysis Of EhHsp90-1 And EhHsp90-2 Genes In Eogystia Hippophaecola Larvae And Construction Of RNA Interference System Of EhHsp90-1

Eogystia hippophaecola(Hua,Chou,Fang & Chen)belongs to the Eogystia genus of Cossidae,Lepidoptera,which is a borer pest that severely destroys the ecological and economic benefits of the artificial seabuckthorn forest in Inner Mongolia,Liaoning and Ningxia so on.The larva bore into sea buckthorn trunksand roots,and they have the strong cold resistance ability that they can overwinter in the root of seabuckthorn.However,heat shock protein 90 is closely related to the tolerance of insects to high and low temperatures.In order to reveal the adaptation mechanism of E.hippophaecola larva to cold tolerance,two Hsp90 genes were selected and cloned based on transcriptome deep sequencing results in this study,and Eh Hsp90-1 and Eh Hsp90-2 were named respectively;q RT-PCR was used to analyze the expressions of two Hsp90 genes in E.hippophaecola larva under low temperature stress;the Eh Hsp90-1 and Eh Hsp90-2 genes with correct sequencing were connected to the prokaryotic expression vectors p ET30 a and the target proteins were successfully expressed;the effects of ds RNA injection dose and interference time on the RNAi efficiency of Eh Hsp90-1 gene were compared.The results are as follows:1.The complete open reading frames and amino acid sequences of Eh Hsp90-1 and Eh Hsp90-2 genes were obtained.The total lengths of Eh Hsp90-1 and Eh Hsp90-2 gene ORFs were 2154 bp and 2346 bp,respectively,encoding 717 and 781 amino acids,and its molecular weight were predicted to be 82.5KDa and 88.8KDa.The two gene sequences contain the characteristic sequences of Hsp90 family.According to the characteristic sequences of Cterminal,they can be divided into cytoplasmic Hsp90 and endoplasmic reticulum Hsp90,and have a high homology with the amino acid sequences of Hsp90 of a variety of lepidoptera,indicating that Hsp90 is highly conserved.2.The expression patterns of Eh Hsp90-1 and Eh Hsp90-2 genes at different temperatures and different times were determined.Under various low-temperature treatments lasting 2h,Eh Hsp90-1 and Eh Hsp90-2 exhibited similar expression patterns,increasing first and then decreasing.Under the treatment of-5? low-temperature stress with different durations,Eh Hsp90-1 was significantly up-regulated after 12 h,whereas Eh Hsp90-2 was up-regulated after just 1h and reached its highest level at 2h,however,the overall degree of upregulation was greater for Eh Hsp90-1.The results indicated that Eh Hsp90-1 gene could play a more important role in the response of E.hippophaecola larvae to lowtemperature stress.3.The accurate expressions of Ehhsp90-1 and Eh Hsp90-2 in vitro were induced.The two genes were connected with the expression vectors p ET30 a and successfully transferred into Escherichia coli.After IPTG induction,recombinant proteins with expected molecular weight were detected in the superfine solution,indicating that the two heat shock proteins were successfully expressed in vitro and were both soluble proteins.The Hsp90 proteins in the E.hippophaecola larvae were detected by western blotting with the polyclonal antibody.4.The effective RNAi system of Eh Hsp90-1 gene was preliminarily constructed.The expression levels of Eh Hsp90-1 gene under different ds RNA injection doses and interference times were compared.It was concluded that the system capable of effective interference with Eh Hsp90-1 gene was the result of injection of 5?g ds RNA and continuous interference for 72 h,which provided a reference for the subsequent analysis of gene function.