Identification,expression Patterns,and Functional Study Of Heat Shock Protein Genes From Lasioderma Serricorne

The cigarette beetle,Lasioderma serricorne（Coleoptera:Anobiidae）,is a major pest insect of stored products with a world-wide distribution.L.serricorne mainly occurs in grain and cash crop in the warehouse,and mainly feeds on crops such as tobacco,soybean,tea,and edible oil crops,which seriously affects the quality of stored crops.The change of temperature in the warehouse will promote the function of a group of special proteins,the heat shock proteins（HSPs）in the L.serricorne.HSPs are highly conserved proteins in insects that are sensitive to temperature changes and can assist insects to adapt to the external environmental temperature to improve the survival rate of insects.At present,the study on HSPs of L.serricorne is scarce.In this paper,13 HSPs genes of L.serricorne were identified by searching the transcriptome database,the sequence characteristics and expression patterns of HSPs were analyzed,and the functions of 2 small HSPs were analyzed.The results are as follows:1.Identification and analysis HSPs genes of L.serricorneA total of 13 HSPs genes were obtained by searching the L.serricorne transcriptome constructed by the research group in the early stage,including 3 HSP90s,6 HSP70s,1HSP60 and 3 small HSPs.The proteins encoded by these genes have the conserved domains of insect HSPs.BLAST comparison results showed that these proteins have high homology with other HSPs of Coleopteran insects.The phylogenetic analysis results also showed that these proteins were most closely related to HSPs of Coleopteran insects.2.Analysis of the expression patterns of HSPs genes of the L.serricorne in different tissuesTreatment of different tissues of 5th instar larva of L.serricorne.The genes Ls HSP21.0,Ls HSP70-3,Ls HSP70-4 and Ls HSP70-5 were all highly expressed in the fat body of L.serricorne.The Ls HSP21.8 gene was expressed at a low level in the fat body,midgut and hemolymph of L.serricorne.The Ls HSP21.7,Ls HSP70-1,Ls HSP70-5 and Ls HSP70-6 gene expression level was higher in hemolymph.The Ls HSP70-4 gene was the most significant difference in the relative expression of HSP70s genes in L.serricorne.3.Analysis of the expression patterns of the L.serricorne HSPs genes under the different temperature conditionsA temperature gradient was set to treat 5th instar larva of L.serricorne,and it was found that Ls HSP21.0,Ls HSP21.7,Ls HSP21.8,Ls HSP60,Ls HSP70-1,Ls HSP70-2,Ls HSP70-3,Ls HSP70-4,Ls HSP70-5,Ls HSP70-6,Ls HSP90-1 Ls HSP90-2 and Ls HSP90-3genes were up-regulated under inducing conditions of high temperature（40℃）.However,under the condition of temperature lower than 26℃,Ls HSP21.0,Ls HSP21.7,Ls HSP21.8,Ls HSP90-1 and Ls HSP90-2 gene expression were significantly down-regulated,while Ls HSP70-5 gene expression remained basically unchanged,while Ls HSP70-1,Ls HSP70-3,Ls HSP70-4 and Ls HSP70-6 gene expression increased to a certain extent.4.Developmental expression pattern analysis of L.serricorne HSPs genesThe gene expression levels of s HSPs and HSP70s were different in different developmental stages of L.serricorne.Among them,Ls HSP21.0 was highly expressed in the pupal stage of L.serricorne,Ls HSP21.7 was highly expressed in the female adult L.serricorne,and Ls HSP21.8 was highly expressed in the male adult of L.serricorne.Ls HSP70-4 gene and Ls HSP70-5 gene were mainly expressed in the larval stage of L.serricorne,while Ls HSP70-3 gene was expressed at a high level in the pupal stage of L.serricorne.Other genes can also be expressed within a certain range.5.Prokaryotic expression and functional analysis of L.serricorne s HSPUsing p Cold II expression vector,the HSP21.7 and HSP21.8 of L.serricorne recombinant proteins were successfully expressed in E.coli by SDS-PAGE,and Western blot showed that the recombinant proteins were soluble.HSP21.8 and HSP21.7 enhanced the tolerance of E.coli cells to high temperature,it indicated that the growth rate(OD₆₀₀absorbance value)of E.coli overexpressing.HSP21.8 and HSP21.7 were significantly increased under high temperature treatment.50°C in controls transformed with p Cold II empty vector.In addition,the bacterial solution after high temperature treatment was spread on the culture plate,and the number of colonies of E.coli overexpressing.HSP21.7and HSP21.8 were significantly more than that of the p Cold II empty vector control group.This result indicated that HSP21.7 and HSP21.8 have thermoprotective functions,which can help L.serricorne resist high temperature stress.In conclusion,based on L.serricorne transcriptome constructed by the research group,this study identified 13 L.serricorne HSPs genes,and carried out expression pattern analysis,prokaryotic expression and recombinant protein analysis of some genes of the HSPs family that could be involved in the life activities of the L.serricorne.The results of this study provides new idea for the biological control of the L.serricorne.