Expression Analysis And Genes Encoding Of Small Heat Shock Protein Genes From Chilo Suppressalis

The rice stem borer,Chilo suppressalis is one of the most damaging insect pests of rice and,is a major pest of rice in Asia,North Africa and southern Europe,occurs in both southern and northern China,and is an important and distributed pest of rice in China.The previous survey showed that small heat shock proteins(sHSPs)were superfamily of proteins that exhibited larger variation in sequence,structure,size,and function unclear.In order to understand the mechanism of action of sHSPs on insects,six sHSPs genes were cloned and their expression patterns were studied and compared the sHSPs genes with the related lepidopteran species.At the molecular level,revealing the relationship between developmental stages and under different temperatures of C.suppressalis.The main results were as follows:Six heat shock protein genes were cloned from C.suppressalis by RT-PCR and RACE,and were named Cshsp17.2,Cshsp21.3,Cshsp22.9b,Cshsp23.9,Cshsp25.7,Cshsp27.3,respectively.The complete cDNA of 6 HSP genes were 813、909、872、844、1513 and 1036 bp respectively,and their respective opening reading frames were447、555、621、663、714 and 729 bp,which encoded 148、184、206、220、237 and242 amino acids,respectively,predicted pI were 9.69、5.97、5.79、4.50、4.55 and 4.94.At the same time,six small HSPs all contained a α-crystalline domain that was characteristic of small heat shock proteins.Sequence analysis found that these six shsps were highly similar to the shsps of other species and were divided into two branches in the phylogenetic tree.None of the six shsps contained introns.The expression patterns of four Cshsps under different tissues,different development stages and temperature stress were studied by real-time quantitative PCR.The results showed that the expression of all six Cshsps could be induced by high and low temperature stress,but the expression patterns were different.Cshsp22.9b andCshsp27.3 were in response to high and cold temperatures.High temperatures could induce the expression of Cshsp17.2,however,it did not respond to low temperature stress.The opposite occured in Cshsp21.3,which low temperatures could induce the expression of Cshsp21.3,but it did not respond to high temperature stress.Cshsp25.7responded to neither high temperature stress nor low temperature stress.Cshsp23.9expression was significantly different,the high temperature stress,the expression was mild.Real-time quantitative PCR indicated that the mRNA expression of Cshsp21.3,Cshsp22.9b and Cshsp27.3 were expressed at highest levels within the fat body as compared to other tissues(head,epidermis,foregut,midgut,hindgut,malpighian tubules,and hemocytes).Expression of Cshsp23.9 and Cshsp25.7 was highest in the hindgut.Only the expression in different tissues of Cshsp17.2 was mild,the expressionof other 5 shsps were differentially.The expression of six shsps could be induced by different development stages.The expression of Cshsp23.9,Cshsp25.7and Cshsp27.3 in female and male pupae were not significantly different.The third instar larvae was the highest expression level of Cshsp25.7,Cshsp27.3 and Cshsp21.3.The first instar larvae was the highest expression level of Cshsp23.9 and Cshsp17.2.The highest expression of Cshsp22.9b was in xiong pupae.HSP22.9b gene was amplified by PCR,ligated to PTYB12(+)exression vector and transformed into Escherichia coli ER2566,induced by IPTG under defferent concentrations.Through SDS-PAGE,the induced fusion protein was successfully expressed.