Cloning And Expression Of Heat Shock Protein Genes In Endoparasitic Wasp,Cotesia Vestalis

Heat shock proteins are ubiquitous existence in organisms and these proteins can protect organisms and cells from stress. Cotesia vestalis is an important parasitoid of the worldwide pest Plutella xylostella. In the study, we cloned and sequenced the full length cDNA encoding four heat shock proteins (hsp40, hsp70, hsc70and hsp90) of Cotesia vestalis and investigated their expression in relation to developmental stage. We also researched the hsps expression of Cotesis vestalis and Plutella xylostella under the thermal stress.1) Cloning and characterization of the hsps in Cotesia vestalisFour ORFs coding hsps involved three HSP families were cloned by the methods of Blast search, PCR, RT-PCR and RACE. The full length cDNA of hsp40, hsp70, hsc70and hsp90were1473bp,2151bp,2327bp and2663bp, contained open reading frames of1068bp,1938bp,1956bp and2172bp, which produced putative protein of355,645,651and721amino acids with calculated molecular weights of39.1kDa,71.0kDa,71.2kDa and82.8kDa, respectively. Two introns were found in Cvhsc70.2) The Cvhsps expression in relation to developmental stageIn the experiment of differential expression corresponding to developmental stage, we analyzed hsp40, hsp70, hsc70and hsp90genes expression in the1st instar larvae, early stage and later stage of2nd instar larvae,3rd instar larvae, pupae, female adults and male adults of Cotesia vestalis by real-time PCR method. The expression profiles of Cvhsp40and Cvhsc70were down-regulated and then up-regulated, but Cvhsp70was up-regulated and then down-regulated at development process, and Cvhsp90was down-regulated and then up-regulated but at last it down-regulated again. Cvhsp40and Cvhsc70had highest expression levels in the female adults; Cvhsp70had a highest expression level in the pupae; Cvhsp90had a highest expression level in the1st instar larvae and3rd instar larvae.3) The Cvhsps and Pxhsps expression in relation to temperatureIn the experiment of differential expression in relation to temperature treated1h at27～42℃ then recovered1h at24℃, we analyzed hsp40, hsp70, hsc70and hsp90genes expression in the1st instar larvae, early stage and later stage of2nd instar larvae,3rd instar larvae, pupae, female adults and male adults of Cotesia vestalis and their host Plutella xylostella by real-time PCR method. The insects were treated at24℃for2h as control. The hsps expression patterns were different in different developmental stages of Cotesia vestalis under high temperature (27℃,32℃,37℃and42℃). The expression levels of Cotesia vestalis hsps were upregulated when the treated temperature increased. But the expression levels of hsp40and hsp90were down-regulated at32℃, and those of hsc70at27℃and32℃were lower than that of the control in the early stage of2nd instar larvae, which might be induced by the effect of parasitism.The hsps expression patterns were different between parasitized and unparasitized Plutella xylostella under high temperatures. The expression levels of hsc70from the early stage of4th instar larvae and hsp70, hsc70and hsp90from the middle stage of4th instar larvae of the parasitized Plutella xylostella were upregulated under the normal condition24℃. In the high temperature treatment, the expression levels of hsps in the parasitized Plutella xylostella and the unparasitized Plutella xylostella were complicated. The temperature for Tmax induction of hsc70expression in the parasitized Plutella xylostella at the early stage of4th instar larvae was5℃higher than those of the unparasitized Plutella xylostella. The temperature for Tmax induction of hsp70, hsc70and hsp90expression in the parasitized Plutella xylostella at middle stage of4th instar larvae were5℃higher than in the unparasitized Plutella xylostella. The temperature for Tmax induction of hsps expression in the other parasitized Plutella xylostella were generally the same as in the unparasitized Plutella xylostella. It suggested that the parasitization induced hsps expression and enhanced the thermal tolerance of parasitized Plutella xylostella at the early and middle developmengtal stages of Cotesia vestalis.