| Chilo suppressalis(Walker)belongs to Lepidoptera,Pyraloidea,which is one of the major pests that affect the stability and high yield of rice in China with the widely distribution and adaptability.Cotesia chilonis belonging to Hymenoptera,Braconidae,and Apanteles,is one of the main parasitoid species of rice stem borer on the field,helping controlling the population of C.suppressalis.However,there were not yet clear that the mechanism of coordinated evolution between C.suppressalis and Cotesia chilonis and the strategy of C.suppressalis in response to the parasitism of Cotesia chilonis.Therefore,this study was compared and analyzed the parasitic transcriptome changes on the host after parasitization to reveal these mechansims initially.The expression characteristics of four heat shock proteins of C.suppressalis in response to the parasitism of Cotesia chilonis were investigated.At the same time,the regularity of their response to temperature was further revealed.These results not only enriched the coordinated evolution theory of host-parasitoids,but also provided theoretical support to prevent and control C.suppressalis with Cotesia chilonis.The results are as follows:1.The transcriptional technology was used to determine the transcriptomes of C.suppressalis that have been parasitized by Cotesia chilonis and those that have not been parasitized.We found that 5685 unigenes were differentially expressed in rice stem borer under the parasitic stress by comparison.The six pathways of insect hormone biosynthesis,oxidative phosphorylation,ribosome,drug metabolism,pyruvate metabolism and metabolic were significantly enriched.There were four heat shock protein genes(hsps)differentially expressed.In fact,two heat shock protein 70 genes(hsp70s)were down-regulated and two small heat shock protein genes(shsps)were up-regulated.2.Three novel hsps were cloned from C.suppressalis,and were named hsp705,hsp706 and hsp21.7c,respectively.Their complete cDNA sequences were 2053,2392 and 870bp,respectively,and their respective opening reading frames are 1896,1905,and 570 bp,which encoded 631,634,and 189 amino acids,respectively.Sequence analysis suggested that HSP705 and HSP706 contained conserved motifs found in HSP70 respective family of other species.At the same time,HSP21.7c contained the conserved features of the small heat shock protein family—α-CAT domains.Phylogenetic analysis showed that two HSP70s showed high homology with other Lepidoptera insects.It was not higly homologous between CsHSP21.7c with other shsps of C.suppressalis,so CsHSP21.7c without introns was a specific small heat shock protein.3.The qRT-PCR(Quantitative real-time PCR)was used to screen and evaluate the stability of nine candidate reference genes of C.suppressalis facing parasitic stress.These genes were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs(NormFinder,The geNorm and BestKeeper)and the comparative△Ct method by qRT-PCR.In general,TUB,EF-1,and NADHD are relatively stable as reference genes.TUB is the most stable gene,while RPS11 is the leaststable gene.Finally,we verified the stability of TUB by the relative expression level of HSP60 gene under the parasitic stress.Last but not least,we confirmed that the reference genes of C.suppressalis under the parasitic stress is TUB.4.Hsp705 was significantly increased in the early parasitizationof C.chilonis,then down-regulated in late period of the parasitization.Hsp706 was significantly down-regulated in the midand late period of the parasitization.Hsp21.7c was significantly up-regulated in the mid-term and was significantly down-regulated in the late period of parasitism.The expression of hsp21.5 in the mid-parasitic stage was also significantly up-regulated.In the transcriptome,two hsp70s(hsp705 and hsp706)were significantly down-regulated,while two shsps(hsp21.5 and hsp21.7)were significantly up-regulated.The expression level of four Cshsps by RT-QPCR were similar with the transcriptome.5.The expression patterns of three no vle hsps under different temperatures from 11 ℃ to 42 ℃ were studied by real-time quantitative PCR.The results showed that the expression of Cshsps could be induced by high and low temperature stress,but the expression patterns were different.Both the expression level of hsp705 and hsp21.7c reached the highest at-6 ℃,while hsp706 reached the highest at-11 ℃.By comparison of relative expression levels,it was found that hsp21.7c was more susceptible to heat induction at the mRNA level whereas hsp705 and hsp706 were more susceptible to cold induction. |
