Regulation Of Heat Shock Transcription Factor On Small Heat Shock Proteins Of Chilo Suppressalis(Walker)

The rice stem borer,Chilo suppressalis(Walker),an important pest,belongs to Lepidoptera,Pyralidae,which widely distributes from south to north in China,and it seriously endangered theyield of rice.In recent years,C.suppressalis has become more serious in some rice growing areas in China under the background of global warming.Our previous study have found that C.suppressalis possessed the strong temperature tolerance.Therefore,the research on the temperature tolerance mechanism of C.suppressalis could reveal the cause of the outbreak from another aspect.The research on the molecular mechanism of C.suppressalis mainly focuse on heat shock proteins at present,however,the mechanism in response to temperature stress is still unclear.Therefore,this study focused on the regulation mechanism of heat shock transcription factor(HSF)and small heat shock proteins(sHSPs)of C.suppressalis to further elucidating the molecular mechanism of temperature tolerance of C.suppressalis.The main results are as follows:1.According to the transcriptomic data,the full-length cDNA sequences of one kind of HSF and two kinds of sHSPs genes were isolated and cloned from C.suppressalis and named Cshsf,Cshsp22.9a and Cshsp23.9a,respectively.Their complete cDNA sequences were 1532,838 and 840,including 1014,603 and 627 bp open reading frames,encoding 337,200 and 208 amino acids,respectively.Genomic validation found that the three genes all contained no introns.CsHSF possessed DBD,HR-A/B,HR-C and CTAD domains,and CsHSP22.9a and CsHSP23.9a both had the ?-crystalline domains,which are typical characeristics of sHSPs family.Phylogenetic tree analysis found that CsHSF had a less relationship to other lepidopteran insect HSF1.Therefore,we suggested that CsHSF did not belong to the conventional HSF1 family.While CsHSP22.9a and CsHSP23.9a clustered together with other C.suppressalis sHSPs,indicating that two CsHSPs shared high homology2.The expression patterns of Cshsf,Cshsp22.9a and Cshsp23.9a were analyzed by RT-qPCR technique.The results found that Cshsf inhibited expression at low temperatures,and the relative expression increased first and then decreased at high temperatures.Cshsp22.9a and Cshsp23.9a had similar expression patterns,which could be induced by high and low temperature,and the relative expression levels reached highest at-11 and 42?.Exposed to continuous high temperature at 42?,the relative mRNA expression of Cshsfwas significantly upregulated at 4 h,and reached the highest after 6 h.While the expression levels of Cshsp22.9a and Cshsp23.9a were upregulated gradually.These results showed that the expression of three proteins were closely related to temperature.3.A prokaryotic expression plasmid of CsHSF recombinant protein was constructed using pTYB12 vector,and the presence of recombinant protein CsHSF was detected by Western Blot.The IMPACT recombinant protein purification system was used to isolate and purify a large amount of recombinant protein CsHSF in vitro,and the concentration of the recombinant protein was 3.120 mg/ml after concentration by ultrafiltration membrane.The promoter sequences of Cshsp22.9a and Cshsp23.9a in lengths of-392-+47 bp and-599-+28 bp were amplified using the GenomeWalker method.And respectively three HSEs of Cshsp22.9a and one HSE of Cshsp23.9a were found in promoter using JASPAR online tools.Finally,the EMSA was used to in vitro verify the binding of CsHSF to Cshsp22.9a and Cshsp23.9a promoter sequences respectively.The results showed that CsHSF could bind to promoter sequences of Cshsp22.9a and Cshsp23.9a4.Using RNAi technique,we successfully silenced the expression of Cshsf gene in C.suppressalis.And we also detected the expression of Cshsp22.9a and Cshsp23.9a at proper temperature(27?)and high temperature stress(42?).The results showed that the interference efficiency of Cshsf expression was 77.42%and 63.42%,respectively at 27 and 42?.After RNAi,the relative expression levels of Cshsp22.9a was significantly upregulated at 27?,while the expression of Cshsp23.9a was not significantly changed.However,under high temperature,the expression of Cshsp22.9a was not significantly changed compared to the control after interference,but,the expression of Cshsp23.9a was significantly decreased.Moreover,the results still found the survival rate of C.suppressalis at high temperature was 63.3%,which was significantly lower than that of the control.These results indicated that Cshsf was related to temperature tolerance of C.suppressalis,and the absence of CsHSF had a significant effect on the expression of Cshsps.