| Small heat shock proteins(sHsps)is an important member of heat shock protein family,which not only plays an important role in biological response to adverse environmental stress,but also is closely related to insect diapause.The orange wheat blossom midge,Sitodiplosis mosellana(Géhin),is a typical obligate diapause agricultural insect,which experiences extreme heat and cold during diapause.In order to clarify the role of sHsps gene in diapause,and to explore the relationship between regulation of diapause and hormones,in this study,the c DN A of SmHsp17.4 and SmHsp20.3 genes in S.mosellana were cloned by RT-PCR,the DNA and promoter sequences were cloned by Genome Walking technologies,and examined their expression in the process of diapause,short-term temperature stresses and ecdysone treatment(20 E)using real-time quantitative PCR.The active recombinant protein was obtained,then the molecular chaperone function was studied by malate dehydrogenase(MDH)from heat-induced denaturation in vitro.The main results as followes:1.We cloned the ORF sequences of Hsp17.4 and Hsp20.3 from S.mosellana,and named SmHsp17.4(Gen Bank accession number: MK714040)and SmHsp20.3(Gen Bank accession number: MK714041),Their full-length of genes are 456 bp and 543 bp,and they encoded 151 and 180 amino acids,respectively.The molecular weight of predicted protein are 17.4 and 20.3 k Da,The theoretical p I are 6.87 and 5.80.the deduced amino acid sequences of the two genes contained conserved α-crystal domain and Ibp A domain of sHsps family.Multiple sequence alignment and phylogenetic analysis indicated that the two proteins was 42.94%,and was most similar(more than 47%)and closely related to sHsps from the Nematocera including Anopheles gambiae.The genomicDNA and c DN A sequence alignment showed that SmHsp20.3 had no intron and SmHsp17.4 contained an intron of 85 bp in length.2.Based on the 5′-end nucleotide sequence of the ORF cDNA of SmHsp17.4/20.3,the promoter sequence of 1272 and 1186 bp were obtained by genome walking technique to investigate the regulatory mechanism of SmHsp17.4/20.3 gene expression.Prediction of transcriptional initiation sites at 189 and 59 bp upstream of SmHsp17.4/20.3 initiation codon by NNPP software.The sequence analysis revealed that the SmHsp17.4/20.3 promoter sequence contains not only the core promoter elements TATA-box and CAAT-box,but also the Heat shock element(HSE),GATA module sequence and the nuclear factor NF-.Simultaneously,the SmHsp17.4 contains two ecdysone response elements DBRC.3.There were significant differences in the expression of SmHsp17.4/20.3 gene in different stages of diapause,but the expression patterns were different in the process of diapause.The expression of SmHsp17.4 was significantly down-regulated after diapause.During diapause,it remained at a low level until October,but the expression increased significantly in November,it means that the transition period of post-diapause,and reached the highest level in January during post-diapause quiescence,then decreased significantly,and decreased to the lowest in the early stage of recovery and development.The change trend of expression level was similar to that of ecdysone(20 E)titer during diapause,and was consistent with that of 20 E response element DBRC in the promoter sequence of the gene,indicating that the expression of ecdysone in diapause may be regulated by 20 E.Different from SmHsp17.4,the expression of SmHsp20.3 in summer of August and winter of December,was significantly higher than pre-diapause,poat-diapause development and other stages of diapause.4.Using qPCR technique to analyze the response of SmHsp17.4/20.3 to over-summering diapausing larvae under extreme high temperature(35～50 ℃)and over-wintering diapausing larvae under extreme low temperature stress(0～-15℃)for 1 h.It was found that over-summering or over-wintering diapausing larvae treated at 35 ℃,40 ℃and-10 ℃ could significantly induce the expression of the two genes,but not induced significantly under extreme temperatures higher than 45 ℃ or lower than-15℃.The expression of the two genes under extreme temperature stress was also related to the duration of stress and the time of recovery after stress,both increased significantly after 30～60 min at35 ℃ and-10 ℃ for 1 h,also recovery of 30 min after heat shock for 1 h at 35 ℃.The SmHsp17.4 was also significantly increased after recovery of 30-240 min at-10℃ for 1 h.5.In order to determine the effect of ecdysone on the expression of Smhsp17.4/20.3 in diapause larva of S.mosellana,the changes in gene expression at different concentrations(0.1～ 0.6 pg /n L)at different times(3,6 and 12 h)after 20 E treatment of diapause larva were analyzed.The results showed that the application of 20 E had no effect on the expression of SmHsp20.3,but could significantly induce the expression of SmHsp17.4,and the change in the expression level was closely related to the concentration and time of 20 E.Among them,the concentration of 0.4 pg /n L had the most obvious induction effect at 6 h after treatment,and the expression level was 3.6 times higher than untreated control.6.The active SmHsp17.4/20.3 recombinant protein was successfully expressed and purified using expression p lasmid p ET28a(+),with concentrations of 36.61 and 4.15 μM,respectively.The molecular chaperone activity analysis showed that the SmHsp17.4/20.3recombinant protein can inhibit MDH from high temperature(43 ℃)degeneration,has significant in vitro activity of molecular chaperone.In summary,the expression o f S.mosellana Hsp17.4/20.3 gene is closely related to the diapause process.The expression of SmHsp20.3 was clearly induced by summer,winter and short term extreme high and low temperature stress,suggesting that it may play a role in heat resistance and cold tolerance during diapause.According to the transcriptional regulatory elements of SmHsp17.4 gene promoter sequence,the expression pattern of SmHsp17.4 gene under diapause and 20 E treatment showed that the expression of the gene in natural diapause was regulated by 20 E,the up-regulated expression during the transition period from diapause to post-diapause can be used as a molecular marker to predict diapause termination.The expression of the gene is also up-regulated under a certain range of short-term extreme temperature stress,which can play a role in coping with extreme temperature stress by inhibiting the accumulation of MDH.This study not only contribute to the understanding of the molecular regulation mechanism of diapause,but also provid e a molecular basis for exploring the adaptation mechanism to stress during diapause and related agricultural prevention. |
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