| In recent years,histone modification,which plays a key role in regulating gene expression,has attracted more and more attention.Histone acetylation is an important post-translational modification of proteins,which is regulated by histone acetyltransferases(HATs)and histone deacetylases(HDACs).HATs can relax chromatin structure and activate gene transcription by adding acetyl groups to histone lysine residues.In contrast,HDACs remove acetyl groups from acetylated histones and inhibit gene transcription.HDAC plays an important role in insect development and stress response,and is a potential target for insecticides.At present,the research on insect HDACs is mainly limited to the model insect Drosophila melanogaster.In this paper,the full-length cDNAs of Tribolium castaneum Class I HDACs(TcHDAC1,TcHDAC3 and TcHDAC8)and two heat shock proteins Hsp70(TcHsp70 and TcHsc70)were cloned.The functions of TcHDAC1,TcHDAC3 and TcHDAC8 in the development of Tribolium castaneum and their roles in regulating the expression of TcHsp70 and TcHsc70 were systematically studied by using RNA interference technique.Furthermore,the insecticidal activity of Class I HDAC inhibitors against.castaneum was analyzed.The results have important scientific significance and application value for further revealing the molecular mechanism of insect HDACs involved in gene transcription regulation and developing new insecticides targeting HDACs.The full-length cDNA sequences of TcHDACl,TcHDACS and TcHDAC8 were successfully amplified by RT-PCR and RACE.Sequence analysis showed that the 1473 bp ORF of TcHDACl encoded 490 amino acid residues,while TcHDAC3 and TcHDAC8 contain a 1296bp ORF and a 1131bp ORF encoding 431 and 376 amino acid residues,respectively.An amino acid sequence alignment shows that TcHDACl shares 96.73%and 81.26%identities with GcHDACI in Gnatocerus cornutus and DmRpd3 in D.melanogaster,respectively.TcHDAC3 is 98.14%and 78.77%identical in pairwise comparisons with GcHDAC3 in G.cornutus and DmHDAC3 in D.melanogaster,respectively.TcHDACS shared 40.25%identity with human hHDAC8.Investigation of the temporal-spatial expression patterns showed that TcHDAC1 and TcHDAC8 had the highest expression level in 3-day-old eggs,while the expression of TcHDAC3 reached the highest mRNA levels in 1-day-old pupae.Intersetingly,a dramatic increase of mRNA expression level was observed from prepupae to 1-day-old pupae for all three T.castaneum HD AC genes.Both TcHDACl and TcHDAC3 had the highest mRNA expression levels in thorax,while the highest expression level of TcHDAC8 was observed in fat body.These results indicate that Class I HDACs may play an important role in the development of.castaneum.The 2-day-old female adults were used for TcHDACs expression analysis under various environmental stresses inliuding high temperature(45℃),low temperature(4℃)and paraquat treatments.The results showed that TcHDACl,TcHDAC3 and TcHDAC8 were down-regulated by 58%,67%and 79%after 2 h 4 h and 2 h at 45℃,respectively.The mRNA expression levels of TcHDAC1 after 2 h,4 h,and 12 h of cold treatment at 4℃ were significantly higher than those in the control,increasing by 3.05-fold,3.13-fold,and 3.37-fold,respectively.Similarly,the expression level of TcHDAC3 was 1.64-fold higher than that of the control after 4 h of cold treatment at 4℃.However,the expression level of TcHDAC8 was down-regulated after 1 h and 2 h at 4℃,but up-regulated after at 4 h at 4℃.The expression levels of TcHDACl and TcHDAC3 increased by 2.44-fold and 4.3-fold,respectively,after 1 h of paraquat treatment.Paraquat treatment also increased the expression of TcHDAC8.These observations suggested that members of Class I HDACs in.castaneum may be involved in the abiotic stress signal pathways.The full-length cDNA sequences of T.castaneum Hsp70,TcHsp70 and TcHsc70,were successfully amplified by RT-PCR and RACE.Sequence analysis showed that the 1941 bp ORF of TcHsp70 encoded 646 amino acid residues,while TcHsc70 contain a 1893bp ORF encoding 630 amino acid residues.Amino acid sequence alignment shows that TcHsp70 shares high identity with the homologous Hsp70 proteins from other insect species including Spodoptera litura(82.04%),Mamestra brassicae(80.80%),D.melanogaster(79.13%).Similarly,TcHsc70 shared 71.76-72.48%identity with Hsc70 of other insect species.The amino acid identity between TcHsp70 and TcHsc70 was 80.96%.Genome structure analysis showed that TcHsp70 had no introns,while TcHsc70 contained two introns.The expression level of TcHsp70 increased by 1081-fold and 1318-fold respectively after 1 h and 12 h of high temperature(45℃)treatment Expression of TcHsc70,however,did not show a significant change in all treatment times examined.These results indicate that TcHsp70 plays an important role in the high temperature stress response of.castaneum.RNAi was conducted to determine the function of Class I HDACs in the development of.castaneum and the role of Class I HDACs in the expression regulation of TcHsp70 and TcHsc70.The results of larval RNAi showed that only 12%of the larvae in dsTcHDACl-treated group succeeded in pupation and all died in pupal stage.Similarly,only 17%of the larvae in dsTcHDAC3-treated group succeeded in pupation and exhibited wing deformity in pupal stage,and only 39%of the pupae could emerge with wing deformity.However,in dsTcHDAC8-treated group,as high as 98.67%larvae succeeded in pupation and emerge normally.On the sixth day after dsTcHDACs injection,changes in expression levels of TcHsp70 and TcHsc70 were determined by RT-qPCR.The results showed that,the knock-down of the expression of TcHDACl decreased the expression of TclHsp70 by 67%,but increased the expression of TcHsc70 by 4.22-fold,respectively.While no significant change was observed for expression of TcHsc70,the RNAi against TcHDAC3 decreased the expression of TcHsp70 by 62%.The knock-down of the expression of TcHDAC8 increased the expression of TcHsp70 and TcHsc 70 by 2.16-fold and 2.91-fold,respectively.Further pupal RNAi was conducted and the dsHDAC-treated adults were exposed to heat shock,and the mRNA expression levels of TcHsp70 and TcHsc70 were measured.The expression level of TcHsc70 in dsTcHDAC1-treated group significantly increased by 4.08-fold,7.21-fold and 37.18-fold after 2 h,4 h and 12 h exposure to heat shock,,respectively,as compared to the control,and dsTcHDAC8-treatment significantly enhanced the induction levels of TcHsp70 by 2.29-fold and 37.18-fold respectively after 1 h and 2 h exposure to heat shock.On the other hand,the knock-down of the expression of TcHDAC3 increased the expression of TcHsp70 by 38%,58%and 64%after 1 h,2 h and 12 h exposure to heat shock,respectively.Furthermore,while the RNAi against TcHDACl inhibited the basal expression of TcHsp70,the expression level of TcHsp70 in dsTcHDAC1-treated group was similar to that of control group after exposure to heat shock.These results further indicate that Class I HDACs play an important role in the development of T.castaneum,and can regulate the expression of Hsp70.Quantitative PCR was used to analyze the effects of trichostatin(TSA)and vorinostat(SAHA)on the expression level of TcHDACs and the insecticidal activity of these two HDAC inhibitors against T castaneum was assayed.Compared with the control group,the expression levels of TcHDAC1,TcHDAC3,TcHDAC6,TcHDAC7 and TcHDAC8 decreased by 70%,75%,65%,80%and 70%,respectively,after 1 day of SAHA treatment,while decreased by 60%,74%,40%,73%and 33%,respectively,after 3 days of SAHA treatment.After 6 days of SAHA treatment,the expression level of TcHDAC7 decreased by 58%,while that of other TcHDACs did not change significantly.The expression levels of TcHDACl,TcHDAC3,TcHDAC6,TcHDAC7 and TcHDAC 8 decreased by 80%,53%,51%,49%and 49%,respectively,after 1 day of TSA treatment,while decreased by 29%,38%,70%,48%and 55%,respectively,after 3 days of TSA treatment.The expression levels of TcHDAC7 decreased by 53%after 6 days of TSA treatment,while the expression levels of other TcHDACs did not change significantly.SAHA and TSA treatment did not inhibit the expression of TcHDAC 11.All larvae died on the second day after treatment with 1 mg/mL TSA or SAHA,while on the fourth day after treatment with deltamethrin.The final survival rates of larvae treated with 100 ug/mL SAHA and deltamethrin were 33%and 37%,respectively,and no significant difference was observed between these two treatment groups.The larvae treated with 100 μg/mL TSA had the lowest survival rate of 17%.The final survival rate of larvae treated with 10 μg/mL SAHA and deltamethrin was 50%.The lowest survival rate of larvae treated with TSA was only 20%,indicating the better insecticidal activity of TSA.These results suggest that HDACs inhibitors had good insecticidal activity against.castaneum. |
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