| Tribolium castaneum(Herbst)(Coleoptera: Tenebrionidae)is an important worldwide stored grain insect.In this study,based on the heat acclimation phenomenon in the actual process of heat treatment,the heat tolerance changes of eggs,larvae,pupae,and adults of T.castaneum at different rates(0,1.25,2.5,5,and 10 ° C / h)from 28 ℃ and exposed at 50 ℃ for different times(0,10,15,20,25,and 30 min)respectively was studied.High-throughput sequencing technology was used to transcriptome sequencing and gene differential expression analysis of T.castaneum before and after thermal acclimation to select the genes related to their heat tolerance.Quantitative Real-time PCR and gene cloning were used to verify the transcriptome sequencing results.To elucidate the molecular response mechanism of the enhanced heat tolerance of T.castaneum after thermal domestication.The present results are in favor of developing an effective heat treatment protocol for pest control.In this study,we found that the thermal tolerance of the larva was stronger than that of the adult.When the space environment temperature was raised at different heating rates to stress the pupa and adult of the egg larvae of the red pseudo-glutinous rice robber,the heat tolerance of each insect state was significantly enhanced compared with that of the control group.The increase of heat tolerance of adults was much higher than that of other insect states,especially when the heating rate was 2.5℃/h.By comparing the effects of different heating rates on the heat tolerance of the T.castaneum,under the stress of slow heating(2.5 ℃/h),the heat tolerance of each insect state of the T.castaneum was higher than that of fast heating(10℃/h).However,when the heating rate was lower than 2.5℃/h,the heat tolerance of each insect state of the T.castaneum decreased.With the acceleration of the heating rate,the heat tolerance of each insect state of T.castaneum shows a trend of increasing first and then decreasing.Using high-throughput sequencing technology,transcriptome sequencing analysis was conducted on the adults of T.castaneum that were heated up to 46 in 2.5 /h(slow heating treatment group)and 10 /h(fast heating treatment group)and 28 thermostatic control group(control group).It was found that there were 7 genes encoding heat shock protein(LOC656270 hsp68 a hsp68b LOC663293 LOC663216 LOC662203)and homologous genes of heat shock proteins including 2 genes encoding Dna J protein(LOC661242,LOC658423)and 4 coding lethal essential protein genes(LOC664276 LOC663240 LOC660153 LOC662237)were significantly upregulated in response to heat stress at different heating rates.It suggested that heat shock proteins play an important role in response to heat stress of T.castaneum.Through the gene cloning verification and RT-q PCR detection of 7 heat shock protein genes with significantly different expressions,it was found that hsp70 a and hsp68 a genes were significantly overexpressed in T.castaneum adults after heat treatment stress at different rates,among which hsp70 a was the most significant,which could be up-regulated by more than 5,000 times.The expression of hsp27 a is very significant,which can be upregulated by more than 1000 times,while the expression of hsp26 is slightly weaker,but there is also a high expression of 20-60 times.HSP90 is not as sensitive as HSP70 and small molecule heat shock proteins in response to high temperature stress.It suggested that HSP68 a and HSP70 A of heat shock protein 70 family and HSP 27 a and HSP 27 b of small heat shock protein family play a significant role in the enhancement of heat tolerance of T.castaneum.After silencing hsp68 a and hsp27 a by RNAi technology,the heat tolerance of T.castaneum was significantly reduced,indicating that the The hsp68 a and hsp27 a genes play a key role in the response of the aldicarb to heat stress. |
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