The Reserch Of Thermal Adaptation Molecular Mechanism Of Tribolium Castaneum (Herbst)

Tribolium castaneum(Herbst)(Coleoptera: Tenebrionidae)is an important worldwide stored grain insect.The changes of heat tolerence of T.castaneum larvae,pupae and adults after exposure to different temperature 28(control),36,and 42°C stress for 0,1,5,10,and 15 h respectively were investigated in the paper.Then,the transcriptomes of larvae,pupa and adult larvae before and after high temperature stress treatment were analyzed through high-throughput sequencing techniques.Differential expressed genes associated with heat tolerance after high temperature stress were screened and further verified by real-time quantitative PCR.Finally,RNAi technology was used to verify critical heat-tolerant gene functions.The results provide theoretical basis for understanding the molecular mechanism of insects adapting to thermal stress and the implementing rational heat treatment programs.The main research results were as follows.The heat-tolerence of the larvae,pupa and adult of T.castaneum experiencing constant sub-lethal high-temperature heat stress singnificantly increased,and the heattolerence of larvae,pupae and adults of T.castaneum was significantly higher than that the temperature of 42°C for 10 h and other treatment groups.IlluminaHiSeq2500 high-throughput sequencing technology was used to perform transcriptome sequencing of larvae,pupae and adults of T.castaneum that had been exposed to 28(control),36,and 42°C heat stress for 10 h,and differentially expressed genes were screened,which were further analized by GO annotation,KEGG pathway and Nr analsis,4 kinds of genes related to heat tolerance of T.castaneum upregulated in reponse to heat stress:(1)Oxidation-related genes: encoding glutathione Stransferasesuperoxide dismutase,CYP450,NADH,acyl-coa dehydrogenase,NADP and ATPase genes.(2)Development-related genes: encoding protein lethal essential for life genes.(3)genes associated with ATP binding: encoding heat shock protein(HSP)genes.(4)various transfer function proteins: encoding transferase genes and so on.RT-qPCR analysis showed that the heat shock protein family genes in the larvae,pupa and adults of T.castaneum were significantly up-regulated after heat stress,including small heat shock proteins(LOC662168,LOC662203,LOC664068)genes,and heat shock protein 70 family(LOC663293,HSP68 a,HSP68b)genes.In addition,a gene encoding the unknown functional protein LOC656546 was also significantly upregulated in the larvae,pupa and adults of T.castaneum in response to heat stress,and the expressed amount of above genes in response to heat stress at 42°C were significantly higher that at 36°C,which was also consistent with the results that the heat tolerence of the larvae,pupae,and adults of the T.castaneum experiencing 42°C heat stress was significantly higher than that of 36°C heat stress.The silencing of HSP68 a,which codes for heat shock protein 70,and LOC656546,which encodes an unknown functional protein,by RNAi technology showed that the heat tolerence of larvae and pupae was significantly reduced.The up-regulated expression of the two genes played an important role in improving the adaptability of larvae and pupae of T.castaneum on heat tolerance.