Cloning and expression analysis of heat shock protein MaltHSP40s gene in Monochamus alternatus
Monochamus alternatus is a significant stem borer in Southern China,and it is also the primary vector for Bursaphelenchus xylophilus,a major forest invasive species in China.To explore the role of heat shock protein HSP40 in M.alternatus'resistance to high-temperature stress,two HSP40 genes of M.alternatus was cloned using RT-PCR and analyzed by bioinformatics,based on the transcriptome data of M.alternatus(GenBank accession number:PRJNA548205).The phylogenetic tree of M.alternatus HSP40 was constructed using MEGA 7.0 software.RT-qPCR was used to detect the expression of HSP40 gene in different tissues of 16-day-old M.alternatus male and female adults under different temperature and time heat shock treatment conditions(35℃,37℃,40℃,42.5℃ and 45℃;0,1 h,2 h and 3 h)and high temperature treatment at 42.5 ℃ for 3 h.The findings showed that two HSP40 genes,MaltHSP40-1(GenBank accession number:MW690168)and MaltHSP40-2(GenBank accession number:MW690169),were cloned from M.alternatus.Their ORF lengths were 1 206 bp and 1 059 bp,encoding 401 and 352 amino acids,respectively.The molecular weights were about 45.24 kDa and 39.25 kDa,and the isoelectric points ranged from 6.73 to 9.07.DNA-J containing conserved HPD motif was found in both sequences.The three-dimensional structure of the protein comprises four a-helices at the N-terminus and a substrate binding domain at the C-terminus.The phylogenetic tree showed that MaltHSP40-1 and MaltHSP40-2 had the closest genetic distance with AglaHSP40-1 and AglaHSP40-2 of Anoplophora glabripennis,respectively.The results of RT-qPCR showed that high temperature stress could induce the expression of HSP40 in M.alternatus.The expression of MaltHSP40-1 and MaltHSP40-2 in male and female adults began to increase at 35℃ and 37.5℃,and reached the peak at 40℃ or 42.5℃.The relative expression and up-regulation of HSP40 in males were higher than those in females.Additionally,MaltHSP40-l and MaltHSP40-2 were expressed in all tissues of male and female adults of M.alternatus.After treatment at 42.5℃ for 3 h,the relative expression levels of MaltHSP40-1 and MaltHSP40-2 in all tissues significantly increased,with the highest relative expression in testes and ovaries.These results could further facilitate an exploration of the role of heat shock proteins in M.alternatus'response to high-temperature stress.They also provide a theoretical basis for revealing the epidemic mechanism of M.alternatus and B.xylophilus disease under the background of climate warming.
Monochamus alternatusheat stressheat shock protein 40heat resistanceauxiliary molecular chaperone
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