Identification And Gene Expression Of Hsp70under Heat Stress In Insecticide-resistance And-susceptible Diamondback Moth(Plutella Xylostella)

The diamondback moth (DBM), Plutella xylostella L., was a major pest of brassicas worldwide, and was an important insect model for research. In this paper, we used DBM as research object, to carry out preliminary study on cloning and mRNA expression of hsp70, and it would lay the foundation for studing the function of HSP70and the anti-adversity in the DBM.In this study, several hsp70gene fragments were cloned from fourth instar larvae and adults of diamondback moth (DBM, Plutella xylostella) through RT-PCR and RACE technique, and finally eight full-length sequences of hsp70gene were obtained.The mRNA expression levels of hsp70gene(one was reported and three were obtained in the study) were detected in insecticide-resistant and-susceptible DBM using Real-time PCR. The results of this study were as follows:(1) Eighteen fragments were cloned from fourth instar larvae and adults of Japanese or Fuzhou DBM under heat stress, and the adults of Japanese or Fuzhou DBM without heat treatment.lt was showed that these fragments were belonged to hsp70gene when they were blast in NCBI web site.(2) Six corresponding full-length hsp70gene sequences can be spliced using the hsp70gene fragments as above mentioned. As blast in NCBI web site, the date demonstrated that the homologies of the full-length hsp70gene sequences were basically all over80%with the other species which the evolution relationship close to DBM. Among them, three629amino acid sequences can be deduced three nucleotide sequences which obtained from Fuzhou DBM, and three664amino acid sequences can be deduced three nucleotide sequences which obtained from Japanese DBM, the homologies of them were91%-94%and more than98%with the HSP70protein sequence that reported, respectively.(3) Two forward primers out of ORF were designed according to one full-length hsp70gene sequence, another two hsp70gene sequences which including ORF were cloned from fourth instar larvae and adults of DBM under heat stress using RACE technique. A669amino acid sequence and a667amino acid sequence can be putated from them, respectively. The homologies of them were98.8%and98.6%with the HSP70protein sequence that reported, respectively.(4) Gene-specific primers which were designed according to the hsp70gene sequences(one was reported and three were obtained from Fuzhou DBM which were described in (2)) were used to amplify hsp70cDNA in Real-time PCR.Results of fluorescence quantitative analysis suggested as follows:Firstly, significantly increased expression of these hsp70mRNAs were observed in fourth instar larvae and adults DBM after heat stress, and the relative expression quantities of these hsp70mRNAs at42℃were far outweigh them at37℃.Secondly, the relative expression quantities of these hsp70mRNAs in adults DBM were more than them in fourth instar larvae significantly, especially the samples after heat shock and in addition to the individual exceptions.Thirdly, between male and female DBM, no significant differences of the relative expression quantities of these hsp70mRNAs were observed in the whole.Finally, the relative expression quantities of these hsp70mRNAs in insecticide-susceptible DBM were higher than them in-resistant strains significantly.