Isolated Of The Heat Shock Transcription Factor(HSP) And Analysis HSP70 By RNAi In Bursaphelenchus Xylophilus

The pine wood nematode Bursaphelenchus xylophilus is a kind of important alien invaded pest in many countries,the pine are destroied seriously because of the causal agent of pine wilt disease.In this paper,the HSP70 of B.xylophilus were analyzed by RNAi,with this base,to know about it's functions in environment adaptation,then,to supply theory support for better understanding the ecological adaptability and mechanisms of B.xylophilus to environment stress,and new method for the control work in future.A couple of specific primers were designed on the basis of the conservative domain of HSF.Then,a fragment that contained 282bp was amplified by RT-PCR in Bursaphelenchus xylophilus.The fragment was identified by sequencing,Blastn and Blastx in the NCBI. New specific primer were designed on the basis of the fragment.With the RACE and RT-PCR technology,the 3'RACE products that contain 1184bp were attained.Through linking the two sequence,we got 1408bp sequence—part of the full-length cDNA of HSF. After analysised of it,the ORF contain 1305bp.The function of HSP70 analysised with the RNAi technology.Firstly,a couple of gene-specific primers were designed according to the bx-hsp-70 in Bursaphelenchus xylophilus(Genbank number is DQ785812) and hsp-70 in C.elegans(Genbank number is NM070667) with DNAman,primer5.0 and oligo.To faciliatae the construction of dsRNA expression vector,the corresponding restriction enzyme sites of the upper stream primer XhoI and the lower primer XbaI were introduced in the end of the 5'extreme.Target products were attained by RT-PCR,then cloned it into the pGM-T vector,after identified by sequencing,the target gene fragment was cloned into the LITMUS28i vectors which contain T7 promotor take advantage of the restriction enzyme site XhoI and XbaI.As a result,we got vectors which can be use of to synthesize dsRNA in vitro.Transformed the vectors into E.coli HT115 which is RNase-Ⅲdeficient,we obtained the E.coli strain which can express dsRNA by induce.dsRNA were synthesized with T7 RNA polymerase in vitro,introduced it into Bursaphelenchus xylophilus in three different concentrations by soaking method after purification.After 48h,the revise death rate was 73.8%,98.19%and 95.59%corresponded to the final concentration 1.5mg/mL,2.0mg/mL and 3.0mg/mL,respectively.As a result,the revise death rate was the max when the final concentration was 2.0mg/mL.After the worm were treated by the concentration of 1.5mg/mL,heat tolerance,productivity experiment were processed.The result show that higher temperature decressed the heat tolerance of Bursaphelenchus xylophilus and influenced the death rate.Pprogenitive ability decline very much,per female worm egg-laying decreased 74.7%and 96.6%after treatment 24 hours and 48 hours,respectively.When the final concentration was 2.0mg/mL,per female worm egg-laying decreased 83.9%and the F1 generation egg-laying decreased 93%after 48h treatment.The result of RT-PCR shows that the target gene was silenced in parts of Bursaphelenchus xylophilus.In the cross RNAi experiment,dsRNA were introduced into C.elegans by feeding the E.coli strains which had expressed dsRNA by induce.Our result indicated that the movement ability of C.elegans obviously loss,and the egg-laying decreased 47%to compared with the control.