松材线虫类毒过敏原蛋白基因的克隆及其dsRNA表达载体的构建

Pine wilt disease in pine trees by pine wood nematode is one of the most devastating forest diseases in the world. In this study, a pair of degenerate oligonucleotide primers was designed according to the conservative regions of cloned venom allergen-like protein genes from different plant-parasitic nematodes. Two fragments of venom allergen-like protein genes were amplified by polymerase chain reaction (PCR) from cDNA of the pinewood nematode Bursaphelenchus xylophilus. Two full-length venom allergen-like protein genes, named Bx-vap-1 and Bx-vap-2, were cloned by means of rapid amplification of cDNA ends from the pinewood nematode Bursaphelenchus xylophilus. Inverted repeat consequences of target gene Bx-vap-1 and Bx-vap-2 were cloned, respectively. Then the two inverted repeat consequences were connected to the downstream of qa-2 promoter in expression vector. After Neurospora crassa electrotransformation, we did PCR to choose positive strains and used QA to induce promoter to express dsRNA. Now we have successfully screened out a strain expressing dsRNA of Bx-vap-1 and Bx-vap-2, which provided reaearch fundament for exploring to establish feeding RNAi method for B. xylophilus.