| Pine wilt disease is a fatal one caused by infection of the pine wilt nematode Bursaphelenchus xylophilus. The disease was first identified in Japan, where an outbreak occurred, and has been since spread to many countries including China, Japan, South Korea USA, Canada, Mexico and Portugal. The pathogenesis of pine wilt disease is not clear, because it is affected by multiple factors including the plant hosts, longhorned beetles, nematodes and the microbes associated with them, as well as abiotic environmental factors. Pine wilt disease has posed enormous pressure on the ecosystem and caused huge economic loss in the disease epidemic countries. Since the beginning of the1980s, pine wilt has emerged firstly and became widespread in China. It has been continuously threatening and destroying the pine ecosystem, and is one of the most dangerous biological disasters in the forest in China. To further revealed the pathogenesis of pine wilt disease, the most direct and effective way is getting the key genetic information in parasitism process, and analysising gene function.At present, it has been recognized internationally that esophageal gland cell secretion of plant parasitic nematodes play a key role in early interaction, genes encoding these secretion has been recognized as pathogenicity-related genes. Venom allergen-like protein gene (Bx-vap-1) expressed specifically in esophageal gland cells has been isolated from Bursaphelenchus xylophilus, it is inferred that Bx-vap-1gene might be pathogenic genes of Bursaphelenchus xylophilus, which plays an important role in early parasitism.In this study, Bx-vap-1gene as the research object, We take positive validation method, Bx-vap-lgene was expressed in insect cells, obtain recombinant VAP protein, to simulate esophageal gland secretion of Bursaphelenchus xylophilus, inoculate three-year old pinus massoniana. Detect pine defense genes expression, terpenes content change and histopathological changes, analyze Bx-vap-1gene function at molecular defense level, physiological metabolism level, histopathological level. Providing fundamental basis for pathogenesis research of pine wilt disease, the main conclusions are as follows:(1) The Bac to Bac baculovirus expression system was used to express Bx-vap-1gene. This expression system is efficient for producing recombinant baculovirus for expression testing in insect cells with high expression, easy screening, exact modification after transcription and no endotoxin toxicity. Fistly, Bx-vap-1gene was cloned into the pFastBacHTA vector, and the recombinant plasmid was transformed into DH10Bac competent E.coli to generate a recombinant bacmid. The recombinant bacmid DNA Was transfected into the Sf9insect cell to generate a recombinant baculovirus with cellfectin. After amplifying, the baculoviral stock was used to infect Sf9insect cells to express the recombinant VAP protein. The expressed recombinant proteins were confirmed by SDS-PAGE and Western blot analysis.(2) According to high conservative amino acids of reported a-pinene synthase gene from pinaceaes, a pair of degenerate primers were designed and partial fragment was obtained by PCR amplification. Based on this known sequence, some new primers were designed, two terminals gene sequence were cloned by3’RACE and5’ RACE respectively. After splicing the fragments of sequence, analyzed the structures and function of the coded protein by bioinformatics softwares. The full-length cDNA of a-pinene synthase gene consists of2103bp, contains a1890bp open reading frame (ORF) encoding629amino acid proteins, including N-terminal transit peptide, metal-binging domain and DDXXD motif, it has the typical characteristics of monoterpene synthase gene. The sequence has been submitted to GeneBank, accessin No. KF547035.(3) Recombinant VAP protein as inoculum to simulate esophageal gland secretion of Bursaphelenchus xylophilus, inoculate three-year old pinus massoniana. Inoculation experiment results showed:recombinant VAP protein can induce a-pinene synthetase gene up-regulated expression. This indicated that:in interaction between Bursaphelenchus xylophilus and Pinus massoniana, VAP protein secreted from Bursaphelenchus xylophilus can be regarded as elicitors to start the host defense response, hence, Bx-vap-1gene of Bursaphelenchus xylophilus which encoded VAP protein is pathogenic gene.(4) Recombinant VAP protein inoculated three-year old pinus massoniana. It appearsd obvious disease symptoms, such as, needles were wilt and droopy, phloem necrosis, xylem pith appeared brown stain. Cytological observation showd recombinant VAP protein can induce plasmolysis and nucleus degradation, this is a typical programmed cell death, this indicates that recombinant VAP protein can causes obvious damage to Pinus massoniana cell. It proved again that Bx-vap-1gene of Bursaphelenchus xylophilus is pathogenic gene. However, recombinant VAP protein didn’t cause cavitation, its formation may needs more elements combined action such as cell wall degrading enzyme. |
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