Cloning And Functional Analysis Of Glutathione S-transferase Gene BxGST3 And BxGST1 In B.xylophilus

Pine wilt disease(PWD),is caused by B.xylophilus,which is a kind of devastating disease.The life span of B.xylophilus was complexity.B.xylophilus can live for a long time under unfavorable environmental conditions.Enzyme GSTs is a important of detoxification enzymein in eucaryotic biologicalthe.In this research,B.xylophilus GST genes were clone and the functions were been analysed to find pathogenic mechanism.The expression of two a GSTs gene BxGST3 and BxGST1 were been tested after B.xylophilus infecting the pine trees through qRT-PCR.And the location analysis by in situ hybridization of the two genes was been tested.The physiological characteristics of B.xylophilus was been tested after RNAi interference of two genes.The main conclusions are as follows:1.Full-length of glutathione acetyltransferase gene BxGST3 and BxGST1 were been cloned and bioinformatic analysis were been tested in Bursaphelenchus xylophilusA total length of two genes was been cloned by RACE amplification technology.Hmology analysis and prediction of three level structure comparison of the two genes named BxGST3 and BxGST1,homology analysis showed BxGST3 and BxGST1 and Guangzhou rhabdias and Meloidogyne 40%-50%.homology similarity analysis showed that the B.xylophilus BxGST3 gene cDNA was 738 bp,the putative protein sequences encoding 207 amino acids from the nucleic acid sequence of BxGST1 cDNA gene of Bursaphelenchus xylophilus was 702 bp from The nucleic acid sequence of putative protein sequences encoding 206 amino acids of.BxGST3 protein predicted theoretical isoelectric point of 6.91;the molecular weight of 23.56 kDa,the predicted molecular formula: C1082H1660N274O306Se;BxGST1 protein isoelectric point: 7.04;molecular weight:23.87kDa;prediction of molecular formula: C1106H1664N274O305S6.protein through a series of physiological and biochemical parameters of the forecast,two protein protein is hydrophilic,located on the membrane surface,is a membrane protein,BxGST3 and BxGST1 protein has many phosphate sites,which proves that it is more receptive to the transmission of external signals.2.Expression level of B.xylophilus GST Genes BxGST3 and BxGST1 after B.xylophilus Inoculation of Pinus thunbergii and analysis of tissue specificityThe results showed that BxGST3 and BxGST1 genes of B.xylophilus increased by the time and reached the peak at 24 h after inoculation.The gene fragment labeled as probes and found the expression of BxGST3 and BxGST1 hybrid cells of mRNA by BxGST3 and BxGST1 in B.xylophilus.BxGST3 and BxGST1 were observed in location of mRNA in tissue cells and the location of B.xylophilus detoxification or antioxidant related organizations.The results showed that markers of deep brown color mainly in B.xylophilus the esophageal gland and genital gland position of the GST genes BxGST3 and BxGST1 of B.xylophilus.That was when the nematode stimulus effect,the body in BxGST3 and BxGST1 gene mainly expressed in esophageal gland and genital gland area,which caused presumably because the nematodes and tree body interaction inthe process of the tree to produce certain substances to resist nematode.Therefore the expressions of BxGST3 and BxGST1 in the esophageal gland and gonad parts is very high.And the expression may increased by intestinal harmful substances decreasing in B.xylophilus.3.Virulence of B.xylophilus after RNAi on GST genesRNA interference gene BxGST3 and BxGST1 of B.xylophilus,results show that there was no difference of B.xylophilus in sterile water and dsGFP immersion in its morphology and Botrytis cinerea on reproduction ability and the ability of pathogenic infection of pines,and after the dsBxGST3 and dsBxGST1 interference solution of B.xylophilus the ability has decreased significantly,while the fluorescence quantitative PCR of interference after nematode and sterile water and dsGFP for the expression of nematodes were compared.The results also confirmed that the interference results in a significant decrease in BxGST3 The expression level of and BxGST1 gene,plays a silencing effect.After interference from the experimental results,the dsBxGST3 and dsBxGST1 interference solution of B.xylophilus morphology did not change significantly,which shows that the disturbance form of these two genes of B.xylophilus had little effect,but after interference of nematodes decreased significantly in the the ability of reproduction and feeding,that has great influence on the silence of two genes on the reproduction of B.xylophilus feeding ability.The interference of B.xylophilus BxGST3 and BxGST1 gene,also has a great influence on nematode pathogenicity in the early stage of pathogenesis,The interference of two genes in B.xylophilus is significantly lower than that in sterile water and soak in dsGFP nematode pathogenicity of many.15 d after dsBxGST3 and dsBxGST1 interference solution of B.xylophilus inoculated Pinus thunbergii has no obvious signs of illness,and the control group of two treatments have obvious lesions and needles of Pinus thunbergii green to yellow;after 32 d dsBxGST1interference solution dsBxGST3 and the emergence of a large number of green and yellow pine needles,but not yellow wilting,while the control group has been completely yellow wilting.Through the experimental results suggested that when the two gene was interfered after silencing of B.xylophilus stress can detoxify Fall,breeding feeding ability also decreased,so that the decline of B.xylophilus pathogenicity.