The Cloning Of 3 Bursaphelenchus Xylophilus Effector Genes

Bursaphelenchus xylophilus was introduced into China more than thirty years,the B.xylophilus causes pine wilt disease(Pine Wilt Disease,PWD)serious damage to our forest ecosystem,causing incalculable damage.Has yet to find effective methods of prevention and treatment of B.xylophilus,B.xylophilus new epidemic continues to increase,and there are all kinds of evidence of epidemic trend of warmer southern climate gradually spread to the cold north,has reached the northernmost Dalian City,Liaoning province and other places.Difficult to control pine wilt disease characteristics associated with its initial occurrence of disease no obvious feature is difficult to detect,and the spread of disease,disease progression is extremely rapid,when obvious symptoms epidemic has been difficult to control.B.xylophilus host plant interaction with the process,destruction by effector host defense system,and the rapid spread of propagation in the host body until the death of the host.Therefore,study of the effects of factors associated with the B.xylophilus pathogenicity to provide a theoretical basis for revealing the pathogenesis of B.xylophilus.Although already completed the genome sequencing,but other plant parasitic nematodes with different strategies,the B.xylophilus gene homologous to find only a small effectors.In this experiment,total RNA was recovered B.xylophilus inoculation pine extract,15 constructed transcriptome libraries,sequenced and analyzed for differentially expressed genes,and then co-expressed and screened GO enrichment analysis candidate effectors and wherein the three candidate effector gene function analysis and verification.The nematodes used in the experiment are the B.xylophilus worm strains Bx2044 and the B.xylophilus strain Bm0617 provided by the Nefu Forest Protection Department nematode&Fungus Laboratory,and the B.xylophilus worm strain Bx 1022 provided by the Chinese Academy of Forestry Sciences.In order to investigate the pathogenicity of the 3 nematode strains,the Pinus thunbergii was selected,P.Massoniana and P.sylvestris var.mongolica as host for inoculation test.The Bx1022,Bx2044 and Bm0617 were inoculated into P.thunbergii,P.massoniana and P.sylvestri.s var.mongolica,and the sensitivity index was calculated to analyze the reproductive capacity of nematode in the sending body.The results showed that Bx1022 to P.thunbergii,P.massoniana and P.sylvestris var.mongolica are the most pathogenic and P.thunbergii is the most susceptible to the disease,the results show that the inoculation of 1 d and 23 d is the two time points that produce a large number of effect factors,while the P.thunbergii and P.massoniana are more susceptible to B.xylophilus and have obvious relationship with each other,Therefore,the Bx1022 inoculated the P.thunbergii and P.Massoniana after the 1 d and 23 d to recover the nematode and extract the total RNA of of the Bx1022 with aseptic water soaked 2 h as CK Group for transcription sequence.By comparing the gene expression level of the inoculation group and the CK group transcriptome,from 15 transcriptome detected a total 17747 gene expression,depending on the difference significant expression analysis(t-text,n=3)results,where Pm-Bx 1022-1d has 10,852 genes differentially expressed not significant,6,895 genes differentially expressed significant;Pm-Bx1022-23d has 9,522 genes differentially expressed not significant,there are 8,225 genes differentially expressed significant;Pt-Bx1022-ld there 10924 gene expression was no significant difference,there are 6,823 genes differentially expressed significant;Pt-Bx1022-23d 8273 has no significant differential gene expression,differential gene expression of 9474 genes significant.By co-expression analysis showed a total of 1,357 genes common in both species exhibit two time points,wherein the 606 gene 2 species 2 time points were significantly upregulated expression,751 genes were down-regulated expression levels.GO enrichment analysis and analysis of the transmembrane domain selected Bx-Efl,Bx-Ef3 is a B.xylophilus and Bx-Ef9 candidate effectors,and functional analysis and gene cloning.In order to further verify the candidate effect factor,gene cloning of Bx-Efl,Bx-Ef3 and Bx-Ef9 by in situ hybridization and RNAi test for gene function analysis.In situ hybridization results showed that Bx-Ef1,Bx-Ef3 and Bx-Ef9 genes in expression in both esophageal glands on B.xylophilus.Effect of nematode factor secretion site in the esophagus gland and gonad and caudal gland and other organs,including esophageal glands as the main organ,the in situ hybridization test results showed that the secretion of parts of the 3 candidate effectors secreted effectors of parts in accordance with the nematode.In order to further verify the gene function RNAi test results show that after RNAi treatment of B.xylophilus corresponding gene expression.Bx-Ef1,Bx-Ef3 and Bx-Ef9 respectively after silencing of normal cultured Bx1022 as experimental control and sterile water as control,RNAi-Ef1,RNAi-Ef3 and RNAi-Ef9 for the RNAi test group was inoculated on lodgepole pine,the results showed that RNAi test group symptom time far later than the pine test even in the control group,experimental control group There has been death situation began to appear symptoms.Bx-Efl,Bx-Ef3 and Bx-Ef9 showed that the gene silencing separately,have influence on pathogenicity of B.xylophilus.By experiments,the function of 3 genes Bx-Efl,Bx-Ef3 and Bx-Ef9 genes associated with pathogenicity of nematode.According to the time of the symptoms and death time of P.thunbergii that RNAi-Ef3 pathogenic RNAi-Ef9>RNAi-Ef1,showed that the effect of each gene on nematode pathogenicity:Bx-Efl>Bx-Ef9>Bx-Ef3.Through the above test results show that Bx-Efl,Bx-Ef3 and Bx-Ef9 gene expression both have characteristics of parts or gene function are in line with the nematode effect factor,so the identification of these 3 genes for gene effect.Through the Q-PCR test for the detection of Bx1022 and Bx2044 were inoculated with P.thunbergii,P.massoniana and P.sylvestris var.mongolica after 23 d expression of Bx-Efl,Bx-Ef3 and Bx-Ef9 genes of the corresponding amount of test results showed that Bx-Ef1,Bx-Ef3 and Bx-Ef9 genes showed in Pm-Bx2044-23d and Ps-Bx1022-23d expression had no significant change(-1<log2<1),the other is significantly increased(log2>1),the expression of Bx-Ef1 at most.The results showed that the pathogenicity of these 3 genes expression and nematode positive correlation.