Screening And Functional Verification Of Regulatory Genes For Early Infection Of Tuber Mustard By Plasmodiophora Brassicae

Clubroot is a soil borne disease of Cruciferae crops.Onceplant is infected,the pathogen will cause the root swelling,affect the absorption of plant nutrients,inhibit plant growth and development;when the disease is serious,it leads to the root rot and necrosis,causing the whole plant to die.After the host plant death,the resting spores can survive in the soil for a long time,and the disease will break out in the next year.As a landmark product of Fuling,tuber mustard(Brassica juncea var.tumida)is seriously damaged by clubroot during its cultivation,which seriously affects its sustainable development.In this study,the morphological observation of the root system of tuber mustard in the early stage of infection by Plasmodiophora brassicae was carried out to determine the different stages of infection and root colonization.The transcriptome sequencing analysis of the process of early infection by P.brassicae was carried out,and the differentially expressed genes in the early stage of infection were screened,and two effector genes were identified,and their biological characteristics were analyzed based on the above analysis,the over expression vector was constructed,and its biological function was preliminarily verified by co-transformation with Bax;the prokaryotic expression vectors of two effector genes were constructed,which were induced by IPTG,and the interaction between the effector proteins extracted by ultrasonic crushing and culturable bacteria in the rhizosphere of vegetable press was analyzed,and the effect of inoculating tuber mustard on the pathogenesis of clubroot was analyzed.The main results are as follows:1.Using fluorescence microscope to observe the root of tuberous mustard inoculated with resting spores of Plasmodiophora brassicae,it was found that the spores of Plasmodiophora brassicae adsorbed on the root surface of tuberous mustard one day after inoculation;three days later,the zoospores larger than the resting spores of Plasmodiophora brassicae could be observed,and the boundary released after germination was clear,and the transparent zoospores in the middle adsorbed on the root hair surface of tuberous mustard and began to infect.2.Six samples were sequenced,and each sample produced 6.61 GB of data on average.A total of 9212 genes were detected by transcriptome sequencing,including9047 known genes and 165 predicted new genes;a total of 5522 new transcripts were detected,including 4779 new alternative splicing subtypes of known protein coding genes,166 transcripts of new protein coding genes,and the remaining 577 transcripts of long non coding RNA.The variation range of phred q30 was 85.54-88.48%,and the average q30 base of each sample was 87.02%.3.The CDS sequences of PBRA＿2565 and 6677 were amplified from the cDNA of Plasmodiophora brassicaes.The fusion expression vectors of PBRA＿2565-pTF-GFP and PBRA＿6677-pTF-GFP were constructed with PTF-GFP as the target vector and BamH Ⅰ as restriction sites.The subcellular localization of the two proteins was in the nucleus,cell membrane and cytoplasm.The expressed effector proteins PBRA＿2565 and 6677 had no virulence,could not directly induce the HR response of programmed cell death,and had no inhibitory effect HR response induced by BAX.4.In the experiment,the CDS sequences of PBRA＿2565 and 6677 were amplified from the cDNA of Rhizopus tumefaciens,which were confirmed by DNA man software after sequencing.Then the constructed vectors were verified by colony PCR and enzyme digestion,and the vectors of PBRA＿2565-pGEX-4T-1 and PBRA＿6677-pGEX-4T-1 were successfully constructed.The results of SDS-PAGE analysis showed that the band of PBRA＿2565 target protein was consistent with the expectation,while the band of PBRA＿6677 target protein existed in the precipitation,insoluble and inclusion body,and the expression effect was better when it was induced at 37 ℃ and 25 ℃ for 4 h.5.The bacteria isolated from the rhizosphere of mustard tuber were coated on Nb plate and inoculated with crude protein PBRA＿2565 and 6677.After 4 days,the results showed that PBRA＿2565 had inhibitory effect on strains T8 and T12,which were fictibacillus enciensiensis and massukua sp.,respectively;the crude protein of PBRA＿6677 had inhibitory effect on strains T2 and C19,which were Bacillus megaterium,respectively Megaterium and Pseudomonas baetical.6.After hydroponics,it was found that compared with the control group,the main root of Plasmodiophora brassicaes inoculation solution and effector protein PBRA＿2565 had less swelling,the lateral root was developed,and the whole plant was relatively well developed;compared with the control group,the main root of Plasmodiophora brassicaes inoculation solution and effector protein PBRA＿6677treatment group had little swelling,the lateral root was relatively less,and the whole plant was relatively short,The total weight of swollen root in the treatment group with effector protein PBRA＿2565 and 6677 was higher than that in the control group,and there was no inhibitory effect on Rhizopus.Incidence rate of incidence rate of pickled mustard tuber was 78%,and the disease index was 58.33.Inoculation of rhizo cyst and PBRA＿2566 effect crude protein were the most common diseases,and the disease index was 22.22.The incidence rate of pickled cabbage with the crude protein and PBRA＿6677 effect crude protein was 22%,and the disease index was 19.44.