| Cabbage(Brassica oleracea var.capitata L.),belongs to Cabbage species of Brassica in the cruciferous family.It is an important vegetable crop in our country.Clubroot disease is a worldwide soil-borne disease caused by clubroot(Plasmodiophora brassicae Woron.),which seriously threatens the growth of all cruciferous plants.Once the field soil is infected with clubroot disease,the pathogen will remain in the soil for a long time and endanger the sustainable and safe production of cruciferous vegetables.We have crossed the highly resistant cabbage hybrid "GZ87" and the highly susceptible cabbage inbred line "263" to obtain the CP population.The extremely disease-resistant pool and the extremely susceptible pool were constructed by combining artificial inoculation identification and molecular markers identification.The resistant and susceptible pools were performed genome re-sequencing and transcriptome sequencing at 0,4,7 and 14 days after inoculation.Finally,through comprehensive analysis,eight candidate resistance genes were abtained in the C7 chromosome cabbage.Based on this research,the following work was carried out in this experiment:(1)The screening and bioinformatics analysis of the candidate resistance genes to clubroot in cabbage.(2)Using Arabidopsis homologous mutants to carry out functional verification of candidate resistance disease genes.(3)Obtaining Arabidopsis plants for overexpressing candidate genes.(4)Transcriptome sequencing of the candidate gene pirl8 Arabidopsis mutant and Arabidopsis wild-type strains 14 days after inoculation and no inoculation,analysis of gene expression differences in the Arabidopsis and resistance metabolic network.The main research results of this experiment are as follows:(1)Screening of candidate resistance genes to clubroot in cabbage: Through the analysis of the transcriptome data and resequencing results of the constructed resistant and susceptible pools in cabbage,eight differentially expressed genes(DEGs)were selected from the QTL region of cabbage C07.There were different expression patterns among the resistant and susceptible pools,and the expression results of eight genes were basically consistent with the transcriptome data by q RT-PCR.The eight DEGs are Bol037115,Bol042270,Bol037119,Bol024340,Bol024384,Bol042144,Bol042142 and Bol042207.The q RT-PCR test results of eight candidate genes showed that the expression of the genes Bol037115 and Bol042270 increased in the resitant pool and dropped in the suscepible pool.The gene Bol042207 had down-regulated expression of different degree in the resistant and susceptible pool.The expression of the gene Bol024384 did not change significantly in the resistant pool,but was up-regulated in the susceptible pool.Moreover,the expression of the remaining four genes in the resistant pool did not change significantly,and they were down-regulated in the susceptible pool.The trend of gene Bol024340 in the resistant pool firstly dropped and then increased,and was down-regulated in the susceptible pool.By analyzing the expression and function annotation,the gene Bol037115 and the gene Bol042270 were selected for candidate resistant genes.(2)Bioinformatics analysis of candidate resistance genes: c DNA sequence of Bol037115 is 507 bp in length,and the gene sequence is completely consistent beween resistant and susceptible varieties,and encoding 168 amino acids.It was named BoMARD2 according to the gene function annotation.Sequence analysis revealed that it carries a zinc lipid structure.Specifically,the zinc finger of FCS type C2-C2 is composed of a shared cysteine sequence.Its function was predicted to relate with the regulation of gene expression;the c DNA sequence of Bol042270 is 1185 bp in length,and the gene sequence is completely consistent between resistant with susceptible varieties,and encoding 394 amino acids.Because its Arabidopsis homologous gene is PIRL8,it is named BoPIRL8.Sequence analysis revealed the existence of a leucine-rich domain.It is in line with the common domain characteristics of most disease-resistant R genes.Its function is related to other transcription factor recognition or signal transduction.(3)Functional verification of candidate resistant genes BoMARD2 and BoPIRL8:a wild-type Arabidopsis,an Arabidopsis mutant with BoMARD2 homologous gene(SALK-125677C)and two BoPIRL8 homologous Arabidopsis mutants(SALK-095144C),SALK-001827C)material was inoculated with No.4 clubroot race.The results of the inoculation showed that the growth of the mutants was better than the wild type,and the incidence of the mutant plants after inoculation was significantly heavier than the wild type.The disease index(DI)of wild-type Arabidopsis thaliana was 48.3,the disease indexs(DI)of the BoPIRL8 homologous Arabidopsis mutant was74.3 and 71.7,respectively,and the disease index(DI)of the BoMARD2 homologous Arabidopsis mutant was Is 68.3.The result of one-way analysis of variance is extremely significant.(4)Obtainment of Arabidopsis plants overexpressing disease resistance genes BoMARD2 and BoPIRL8: Overexpression vectors of BoMARD2 and BoPIRL8 were constructed and successfully introduced into wild-type Arabidopsis by inflorescence transfection.(5)Research of molecular network for disease resistance: on 14 days after inoculation of Arabidopsis thaliana mutants with BoPIRL8 homologous gene and wild-type,transcriptome sequencing was performed for three repetitions,and a total of12 samples were sequenced,and 76.86 Gb Clean Data was obtained.Each sample was Clean.Data reached 5.84 Gb or more,and the percentage of Q30 bases was 95.44%and above.A total of 26,853 expressed genes were detected in this analysis,including26,616 known genes and 237 new genes;55,729 expressed transcripts,including45013 known transcripts and 10716 new transcripts.Sequencing results showed that compared with wild-type pirl8 Arabidopsis mutants of inoculation,GO analysis showed that Arabidopsis mutants triggered related defense responses and regulation of stress,and also stimulated a large number of responses related to reactive oxygen species.KEGG metabolic pathway analysis showed that it is speculated that the gene PIRL8 may be related to the five pathways of plant hormone metabolism pathway,cyanoamino acid metabolism pathway,glutathione metabolism pathway,phenylpropane metabolism pathway and MAPK cascade regulatory network.It is mainly related to seven types of substances including reactive oxygen species,lignin,auxin,cyanide,glutathione,MPK4 and PP2 C.Under the induction of PIRL8,the expression of MPK4 and PP2 C proteins of the MAPK pathway are inhibited,so that disease-resistant related proteins and plant resistance can proceed immediately,and at the same time,the inhibition of reactive oxygen species in certain cells that need to synthesize lignin in the roots is relieved.In some cells that have been infected with clubroot,the respiratory burst promotes the programmed death of the infected cells.PIRL8 can also promote the maintenance of GSH content to regulate the active oxygen content in cells in a timely manner to avoid unnecessary "injury".In terms of hormones,it can reduce the response level of growth hormone by inhibiting the transmission of growth hormone signal GH3,and inhibit cell overgrowth.The endogenous hydrogen cyanide of Arabidopsis also plays a certain role in the path of disease resistance.According to the results of other researchers,it is predicted that it can lead to programmed cell death to achieve the defense function. |
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