Cloning And Functional Analysis Of Clubroot Resistant Genes In Resynthesized Brassica Juncea L.

Rapeseed is an important oil crop in China.Currently,a series of industrial chains have been formed around its multifunctional uses,which plays an irreplaceable role in China’s economic development.In recent years,clubroot has become an important disease that restricts rapeseed production,and the loss of production presents a trend of increasing year by year.Chemical or biological controls are often accompanied with high cost,less effective and environmental pollution.Cultivating new resistant varieties is the most effective measure to control the clubroot.Therefore,it is of great significance to obtain resistance genes to clubroot and study the molcular mechanism of resistance genes.In this experiment,we used CT19,a resynthetic clubroot resistant mustard,and a yellow mustard in northern Shaanxi,a susceptible rapeseed,as parents.Quantitative trait loci（QTL）for clubroot-resistant gene were identified based a F₂ genetic population through reduced-representation sequencing combining resistance and susceptibility phenotypes.And the identified candidate genes were screened by combining the QTLs and transcriptome sequencing data from resistant and susceptible parents.In order to verify the function of candidate genes,we transformed the candidate genes into model plant,Arabidopsis,and explored the molecular mechanism of candidate genes through transcriptome sequencing of transgenic materials.Finally,some possible molecular mechanisms were tested and verified,providing theoretical basis for cultivating new resistant varieties for clubroot.The main results of the study are as follows:1.Mining of genes for resistance to clubroot:to mining new resistant genes for clubroot,170 F₂ population was constructed with CT19 and the yellow mustard in northern Shaanxi as parents,we obtained three significant QTLs（q CRa3-1,q CRa5-1and q CRb2-1）,q CRa3-1 was 25.09Mb-25.54Mb segments on A03 chromosome,with a phenotype contribution rate of 74.30%,there are 66 annotated genes in this region;q CRa5-1 was 35.28Mb-35.84Mb segment on chromosome A05 with a phenotype contribution rate of 2.5%,with 87 annotated genes in the region;q CRb2-1 was55.69Mb-56.04Mb segments on chromosome B02 with a phenotype contribution rate of 6%,there are 56 annotated genes in the region.The clubroot resistant QTL q CRb2-1 and q CRa5-1 on B02 and A05 are reported for the first time.Combining parental transcriptome data,we screened 22 genes with differentially expression genes in the QTL region of A03 chromosome at 7 or 14 days after inoculation（dai）,and finally two candidate genes with TNL（Toll interleukin-1 receptor-like nucleotide-binding site leucine-rich repeat）structure,Bju VA03G52300 and Bju VA03G52310 was obtained,which may be key genes for resistance to clubroot.2.Functional verification of BjuVA03G52300:By analyzing the sequence of Bju VA03G52300,it was identified that Bju VA03G52300 corresponds to two different genes in Chinese cabbage,Bra019412 and Bra019413.The overexpression of Bju VA03G52300-RS1,a homologous copy of Bra019412 possesses in both resistant and susceptible parents,can alleviate the symptoms of clubroot in Arabidopsis.The overexpression of Bju VA03G52300-R1,a homologous copy that is unique to resistant parents,enhances susceptibility to clubroot in Arabidopsis.The unique copy of Bju VA03G52300-R2,homologous with Bra019413,in disease-resistant parents can also alleviate the symptoms of clubroot.In order to explore the mechanism of Bju VA03G52300-R1 regulating on susceptibility to clubroot,transcriptome profile on wild-type,Bju VA03G52300-R1OE,and Bju VA03G52300-RS1OE lines were analysed at 14dai.The results indicated that multiple aspartic protease expressions were induced in Bju VA03G52300-R1OE lines.In order to explore whether aspartic protease has a regulatory effect on resistance to clubroot,Arabidopsis double mutants asp2-4encoding aspartic protease genes（AT4G22050 and AT1G03220）were constructed using CRISPR technology.asp2-4 enhanced susceptibility to clubroot.Transcriptome sequencing was performed on asp2-4 at 14 dai.We found that there were 2751common differentially expressed genes between asp2-4 and Bju VA03G52300-R1OE after inoculation compared with control.Therefore,we speculate that AT4G22050 and AT1G03220 may be the reasons that affect the sensitivity of Bju VA03G52300-R1 to clubroot.3.Functional verification of BjuA014074:Through analysis of parental transcriptome data,it was shown that several PR proteins in susceptible parent were induced compared with resistant parent.We found that seven of the PR genes were homologous to At CAPE3 in Arabidopsis,and the mutant of At CAPE3 was more sensitive to clubroot.By analyzing the inoculation of BjuA014074 Arabidopsis overexpressing lines,the PR protein family genes were significantly higher in the overexpression of Bju A014074-R than that of wild-type and overexpression Bju A014074-S lines after inoculation of P.brassica,the synthesis of suberin,ROS burst and hormone related genes also shared the simlar trend.In a word,Bju A014074-R may interact with other PR proteins and activate the synthesis of suberin,ROS burst and hormone related genes to enhance resistance to clubroot.We transformed Bju A014074-R into a backbone parent of Brassica napus（530c）in our laboratory and obtained a total of 12 T₀ positive plants.4.Based on a comprehensive analysis of transcriptome data from Bju VA03G52300-RS1OE,Bju VA03G52300-R1OE,Bju A014074-ROE,Bju A014074-SOE,asp2-4,and wild type Col-0 at 14dai,we found that fatty acids,suberin and phytohormones may play an important role in the infection of P.brassica.Therefore,GUS staining was used to analyze the expression patterns of key genes in related pathways,and the results showed that suberin synthesis and fatty acid pathway related genes can be regulated by P.brassica.Through the inoculation test of mutants in the suberin pathway,we found that suberin may be inhibited in the early stage of infection and induced in the late stage of infection.The inoculation test of key genes in the fatty acid pathway showed that fatty acids play a negative regulatory role in P.brassica infection.At the same time,gas chromatography was used to analyze the inoculated mutant roots,the results suggested that linoleic acid（18:2）may be the key fatty acid obtained by P.brassica from the host plant.5.Ethylene plays an important role in the infection process of P.brassica:Mutant inoculation test of EIN3/EIL1,the key transcription factor of ethylene signalling,and exogenous ethylene treatment,showed that ethylene may play a positive regulatory role in the early stage of infection,and play a negative regulatory role in the late stage of infection.Through quantitative analysis and promoter analysis,we found that WRKY75 may be directly regulated by EIN3,which positively regulated the resistance to clubroot.At the same time,WRKY75 may enhance the resistance to clubroot by regulating salicylic acid,jasmonic acid and reactive oxygen species.In our study,by using the mapping,cloning and functional verification of the clubroot-resistant genes in resynthetic clubroot resistant mustard,new QTLs locus for resistance to clubroot disease was excavated,and the molecular mechanism of resistance to clubroot disease was analyzed,which provided provide the genetic basis for improving the efficiency of breeding for clubroot-resistance.