Construction Of Suppression Subtractive Hybridization (SSH) Library Of Brassica Juncea Var. Tumida Infected By Plasmodiophora Brassicae And Identification Of Differentially Expressed Genes

Brassica juncea var. tumida Tsen (Cruciferae), known as Fuling mustard, which isspecial vegetable in China. Fuling mustard is the raw material for one of the pickles inthe world. The picked mustard is very popular for their special flavour and nutritionalvalue. Fuling pickle is the largest production scale, the most powerful supportingindustries and competitive industries in Fuling District Area. But in recent years, theyield of Fuling mustard has been seriously affected by clubroot disease caused byPlasmodiophora brassicae, which has caused serious yield losses. In view of B. junceavar. tumida is a kind of meet a single genus living obligatory parasitism fungus,uncultured in vitro, the whole genome is unsequenced, little known about the functionalgene, low homology with others pathogenic fungi and lack of resistance veriety, so it isdifficult to breeding for disease resistance. Breeding of clubroot resistance cultivars(CR) is the top priority for the disease control. But the CR is very scarce and the diseaseresistance may be overcome by new virulent physiological isolates of P. brassicaestrains. In order to breeding resistant veriety, we need to know the interaction between P.brassicae and B. juncea var. tumida and illuminate the pathogenesis of clubroot.In the research we proceed with the molecular interactions between P. brassicaeand B. juncea var. tumida explored differential expression gene of B. juncea after P.brassicae infection, illuminated the pathogenesis of clubroot acquire the diseaseresistance-related genes, and lay the foundation for disease prevention. The research hasmade the following achievement through years of work.1）Suppression subtractive hybridization (SSH) library of B. juncea var. tumidawas establish: The seeds of B. juncea var. tumida were sown with perlite powdersupplemented with MS nutrient solution in a light incubator. The mustard growed tofour true sativa leaves after two weeks and provided enough seedings for inoculationand interactions between pathogen and host. Suppression subtractive hybridization(Clonetech, USA) technology was adopted to construct the forward subtracted libraryusing clubroots as tester and healthy roots as driver. A total of224positive spots wereobtained after being verified by blue-white selection, PCR and cDNA array dot blotting.The cDNA of the positive spots was sequenced, we obtained196high-quality sequenceswith an average length of332bp. The173ESTs were compared to the NT-and EST-database from NCBI using the BLASTN algorithm. Whereas146ESTs (84.4%of the total) were significantly similar to known sequences in plants and1EST similar to P.brassicae after making a blast search against the Nt-and EST-database, the remaining22(12.7%of the total) belonged to P. brassicae sequences and3ESTs origined fromother microorganisms by PCR using the resting spores of P. brassicae as template and1EST was unclear due to the too short sequence. The unique gene sequences were alsocompared to the NR database from NCBI using the BLASTX algorithm. Of these,58unigenes (33.5%of the total) were significantly similar to genes encoding knownproteins，and15ESTs (8.7%of the total) were significantly similar to genes encodinghypothetical proteins. The remaining100ESTs (57.8%of the total) had no similarity toknown protein-encoding genes. The proteins encoded by the genes were involved invarious cell and molecular processes such as stress response and defense regulation,regulation of transcription, energy metabolism, transportation and ribosomal structureand/or function.2）Screening and verification of B. juncea var. tumida SSH library differenitialgene:The expression levels in diseased and healthy roots of four up-regulated genesrandomly chosen in the library were analyzed using RT-qPCR, which was ATP synthaseepsilon chain gene JK616040,40S ribosomal protein gene JK616031, cytochrome-b5reductase gene JK615948and universal stress proteins gene JK615982respectively. Thefour genes which all showed up-regulated in diseased roots compared to the healthyroots indicated a very high reliability of differentially expressed ESTs.3）Dynamic expression analysis of important B. juncea var. tumida differentiallyexpressed genes: We made bioinformatics analysis of the ESTs obtained by the libraryand found seven genes most likely involved in diseases resistance and/or stress response.The expression level of the ethylene responsive transcription factor (ERF) gene wasreduced at10,15dpi in diseased roots compared with the healthy ones and was almosthigher than in healthy roots after20dpi. The transcription of the abscisic acid receptorgene in diseased roots decreased relative to the healthy ones before25dpi and wasmuch higher than in healthy roots at30,40dpi. The amount of transcript of thecalcium-dependent protein kinase5gene (CDPK) in diseased roots was up-regulated indiseased roots compared with healthy ones from10dpi to25dpi and down-regulated at30,40dpi. The expression level of quinone reductase gene decreased in infected rootscompared with the healthy ones between10dpi and30dpi and only higher than inhealthy ones at40dpi. The quantity of transcription of the MYB gene (JK616047) andthe heat shock transcription factor gene (JK616021) was not significantly different in inoculated and control tissues before15dpi and was up-regulated in diseased roots from20to40dpi compared with healthy roots. The expression level of thepathogenesis-related protein gene (PR4) was always higher in diseased roots than inhealthy ones and was significantly increased in diseased roots at40dpi. Among thesegenes, ethylene responsive transcription factor, calcium-dependent protein kinase5,heat shock transcription factor gene and pathogenesis-related protein gene may beinvolved in the plant resistance and abscisic acid receptor, a quinone reductase gene andMyb factor may act in the progress of the disease.4）Measure of hormone induce by B. juncea var. tumida defense response: Aftertreatment with various phytohormones such as abscisic acid (ABA), methyl jasmonate(MeJA), ethylene (ET) and salic acid (SA), the result showed that CDPK(calcium-dependent protein kinase) could be induced by ABA, while be repressed byMeJA, ET, SA and PR4(pathogenesis-related protein4) could be induced by SA, whilebe repressed by MeJA, ET, ABA.5）Cloning and characterization of full-length gene with important function:Full-length cDNA sequence of ERF (ethylene responsive transcription factor) gene andpathogenesis-related protein gene was acquired by RT-PCR and RACE. The proteinstructure and function of deduced amino acid was predicted using biological softwareon line. Analysed by ORF Finder,943bp ERF cDNA sequence was composed of666bpORF and277bp3’noncoding region and encode221amino acids;584bp PR4cDNAsequence was composed of423bp ORF and161bp3’noncoding region and encoded140amino acids. The molecular weight of the encoded ERF protein was24413.1Da and thetheoretical pI was6.35; the molecular weight of the encoded PR4protein was15.5kDand the theoretical pI was8.13. There was no signal peptide and transmembrane domainin the encoded ERF protein and the encoded ERF protein was hydrophilic; there wassignal peptide in the encoded PR4protein and no transmembrane domain in PR4proteinand the encoded PR4protein was hydrophilic. The ERF protein contained a conservedAP2/ERF DNA binding domain and the PR4protein contained a conserved DPBB-1super family structure domain.Phylogenetic tree showed that ERF of B. juncea var. tumida had higher geneticrelationship with brassica oleracae and brassica napus than other crops and PR4of B.juncea var. tumida had higher genetic relationship with Brassica rapa and Sambucusnigra than other crops.On the basis of clone and identify the ERF gene, we have constructed ERF over-expression vector, according to the Agrobacterium tumefaciens-mediatedtransformation (ATMT), we have obtain2arabidopsis Kanamycin resistance plants. ThePCR verification of positive transgenosis plants, the expression analysis of exogenousgene, functional verification by inoculate is ongoing.Conclusion: Based on the research of EST of the B.Juncea var. Tumida SSH library,We screen and identify some important up-regulated pathogenesis-related genes in theearly course of disease which were involved in the plant defense response. Thepreliminary research suggest that ethylene responsive transcription factor (ERF) gene、calcium-dependent protein kinase5gene (CDPK)、pathogenesis-related protein gene(PR4) and heat shock transcription factor gene may be related to plant resistance andabscisic acid receptor gene、a quinone reductase gene、a transcription factor belongingto the Myb family may be involved in the progress of club disease.