| The fungal pathogen Sclerotinia sclerotiorum (Lib) de Bary causes a destructive disease, one of the main diseases in oilseed rape (Brassica napus). Use of resistance varieties is economic and effective measures for control of the disease. However, varieties with high resistance are at present lack, and the molecular mechanisms are beyond understanding, which have limited discovery of key resistance genes for resistance improvement. Therefore, it is significant that, by development or transfer of the innovative technologies developed in the model plant Arabidopsis thaliana, cloning and investigation of functional genes for resistance improvement in oilseed rape.In this study, four resistance-related genes were selected from our Unigene/cDNA librarybased on gene annotation and microarray expression profiling, their complete sequences were cloned if they are not in full length in the library, and their functions were primarily identified by analysis of expression induced by the pathogen or by transgenics (silencing and overexpression). Main results were as follows.1. Cloning of complete BnEIN3 and induced expression by the pathogen. Plant ethylene insensitive 3(EIN3) had been implicated in plant defense as a key regulator in the ethylene signaling pathway. In the study, we cloned the complete sequence a Brassica napus EIN3 (BnEIN3), a 1947 bp DNA fragment which showed 82% identical to Arabidopsis thaliana EIN3. Its ORF encodes 614 amino acids containing an EIN3 domain. BnEIN3 expression in highly resistant oilseed rape variety increased in late stage of S. sclerotiorum infection, and was significantly higher than that in susceptible or middle resistance varieties when BnEIN3 expression was suppressed by S. sclerotiorum in middle resistant and susceptible varieties. The inhibition in the susceptible variety was higher than that in the middle resistance variety. It can be concluded that BnEIN3 may play a role in resistance to S. sclerotiorum in oilseed rape.2. Functional analysis of Brassica napus homologs At3A06,At6C02 and AtEIN3. Interference of artificial microRNAs was an effective high-throughput technology to knock down gene expression for gene function analysis. In this study, functionally unknown Brassica napus genes Bn6C02, Bn3E09, Bn3A06 and BnEIN3 which were obviously upregulated or downregulated in our experiments of cDNA microarray profiling after S. sclerotiorum infection were selected from our library for gene silencing study. Their homologs At3A06(AT2G43945), At6C02(AT2G33860) and AtEIN3(AT3G20770) from Arabidopsis thaliana were used to design target sequences for construction of artificial microRNAs interference vectors amiRNAi-3A06, amiRNAi-6C02 and amiRNAi-EIN3. Then these vectors containing a gene bar conferring resistance to herbicide glufosinate ammonium were transformed into Arabidopsis. Three or more T1 transgenic plants of each vector were obtained with herbicide resistance, and identified as positive transgenic plants by PCR. The expression of At3A06, At6C02 and AtEIN3 were detected by real-time quantitative PCR. The results indicated that compared with non-transgenic plants, the expressions of target genes in corresponding transgenic plants were significantly inhibited. In T2 transgenic plants with At3A06 which was significantly inhibited, test of resistance to S. sclerotiorum showed that the transgenic plants exhibited higher susceptibility than that of the non-transgenic plants, suggesting that At3A06 gene may positively regulated plant resistance to S.sclerotiorum.3. Overexpression of genes Bn6C02 and Bn3E09. Based on our experiments of cDNA microarray profiling, resistance-related genes Bn6C02 and Bn3E09 were selected to construct overexpression vectors, pG6C and pGB3E, for gene function indentification through transgenic method. Positive transgenic plants identified by herbicide resistance and PCR were obtained and provided for next step study of the genes in our lab. |
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