Function Analysis Of Resistance-related Genesagainst Sclerotinia Sclerotiorum In Brassica Napus

The fungal pathogen Sclerotinia sclerotiorum (Lib) de Bary causes a destructive disease, one of the main diseases in oilseed rape (Brassica napus). Based on the microarrays results of gene expression profile in Brassica napus, we used real-time quantitative PCR to detect some disease-related gene expression patterns of the cDNA microarray. Using the microarrays results as a basis, three candidate genes were selected and constructed into RNAi vectors. Further studies on expression patterns after inoculation of S.sclerotiorum and functions of the transgenic plants, the results indicated these three genes were related to disease resistance. The main results are as follows:1. The verification of cDNA chip.The real time PCR results of the selected gene expression patterns were basically consistent with cDNA microarray gene expression. It indicated cDNA chips was reliable.2. Functional analysis of gene AT-29. AT-30 and AT-10 by artificial miRNA.Interference of artificial microRNAs was an effective and high-throughput technology to knock down gene expression for gene function analysis. Based on resistance to S.sclerotiorum cDNA microarray, we selected three genes which were up-regulated when induced by S. sclerotiorum. The three homologous genes in Arabidopsis thaliana were used as target sequences, and then constructed amiRNA interference vectors by overlapping PCR. With the methods of agrobacterium-mediated transformation of A. thaliana inflorescence dip, three or more Tl transgenic plants of each vector were screened with the herbicide and identified as positive transgenic plants by PCR verification. Compared with non-transgenic plants, the expression of target genes in corresponding transgenic plants was inhibited nearly 50% and almost nearly 88%. We detected PDF1.2 (dependent on the jasmonic acid-associated signaling pathway defensin) and PRI (dependent on the salicylic acid-associated signaling pathway) expression level in transgenic plants, the expression of PDF1.2 and PRI in corresponding transgenic plants was respectively reduced 50% and almost nearly 90%. The T2 transgenic plants displayed hypersusceptibility to S.sclerotiorum. These results suggest that the AT-29, AT-30 and AT-10 genes may be involved in plant defense responses, especially resistance to S.sclerotiorum.