The Induced Expression And Identification Of Resistance To Virue On BN5 And BN10 Of Oilseed Rape And Genetic Transformation Of Arabidopsis Thaliana

Rapeseed (Brassica napus) is one of the most important oilseed crops in China. Virus disease is the main disease in oilseed rape. Turnip mosaic virus(TuMV), Cucumber mosaic virus(CMV), Youcai mosaic virus(YoMV) and Tobacco mosaic virus(TMV) are major virus that infecte oilseed rape. At present, the cultivation of resistant varieties is the best way to resist virus diseases. But we are lacking in highly resistant or immune varieties of rape and the molecular mechanism of resistance to virus is also lagging behind.In this research, we selected two genes (named BN5 and BN10) form oilseed rape genome based on UniGene libraries and cDNA microarray induced by disease defense response signaling molecules on leaves. BN5 and BN10 genes were transformed into Arabidopsis thaliana by way of floral-dip mediated by Agrobacterium tumefaciens; The difference in expression of BN5 and BN10 genes in different tissues were detected by fluorescence quantitative PCR(Q-PCR). Viruses and chemical treatment were also analysed. The main results are as following:1. The plants of Huaza No.6 which is a hypersensitive variety showd mosaic, mottle, blister spot, clear vein and shrinking symptoms infected by TuMV, CMV and YoMV. Majority of positive transgenic plants of pRNAi-BN5 were more susceptible to the viruses than the non-transgenic ones after inoculated with Cucumber mosaic virus (CMV), TuMV and YoMV viruses. Furthermore, transgenic plants were more susceptible to CMV than to YoMV and TuMV.2. Gene expression profiles of transgenic plants transformed with BN5 RNA interference vector were detected by real-time quantitative PCR. The results showed that the expression level of BN5 gene was inhibited. The relative expression of BN5 gene in transgenic plants was only 22%-45% relative to the non-transgenic control. BN5 can be detected in roots, stems and leaves of transgenic plants and non-transgenic ones with the highest expression level in stems and the lowest in roots. TuMV, CMV and YoMV can induce up-regulated expression of BN5 gene in transgenic and non-transgenic plants although their degree is various. The expression of BN5 gene was highest and first reached the peak and then declined rapidly induced by CMV infection than by YoMV and TuMV. This result showed that BN5 gene was hypersensitive to CMV infection. BN5 gene may have important function in resistant to viruses.3. BN10 can be detected in roots, stems and leaves in non-transgenic oilseed rape plants with the highest expression level in roots and the lowest in leaves. Methyl jasmonate (MeJA) and oxalic acid (OA) can induce up-regulated expression of BN10 gene in leaves analyzed by quantitative fluorescent PCR. The results suggested that BN10 gene expression up-regulated firstly and then decline induced by mathyl-jasmonate treated. BN10 had the highest expression level at 24h (2.58 times before the induction), downregulated to 72h (1.13 times before the induction). After oxalic acid induced, BN10 gene expression reached the maximum at 12h (2.5 times before the induction), dropped to the lowest at 72h (only 25% before the induction). These data suggested MeJA and OA could induce BN10 gene expression. It can be inferred tha BN10 may play a role in stress resistance pathway.4. RNA interference vectors and plant over-expression vectors of BN5 and BN10 genes (namely pRNAi-BN5, pG4A-BN5, pRNAi-BN10 and pG4A-BN10) were transformed into Agrobacterium tumefaciens EHA105 by using CaCl2 treated method.20, 10,7 and 10 of TO generation of transgenic Arabidopsis thaliana plants of four vectors were obtained tested by herbicide screen and PCR test.