Identification Of Virus Strains, Resistance-related Genes And Transgene Drift In Oilseed Rape

Virus disease is one of the main diseases in oilseed rape (Brassica napus). It can cause serious economic losses in the epidemic years. Viruses that infect oilseed rape can be divided into different strains and the virulence is different among these strains. Use of resistance varieties is economic and effective measures for control of the disease. However, varieties with high resistance or immunity to viruses are at present lack. Molecular mechanisms of resistance to viruses are beyond understanding and there is a demand for discovering key resistance genes for resistance improvement. So to find the key resistance genes and to breed new transgenic oilseed rape varieties are the effective approaches to control the virus diseases. However, one of the most pressing issues surrounding transgenic oilseed rape cultivation is the potential impact of the genetically modified (GM) genes entering neighboring non-GM oilseed rape plants, related weeds or closely related wild relatives. Although transgenic oilseed rape is not commercialized in China, many organizations are developing transgenic oilseed rape for commercialization and China imports a large amount of transgenic rapeseed from Canada. Therefore there is an urgent need to conduct research and to accumulate data on transgenic environmental risks under the China natural environments. The lack of this line of research is clearly evident in China.In this study, we surveyed the viruses in oilseed rape fields and separated several isolates to identify the resistance of 13 cultivars and 14 germplasms of oilseed rape to the virus. The virulence of these isolates was tested to definitude the variance of viruses infected oilseed rape varieties. Three genes with putative function for disease resistance were selected from a resistance-related oilseed rape UniGene library and were transformed into oilseed rape in order to elucidate experimentally gene functions and their value for breeding. Then pollen-mediated gene flow from herbicide-resistant transgenic oilseed rape variety to the Chinese non-transgenic ones was investigated under the China's environmental conditions. Gene flow rates and factors affected pollen dispersal were monitored. These results provided scientific evidence for the effective commercialization and safe management of transgenic oilseed rape. The main results obtained are listed as follows:1. The main virus that infected oilseed rape and the variance of its related isolates were confirmed. The virus diseases of oilseed rape were surveyed in 12 cities and counties of Hubei and Anhui Provinces. Serological diagnosis of the viruses revealed that the proportion of Turnip mosaic virus (TuMV), Cucumber mosaic virus (CMV) and Youcai mosaic virus (YoMV) was 90.7 %, 8.9 % and 0.8 %, respectively, in 258 diseased samples tested by indirect enzyme linked immunosorbent assay (ELISA). Resistance of 13 cultivars and 14 germplasms in oilseed rape to TuMV were evaluated by artificial inoculation. 13 cultivars, which were currently applied in oilseed rape production, were all susceptible to TuMV. However, their disease indexes were lower than control cultivar Zhongyou 821. Among 14 oilseed rape germplasms tested, 3 showed high or medium resistance to TuMV. The results of TuMV isolates infected oilseed rape varieties showed that there were significant differences in the virulence of tested isolates. However, the virulence of TuMV isolates was not related with their origin places. Both strong and weak virulence of isolates were found in every sampling place. We obtained 18 cp gene sequences of TuMV isolates. Sequence comparison and phylogenetic analysis indicated that sequence identity of 17 cp gene was more than 97% with representative of MB group and all belonged to MB group. An isolate WRS1 originating from radish had identity of 95.4%~98.7% with representatives of MR group and belonging to MR group. Phylogenetic analysis indicated that another radish isolate WRS2 formed as ingle branch in MB group, possibly WRS2 was the offspring of a recombination between MR and MB groups.2. A rod-shaped virus infected oilseed rape was identified as a strain of YoMV. In China, a rod-shaped TMV-like tobamovirus infecting oilseed rape was wrongly considered as Tobacco mosaic virus (TMV) for a long period. Recently, a tobamovirus had been isolated from oilseed rape in China. Its characteristics on biology, seriology and molecular biology were studied. It had strong infectivity to Cruciferae but less to Solanaceae tested by artificial inoculation. Its host range was different from TMV. Purified virions were rod-shaped and particle was 295 nm in length and 15 nm in width. Virion morphology of the virus was consistent with those of the tobamoviruses. However, the virus was serologically distinguishable from TMV. Sequence analysis of the 3' terminal 2283 bp showed that sizes of mp, cp genes and 3' non-coding region (NCR) were 798 bp, 474 bp and 235 bp, respectively, with overlapped 77 bp between mp and cp genes. Sequence identity of mp, cp genes and 3' NCR is all less 60% to those of TMV, higher than 80% to those of Tobamoviruses infecting Cruciferae plants, and higher than 90% to those of YoMV strains. So the virus was belonged to a strain of YoMV and named as YoMV-Wh. The study of characteristics of YoMV-Wh can help people take measures to control the virus and provided evidence for the right nomenclature and taxonomy of rod-shaped tobamoviruses that infected oilseed rape.3. Transgenic oilseed rape plants which resistant to the viruses were obtained in this study. Three genes with putative function for disease resistance were selected from oilseed rape UniGene library and named as BN1, BN5 and BN10, respectively. Complete sequence of non full length gene was cloned by using 5'-RACE Kit. Plant over-expression vectors and RNA interference vectors of three target genes were constructed and transformed into B. napus L. by using the pollen tube-mediated methods at 8-12 h after artificial pollination. Four transgenic plants of three target genes were obtained tested by herbicide screen, PCR test and southern blot identification. In the progeny of transgenic oilseed rape, a male sterile mutant with yellow-white coloured flowers and a plant chimera with yellow-white double coloured flowers were found. Gene expression profiles of transgenic T1 generation 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, but the inhibited degrees were different in different transgenic plants. The lowest relative expression of BN5 gene in transgenic plants was only 20% relative to the non-transgenic control. The herbicide resistant gene bar and target gene BN5 were co-segregated in transgenic T1 generation plants. Majority of positive transgenic plants 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. According to this characteristic we concluded that BN5 gene had the natural resistance to the viruses and has significant value for breeding.4. In this report the rate and the distance of pollen-mediated gene flow were first reported under the China natural environments. Herbicide resistance bar gene was used as selectable marker to test the pollen dispersal. The results indicated that pollen dispersal occurred up to 2000 m and its rate sharply decreased as the distance from the pollen source increased. However, the rate of long-distance pollen dispersal from 33.5 m to 2000 m was less than 0.015% and did not present a gradual decrease, indicating the randomization of dispersal and pollination. Most pollen dispersed within a 4.5 m area around the transgenic pollen source plot with a maximum of 1.19% at the sampling site of 1.4 m. Wind direction significantly affected the direction and the distance of pollen dispersal. However, the number of potentially pollinating bees was not correlated with direction and distance from the pollen source plot, suggesting that bee density and distribution would not contribute to the differences in pollen dispersal among different directions. This study is the first report on research in the rate and the distance of pollen-mediated gene flow under the China's environmental conditions. The results provided scientific evidence for the effective commercialization of transgenic oilseed rape and its safe management and also provided important data on pollen spatial distribution pattern.