| Rapeseed (Brassica napus L.) is an important oil crop, providing a wealth of edible oil with high nutritional values for human.However, rapeseed often suffers from a variety of diseases, including sclerotinia, viral diseases, downy mildew, white rust and bacterial black spot, soft rot, black spot and so on.In every year plant diseases may cause a global crop production loss by 12%. Downy mildew is an important disease in Brassica napus L. which may occur in the whole growth period. In some areas, downy mildew is a serious disease in rapeseed.RPW8, a broad-spectrum disease resistance gene from Arabidopsis thaliana, confers good resistance to powdery mildew, downy mildew, and other viral diseases. The RPW8 locus contains two resistance genes:RPW8.1 and RPW8.2. Both of the genes RPW8.1 and RPW8.2 can express resistance to downy mildew in Arabidopsis thaliana. It is known that Arabidopsis thaliana and Brassica napus L. are classified in the same family Brassicaceae, sharing a common ancestor. They have a close genetic relationship.In present study, the RPW8.1 gene in a plasmid vector was transformed into Brassica napus L. by floral-dip method. A number of transgenic plants were obtained and tested with the PCR method. They were also inoculated with downy mildew pathogens to observe the phenotypes of the transgenic lines. The transcription levels and the sub cellular expression locations of the gene were analyzed.The main results obtained are as the following:1.A broad-spectrum disease resistance gene RPW8.1 from Arabidopsis thaliana was used to transform Brassica napus L. by floral-dip method and transgenic plants were successfully obtained. It is shown by a double primer PCR detection that the exogenous resistance gene RPW8.1 has been successfully introduced into the Brassica napus genome.2. According to the preferrable ratio of transformation suspension:Ms medium+sugar (15%mg/L) +6BA(0.01mg/ml)+silwet-77 (0.05%), added with the Agrobacterium containing the plasmid vector, the best floral-dip time was at about 10:00. The material with the highest transformation rate was MY15-1-1. The transformation rate was very close to 2‰. processing times higher than other materials and other processes. When processing materials are different but processing times are the same, the conversion rate is not the same for each material, indicate the floral-dip method for the conversion of different materials is not the same, there is no stable conversion ratio; Further, the processing results of different times in the same material also shows the effect of three inflorescences dip higher than twice and one dip, dip repeatedly can significantly increase the conversion ratio of the material.3. Phenotypic observation of the transgenic plants after inoculation of downy mildew indicated that some transgenic plants were resistant to downy mildew, but they have varable degrees of resistance. Some had high resistance, some had low resistance, and some transgenic lines did not produce any resistance to downy mildew. The causes of these phenomena may be that the exogenous gene was inserted into different positions in the genome, resulting in significantly different transcription levels of exogenous gene, even silencing or no expression of the exogenous gene.4. Resistant transgenic plants had higher RPW8.1 expression levels. Total RNA was extracted after the first strand cDNA reverse transcription by qRT-PCR analysis, indicating that the RPW8.1 gene transcription and translation of the protein can be expressed, offering the resistance of the transgenic lines.5. Differences in growth of downy mildew in the wild-type and the transgenic lines were analyzed and compared at the cellular level after one week of inoculation under microscope. It was found that RPW8.1 gene in rapeseed tissue cells produced a significant inhibition to downy mildew. RPW8.1 gene by inhibiting the growth of downy mildew haustorium, cut off sources of nutrients and moisture for downy mildew, eventually leading to death of downy mildew, so that the transgenic lines exhibited resistance to downy mildew.6. After the test of T2 generation of the transgenic lines it was shown that the segregation ratio of the transgene RPW8.1 was 3:1, suggesting that the copy number of the transgene into B. napus was a single copy. |
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