| Oilseed rape (Brassica napus) is the third largest oilseed crop in the world and provides approximately 13% of the world’s supply of vegetable oil. However, oilseed rape can be seriously infected by Sclerotinia sclerotiorum, which causes the rotting of stems and leaves, resulting in tremendous yield loss worldwide. Due to the lack of endogenous resistance resources in cruciferous plants, little progress has been achieved in the breeding of Sclerotinia resistance in oilseed rape by applying traditional breeding methods. Chemical control of S. sclerotiorum is expensive, often ineffective, and causes environmental pollutio. Therefore, the transgenic modification of the crop shows promise for the production of new oilseed rape lines with greater resistance to Sclerotinia.In previous work, LJAMP2, an antifungal protein gene, was isolated from the seeds of motherwort (Leonurus japonicus) and LJAMP2 overexpression significantly improved the resistance to S. sclerotiorum. At the same time, "Gene-deletor" system successfully deleted the exogenous gene in tobacco and addressed the biosafety of the transgenic plants. In the present study, we first constructed a plant expression vector pBGP-FLP-LTP-GN contains LJAMP2 and then "Gene-deletor" system was transferred into oilseed rape via Agrobacterium-mediated transformation. "Gene-deletor" system might provide a technique to control GM crops that may be applied to the field production. The results are as follows:1、Construction of plant expression vectorsLJAMP2 was amplificated by PCR and inserted into pCXSN vector. The resultant pCXSN-LJAMP2 and pBGP-FLP-GN were digested with Hind III and EcoR I, then ligated together and generated the vector pBGP-FLP-LTP-GN. After sequencing, the plant binary vector pBGP-FLP-LTP-GN was transformed into Agrobacterium EHA105.2、Transformation and PCR identification of transgenic plants We transferred LJAMP2, pBGP-FLP-GN and pBGP-FLP-LTP-GN into B. napus via Agrobacterium-mediated methods, espectively, and a large number of putative plants with kanamycin resistance were obtained. GUS staining and PCR anaylsis showed that the foreign gene had been integrated into the genome of the transformants. Reverse transcription-PCR (RT-PCR) confirmed that expression of LJAMP2 and FLP genes was detected in transgenic plants.3、Disease-resistance assays in transgenic LJAMP2 oilseed rapeIn vitro experiments revealed that the mycelial growth of S. sclerotiorum was significantly inhibited when supplied with crude leaf extracts from transgenic LJAMP2 plants. Furthermore, in vivo studies showed that transgenic oilseed rape plants that overexpressing LJAMP2 had enhanced resistance to S. sclerotiorum. Semi-quantitative reverse transcription PCR analysis showed that the LJAMP2 gene was transcribed in the transformed plants. In addition, the transgenic LJAMP2 plants constitutively activated the defense-re la ted gene PR-1 and increased H2O2 production, but did not enhance PDF1.2 expressio. Our results suggested that the constitutive expression of the LJAMP2 gene from motherwort seeds might be exploited to improve the resistance of oilseed rape against S. sclerotiorum.4、Deletion efficiency analysis of transgenic pBGP-FLP-GN oilseed rape Compared with the wild-type and transgenic LJAMP2 rapeseed plants, GUS staining showed that most of these pollen grains with the vector pBGP-FLP-GN had yellow color, suggested that all forigen genes were excised from transgenic pollen grain carrying "Gene-deletor" system. Furthermore, statistic analysis showed the deletion efficiency in trasnsgenic pollen with pBGP-FLP-GN was 82.51%. All the results suggested that the delection efficiency in transgenic oilseed rape plants containing vector pBGP-FLP-GN reached an apparent deletion effect. |
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