Organizations And Development-specific Expression Of Disease-resistant Gene Transformation Of Brassica Napus And Disease Resistance

Sclerotinia stem rot is the major disease of Brassica napus in south China.lt is caused by Sclerotinia sclerotiorum which invades into senescence leaves directness or mediated by the petals droped on the leaves. Chitinase degrades fungal cell wall structural polysaccharides to inhibit fungal growth.Oxalate oxidase degrades oxalic acid which is harmful to rape cultivars.We constructed the chitinase gene and the oxalic oxidase gene under a sagl2 promoter respectively and transformed into B. napus so that the genes can only express in senescense leaves. Because of synergistic antifungal properties and specific prometer, combinations of these defense transgenes exhibited tissue and development specific expression and increased levels of protection against infection by fungal pathogens.Genetic transformation with the in planta method has greatly promoted researches of functional genomics in model plant, Arabidopsis. However few of crops were reported being transformed with the method. Via in planta method, genetic transformations of different cultivers of Brassica napus, ie. Zhongyou 821, Xiangyou 13, Ning RS-1, S2501-1, Huashuang 3 were mediated by Arobacterium containing plasmid ApSCO. Under the suitable condition, the floral branches of Brassica napus were dipped in the suspension of Agrobacterium for 10 to 15 days. The 100,000 seeds obtained by in planta method were selected on selective plates supplemented with Kanamycin 270mg.L"1. The plants with Kanamycin resistance were analyzised by PCR and southren blot. The result indicated that the transgenes were transferred into all the five cultivers and integrated into the plants genome.The transformation frequency, which was 0.1% or so, was very close between different cultivers of Brassica napus. The success of in planta method in B. napus showed that the foundation of the higher efficiency transformation method and the genomics study oiBrassic napus had been laid.The disease resistance of the plants which were confirmed by PCR and southern blot were observed by three different method, detached leaves inoculation,oxalic oxidase solution marinaton and detached petals inoculation.The disease index of 19 independent transformants of Tl generation is less then 70% by the detached leaves inoculation and the minimization lesion is 1.32cm( 1.25cm less than the nontrangenic control).The detached leaves inoculation during different developmental period were also observed. The lesions diameter of senescence leaves is 0.64cm less than the young leaves in average. The results indicate the chimeric gene expression was developmental specific and the further confirmation was also obtained by RT-PCR.