Transformation Of Rape(Brassica Napus L) With MSI-99m Gene And Analysis On Disease Resistance Of Transgenic Plants

Rape, as an important interesting crop, can provide protein edible vegetable oil.Its yield and quality has been affected by sclerotium disease seriously. At present control measures are mainly the application of chemical pesticides. But environmental pollution and drug residues could cause damage to human healthy and make the people urgently need a new kind of economic and environmental ways to prevent from sclerotium disease. Plant breeders have always been trying to create stable and high varieties to solve this problem.MSI-99m is a synthetsized Magainin II analogue with high inhibitory effects to a wide spectrum of microbial organisms, including bacteria, fungi and viruses. Synthetsized MSI-99m modified at three amino acid sites, can be stably expressed in plants in vivo without degradation.In this study MSI-99m gene was transferred into Brassica napus cultivars "Zhongyou821" using Agrobacterium tumefaciens-mediated method and the resistance of transgenic lines to rape Sclerotina rot was evaluated. The results are as follows:In total,25independent Kan-resistant putative transformants were regenerated,9of which were confirmed to be PCR-positive. Expression of MSI-99m gene in the7transgenic lines was analyzed using quantitative RT-PCR. MSI-99m was expressed in all the transgenic lines but the levels of transcripts among the transgenic lines were a lot different (line M14is9times of line M10). No expression of MSI-99m was observed in the wild-type control.For evaluating the resistance of the transgenic lines to S. sclerotiorum, three methods, detached leaves test, young plant test and mature plant test, were applied in this experiment. After4days of co-culture with S. sclerotiorum, the disease index of all transgenic lines were lower than that in the wild-type controls, even big difference presented among the transgenic lines. Bioassay studies of disease resistance in young plant stage and flowering period respectively showed several lines expressing MSI-99m developed significantly fewer disease symptoms caused by S.sclerotiorum than the controls. The line M14with the lightest symptom presented most tolerance and could grow normally.Our results demonstrated that high expression level of MSI-99m will be necessary to enhance resistance against Sclerotinia sclerotiorum.