Construction Of Antisense Bivalent TuMV-Nib And LFY Gene Of Plant Expression Vector Into Spring Chinese Cabbage By Agrobacterium Tumefaciens

Althoung Chinese cabbage is important to vegetable crop of orgin of our country produce,but internal spring Chinese cabbage start breeding later,the greater part use seed from Japan and Retublic of Rorea for in production.Even if there is determinate antrireversion force(late-bolting),it don't anti-morbus virosus.In Chinese Cabbage produce,antephase bolting and serious morbus virosus of later period already is anxious wait for solving issue.In breeding late-bolting and anti-morbus virosus of Chinese cabbage variety adopting method of convention breeding,the time limit is long and the efficiency is low,but it hard to be given to both disese resistance.Therefore,using the gene engineering technology to improve the disease resistance of Chinese cabbage has a great importance of economic values and current significance.The foundation of clone TuMV-Nib and LFY gene have not only constructed double valence anti-expression plant vector,through regeneration and transformation the research of tissue culture.Transgenic plants have been got.The main results are as follows:1。The utilization of clone TuMV-Nib and LFY gene have obtained the engineering clone,through enzymatic,cleavage,ligation and transforrnation,PCR-amplification of recombinant plasmid and verification of enzymatic cleavage,so a plant expression vector of antisense with TuMV-Nib and LFY gene was constrcucted.The method of adopted fizing and thawing transformate LBA4404 and gain engineering bacteria.2.The high frequent regeneration was limited by the genotype.During best differentiation culture medium:Ms+6-BA 2mg/L+NAA0.5mg/L+TDZ0.1mg/L+AgNO₃4mg/L,5～6d aseptic plantlets with cotyledon of stem cultivate in the light/dark at 25℃,16/8hr.The differentiation frequency of indefinite buds average above 85%,especially 'Qing 3 fushan'.3.The agrobacterium-mediated transformation which contained 10 mg/L Km and OD of Agrobacterium is 0.5 apply for Chinese cabbage genetic transformation.Obtaining the best advantageous transformation condition:in the first cutivating the explant with 2～3d in advance, in the second being infected by the agrobacerium-medium in 2mine,in the third being cultivated in 4～6 weeks,in the last taking seedling to the root culture medium.In the final have obtained eleven anti-plants,especially "Qing3 Fushan',obtaining transformation efficiency is 3.3‰.