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以8个大白菜亲本材料无菌苗为实验材料,从近生长点处切下无菌苗子叶,在M S+1 m g/L 2,4-D培养基上预培养48 h,以携带豇豆胰蛋白酶抑制基因(该基因可赋予大白菜抗菜青虫和小菜蛾等昆虫的抗性,Cow peaT ryps in Inh ib itor gene,CpT I)的质粒pB inΩSCK为载体,通过OD600值约0.3~0.4的根癌农杆菌LBA 4404侵染3m in,在M S+2 m g/L BA+0.5 m g/L NAA+5 m g/L硝酸银+2%蔗糖+8 g/L琼脂培养基上共培养48 h,将其转到含有卡那霉素和头孢霉素的筛选培养基中,约4周出现大量转化体,经分子杂交检测,证明了豇豆胰蛋白酶抑制剂基因整合到了大白菜基因组中,室内和田间的抗虫试验也表明,豇豆胰蛋白酶抑制剂基因赋予了转基因大白菜较强的抗虫能力.本研究还对影响农杆菌遗传转化效率及植株再生的各种因素进行了优化.
Aseptic seedlings of eight Chinese cabbage parents were used as experimental materials, and the leaves of sterile seedlings were excised from near growth point and pre-cultured on M S + 1 mg / L 2,4-D medium for 48 h to carry cowpea Trypsin inhibitor (the gene can confer resistance to insects such as Chinese cabbage Pieris rapae and Plutella xylostella, Cowpea ryps in Inhb gene, CpT I) plasmid pBinΩSCK as a carrier, by OD600 value of about 0.3 ~ 0.4 Agrobacterium tumefaciens strain LBA 4404 was infected for 3 mins and co-cultured for 48 h with M S + 2 mg / L BA + 0.5 mg / L NAA + 5 mg / L silver nitrate + 2% sucrose + 8 g / L agar , Which was transferred to kanamycin and cefotaxime screening medium, a large number of transformants appeared in about 4 weeks. The results of molecular hybridization showed that cowpea trypsin inhibitor gene was integrated into Chinese cabbage genome, Insecticidal tests in the field also showed that the cowpea trypsin inhibitor gene confers strong resistance to transgenic Chinese cabbage.The factors that affect the efficiency of Agrobacterium transformation and plant regeneration are also optimized.