豇豆胰蛋白酶抑制基因改良大白菜抗虫性的研究

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MS＋1 mg/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,Cowpea Trypsin Inhibitor gene,CpTI）的质粒pBinΩSCK为载体,通过OD600值约0.3～0.4的根癌农杆菌LBA4404侵染3min,在MS＋2 mg/L BA＋0.5 mg/L NAA＋5 mg/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.

Insect Resistance of CpTI Gene-improved Chinese Cabbage

Cotyledons were cut from aseptic seedlings of 8 Chinese cabbage materials near their growth points and pre-cultured on the medium of MS+1 mg/L 2,4-D for 48 hours. Then they are infected by A.tumefaciens LBA4404 whose OD600 ranged within 0. 3 ～ 0. 4 for three minutes by employing plasmid pBINΩSCK carrying CpTI gene and then cultured on the medium of MS+2 mg/L BA+0.5 mg/L NAA+ 5 mg/L silver nitrate+2 % sucrose+8 g/L agar for 48 hours. After this, they were transferred to the screening medium containing kanamycin and cefetaxime and produced a lot of transformants four weeks later. The transformants were proved by molecular hybridization that CpTI gene had been integrated in the genome of Chinese cabbage and indoor and field experiments also proved that CpTI gene had bestowed Chinese cabbage with a strong insect resistance. In addition, this study optimized various factors concerned with the agro bacterium transformation efficiency and plant regeneration.