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Bt cry1Ah基因是本实验室从苏云金芽胞杆菌(Bacillus thuringiensis,Bt)菌株BT8中克隆的一种新型杀虫蛋白基因。在前期工作中已根据植物密码子偏好性对cry1Ah基因进行过一次优化。根据玉米密码子偏好性对cry1Ah基因进行第二次优化,并将两次优化的cry1Ah基因分别构建了pAhmG和pmAhb植物表达载体,利用农杆菌介导法转化玉米自交系综31,分别得到10株和9株阳性转基因玉米植株。通过ELISA、Westernblot检测及生物活性检测,比较不同优化的基因在玉米中的蛋白表达量及抗虫情况。初步结果表明外源基因在玉米植株中可以稳定表达并可以遗传。二次优化基因比一次优化基因在玉米中的蛋白表达量更高,抗虫性更好。表明密码子优化有利于提高外源基因的表达。
The Bt cry1Ah gene is a novel insecticidal protein gene cloned from BT8 strain of Bacillus thuringiensis (Bt) in our laboratory. The cry1Ah gene was optimized once based on plant codon bias in previous work. The cry1Ah gene was optimized for the second time based on the codon preference of maize. The two optimized cry1Ah genes were used to construct pAhmG and pmAhb plant expression vectors respectively. Agrobacterium-mediated transformation of maize inbred lines was used to obtain 10 Strains and 9 positive transgenic corn plants. By ELISA, Western blot and bioactivity detection, compared different optimized genes in maize protein expression and insect resistance situation. Preliminary results showed that foreign genes can be stably expressed in maize plants and can be inherited. The results showed that the secondarily optimized gene expressed more protein and better insect resistance than the first optimized gene in maize. It shows that codon optimization is helpful to improve the expression of foreign genes.