Zhou Zongliang, Lin Zhimin, Geng Lili, Su Jun, Shu Changlong, Wang Feng, Zhu Yanming, Zhang Jie
College of Life Sciences, Northeast Agricultural University, Harbin 150030, Heilongjiang, China.
Sheng Wu Gong Cheng Xue Bao. 2012 Oct;28(10):1184-94.
cry1Ah1, one of holo-type cry genes, cloned in this laboratory from Bacillus thuringiensis strain has been patented in China, and it encoded a protein with strong insecticidal activity against certain lepidopteran insect pests, such as Chilo suppressalis. cry1Ah1 gene is exhibiting good application prospects. In order to improve the expression level of cry1Ah1 gene in rice, and investigate the effect of codon usage preference of gene expression, we designed five different optimized schemes for cry1Ah1 insecticidal critical fragment in accordance with bias of rice codon, to improve G+C content, removed the shear signal and unstable factors. Optimized cry1Ah1 genes were transformed into Escherichia coli Rosetta (DE3) respectively, and 65 kDa polypeptides was expressed normally in inclusion body separately. All of these expressed polypeptides showed insecticidal activity against 2nd-instar larvae of Plutella xylostella and neonate of Chilo suppressalis. After transformation with modified cry1Ah1 genes into Var nippobare, the transgenic rice seedlings were detected by PCR, the positive rate containing target gene was more than 87%. Afterwards, the results of real-time RT-PCR and ELISA assay indicated that the highest expression level of five modified cry1Ah1 genes was that using the highest frequent codons. Average expression amount of Cry1Ah1 polypeptides was 0.104% of total soluble proteins from the positive transgenic rice.
cry1Ah1是本实验室从苏云金芽孢杆菌菌株中克隆的全型cry基因之一，已在中国获得专利，它编码一种对某些鳞翅目害虫具有强杀虫活性的蛋白质，如二化螟。cry1Ah1基因展现出良好的应用前景。为提高cry1Ah1基因在水稻中的表达水平，并研究基因表达的密码子使用偏好性的影响，我们根据水稻密码子偏好性设计了五种不同的cry1Ah1杀虫关键片段优化方案，以提高G+C含量，去除剪切信号和不稳定因素。将优化后的cry1Ah1基因分别转化到大肠杆菌Rosetta(DE3)中，65 kDa多肽分别在包涵体中正常表达。所有这些表达的多肽对小菜蛾二龄幼虫和二化螟初孵幼虫均表现出杀虫活性。将修饰后的cry1Ah1基因转化到日本晴中后，通过PCR检测转基因水稻幼苗，含目标基因的阳性率超过87%。随后，实时RT-PCR和ELISA检测结果表明，五个修饰后的cry1Ah1基因中表达水平最高的是使用最频繁密码子的方案。Cry1Ah1多肽的平均表达量为阳性转基因水稻总可溶性蛋白的0.104%。
