改造Cry1Ac蛋白C端对转基因水稻抗二化螟虫的影响

Influence of the C-terminal Fragment of Cry1Ac Protein Transformation on Transgenic Rice Resistance to Striped Stem Borer

二化螟虫的泛滥严重影响水稻的产量,cry1Ac基因是目前世界上应用最广泛和最高效的抗虫基因之一,但二化螟虫通过进化会逐渐对其产生抗性。通过改变蛋白结构来构建新的Cry蛋白是解决这一问题的有效途径之一。为分析改造Cry1Ac蛋白C端能否对转基因作物抗虫性产生影响,本研究采用Cry1Ja蛋白的C端替换Cry1Ac蛋白的C端,重组获得CryFLAc蛋白。将cry1Ac基因和编码CryFLAc蛋白的cryFLAc基因分别构建到pTF101.1-ubi植物表达载体上,利用农杆菌介导方法转化到吉林省水稻品种吉粳88中;采用PCR、RT-PCR、免疫检测试纸条检测的方法确定cry1Ac、cryFLAc基因整合和表达情况;通过室内和田间抗虫性测试评价CryFLAc和Cry1Ac蛋白对T1代转基因水稻抗虫性的影响。研究发现:cry1Ac、cryFLAc基因均成功整合到水稻基因组中,并稳定表达;转cryFLAc基因水稻抗二化螟水平达到抗性级别,转cry1Ac基因水稻达到高抗级别;转cry1Ac基因水稻二化螟抗性高于转cryFLAc基因水稻。结果表明:Cry1Ja蛋白的C端替换Cry1Ac蛋白的C端不会提高转基因水稻的抗虫性。上述研究为人工设计合理化改造Cry蛋白提供了新的理论依据。

The rice stem borer （Chilo suppressalis Walker） is one of the most detrimental pests affecting on rice crops. Although crylAc gene had been explored as the most extensive and efficient way to control this pest, the evolution of the rice stem borer would gradually produce resistance. In oder to analysed whether the C-terminal fragment of CrylAc protein had a marked impact on insect resistance of transgenic crops, changing the protein structure to construct a new Cry protein was one effective way. In this research we took the C-terminus of CrylJa protein place of C-terminus of CrylAc protein, to obtained a novel protein CryFLAc. CryFLAc gene, coding CryFLAc protein, and c rylA c gene were constructed into pTF101.1-ubi plant expression vector respectively, and then transformed into Jilin rice cv. Jijing 88 by using agrobacterium mediated method. The integration and expression of the crylA c and cryFLA c genes were determined by PCR, RT-PCR and immunoassay test strip test, the effects of CryFLAc and CrylAc protein on insect resistance of T1 generation transgenic was tested by indoor and field insect resistance test. The results revealed that crylA c and cryLA c genes were successfully integrated into the rice genome and stably expressed, the cryFLAc transgenic rice exhibited resistance against striped rice borer larvae, but lower resistance than crylA c transgenic rice lines. Replacement C-terminus of CrylAc protein counld not improved the insect resistance oftransgenic rice. It has improvd enlightenment significance for developing new high resistant protein.