改造Cry1Ac蛋白C端对转基因水稻抗二化螟虫的影响
发布时间：2018-11-07 17:02
【摘要】：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。
[Abstract]:Cry1Ac gene is one of the most widely used and highly effective insect-resistant genes in the world, but the resistance of Chilo suppressalis is gradually produced through evolution. One of the effective ways to solve this problem is to construct a new Cry protein by changing its structure. In order to analyze whether the C terminal of modified Cry1Ac protein can affect the insecticidal resistance of transgenic crops, the C terminal of Cry1Ja protein was replaced by C terminal of Cry1Ac protein, and the CryFLAc protein was obtained. Cry1Ac gene and cryFLAc gene encoding CryFLAc protein were constructed into pTF101.1-ubi plant expression vector, and transformed into Jilin rice variety Jijing 88 by Agrobacterium tumefaciens. The integration and expression of cry1Ac,cryFLAc gene were determined by PCR,RT-PCR, immunoassay, and the effects of CryFLAc and Cry1Ac proteins on the insecticidal resistance of T1 generation transgenic rice were evaluated by indoor and field insecticidal tests. The results showed that cry1Ac,cryFLAc gene was successfully integrated into rice genome and expressed stably, and transgenic rice with cryFLAc gene reached resistance level, and transgenic rice with cry1Ac gene reached high resistance level. The resistance of transgenic rice with cry1Ac gene was higher than that of transgenic rice with cryFLAc gene. The results showed that the C terminal substitution of Cry1Ja protein for Cry1Ac protein did not improve the insecticidal resistance of transgenic rice. These studies provide a new theoretical basis for the rationalization of artificial design and modification of Cry protein.
【作者单位】： 吉林省农业科学院农业生物技术研究所吉林省农业生物技术重点实验室;东北师范大学;哈尔滨师范大学生命科学与技术学院;吉林农业大学生命科学学院;
【基金】：吉林省农业科技创新工程(吉财教[2012]1072) 吉林省科技发展计划项目(20160520060JH);吉林省科技发展计划项目(20160204033NY);吉林省科技发展计划项目(20140204014NY)共同资助
【分类号】：S435.112.1
[Abstract]:Cry1Ac gene is one of the most widely used and highly effective insect-resistant genes in the world, but the resistance of Chilo suppressalis is gradually produced through evolution. One of the effective ways to solve this problem is to construct a new Cry protein by changing its structure. In order to analyze whether the C terminal of modified Cry1Ac protein can affect the insecticidal resistance of transgenic crops, the C terminal of Cry1Ja protein was replaced by C terminal of Cry1Ac protein, and the CryFLAc protein was obtained. Cry1Ac gene and cryFLAc gene encoding CryFLAc protein were constructed into pTF101.1-ubi plant expression vector, and transformed into Jilin rice variety Jijing 88 by Agrobacterium tumefaciens. The integration and expression of cry1Ac,cryFLAc gene were determined by PCR,RT-PCR, immunoassay, and the effects of CryFLAc and Cry1Ac proteins on the insecticidal resistance of T1 generation transgenic rice were evaluated by indoor and field insecticidal tests. The results showed that cry1Ac,cryFLAc gene was successfully integrated into rice genome and expressed stably, and transgenic rice with cryFLAc gene reached resistance level, and transgenic rice with cry1Ac gene reached high resistance level. The resistance of transgenic rice with cry1Ac gene was higher than that of transgenic rice with cryFLAc gene. The results showed that the C terminal substitution of Cry1Ja protein for Cry1Ac protein did not improve the insecticidal resistance of transgenic rice. These studies provide a new theoretical basis for the rationalization of artificial design and modification of Cry protein.
【作者单位】： 吉林省农业科学院农业生物技术研究所吉林省农业生物技术重点实验室;东北师范大学;哈尔滨师范大学生命科学与技术学院;吉林农业大学生命科学学院;
【基金】：吉林省农业科技创新工程(吉财教[2012]1072) 吉林省科技发展计划项目(20160520060JH);吉林省科技发展计划项目(20160204033NY);吉林省科技发展计划项目(20140204014NY)共同资助
【分类号】：S435.112.1
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