3个抗虫耐盐基因载体对烟草的遗传转化及表达分析

发布时间：2018-08-30 16:05

【摘要】：病虫害和土壤盐渍化是影响烟草生长发育的重要因素,转基因技术已成为改善这一现状的有效手段。本研究将部分改造的Bt基因Cry1Ac基因、Cry3A基因及NTHK1基因构建在同一载体上,利用农杆菌介导法转化野生烟草植株。经PCR检测,获得10个转基因株系。经荧光定量PCR检测,Cry1Ac基因表达量在104~10~5数量级,1号转录丰度1.58×10~5为最高,而Cry3A基因表达量在10~5~10~6数量级,10号转录丰度1.34×10~6为最高;10个转基因株系双Bt基因均得到表达且存在表达差异,而对照烟草没有Bt基因表达。经ELISA检测,转基因烟草Cry1Ac蛋白平均表达量为150.441 ng/g,变化在0.86~201.74 ng/g;Cry3A蛋白平均表达量为1 192.592 ng/g,变化在23.33~2413.47 ng/g。经过对烟草斜纹夜蛾的虫试,初步证明转基因植株获得了抗虫性。组培苗耐盐试验表明,不同盐浓度条件下,转基因株系的分化状况优于对照。证明抗虫耐盐多基因的一次性转化,可提高烟草的抗虫能力和耐盐性。
[Abstract]:Insect pests and soil salinization are important factors affecting tobacco growth and development. Transgenic technology has become an effective means to improve this situation. In this study, the modified Bt gene, Cry1Ac gene, Cry3A gene and NTHK1 gene were constructed on the same vector and transformed into wild tobacco plants by Agrobacterium tumefaciens. Ten transgenic lines were obtained by PCR detection. The expression of Cry1Ac gene was detected by fluorescence quantitative PCR in the order of 10 ~ 10 ~ 10 ~ 5, 1. 58 脳 10 ~ (-1) was the highest, while the expression of Cry3A was in the order of 10 ~ 10 ~ 10 ~ (6) and 10 ~ (10) 脳 10 ~ (6). The double Bt gene of 10 transgenic lines were all expressed and expressed differently. There was no Bt gene expression in control tobacco. By ELISA analysis, the average expression of Cry1Ac protein in transgenic tobacco was 150.441 ng/g,. The average expression level of Cry1Ac protein was 1 192.592 ng/g, at 0.861.201.74 ng/g;Cry3A. The average expression level of Cry1Ac protein was 23.33102413.47 ng/g.. The insect resistance of transgenic plants was preliminarily proved by the insect test of Spodoptera litura. The salt tolerance test of tissue culture seedlings showed that the differentiation status of transgenic lines was better than that of control under different salt concentrations. It was proved that one-time transformation of insect-resistant polygenes could improve the insect resistance and salt tolerance of tobacco.
【作者单位】：河北农业大学林学院;廊坊市农林科学院;
【基金】：国家自然科学基金项目(31370663) 国家“863”计划项目(2013AA102703)共同资助
【分类号】：S572;Q943.2

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