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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。
Pests and diseases and soil salinization are important factors that affect the growth and development of tobacco. Transgenic technology has become an effective means to improve this situation. In this study, some of the transgenic Bt Cry1Ac, Cry3A and NTHK1 genes were constructed on the same vector. Agrobacterium-mediated transformation of wild tobacco plants was performed. After PCR detection, 10 transgenic lines were obtained. The expression of Cry1Ac gene was detected on the order of 104-10-5 by fluorescence quantitative PCR. The highest transcriptional abundance of Cry1Ac was 1.58 × 10-5, while the expression of Cry3A gene was in the range of 10-5-10-6. Degree 1.34 × 10 ~ 6 was the highest. The double transgenic Bt genes were expressed and there were differences in expression, while the control tobacco had no Bt gene expression. The average expression level of Cry1Ac protein in transgenic tobacco was 150.441 ng / g, ranging from 0.86 to 201.74 ng / g by ELISA. The average expression level of Cry3A protein was 1 192.592 ng / g, ranging from 23.33 to 2413.47 ng / g. After the test of tobacco Spodoptera litura, preliminary proof transgenic plants obtained insect resistance. Salt tolerance test of tissue culture seedlings showed that under different salt concentrations, the differentiation status of transgenic lines was better than that of the control. It proved that one-time transformation of insect-resistant and salt-tolerant multi-gene could improve the insect resistance and salt tolerance of tobacco.