转Bt基因烟草中Cry1Ac基因的叠加效应  

Superposition Effect of Cry1Ac Gene in Transgenic Bt Tobacco(Nicotiana tabacum)

作　　者：任亚超[1,2] 董研 张文林[1,2] 杨敏生 

机构地区：[1]河北农业大学林学院,保定071000 [2]河北省林木种质资源与森林保护重点实验室,保定071000

出　　处：《农业生物技术学报》2017年第12期1903-1917,共15页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31370663和No.31660211);河北省研究生创新资助项目(No.1099009)

摘　　要：植物转基因研究发现,多拷贝插入往往导致外源基因表达量下降,但在构建载体时将同一外源基因串联构建于同一载体上,转化植株后是否会提高外源基因的表达量尚未报道。为了提高转基因植物中外源目的基因的表达量,探索同一目的基因叠加对基因表达及转基因植株生长的影响,本研究利用农杆菌(Agrobacterium tumefaciens)介导的叶盘法分别将携带单、双苏云金芽孢杆菌(Bacillus thuringiensis,Bt)基因Cry1Ac的植物表达载体转化烟草(Nicotiana tabacum)组培苗,并对转基因株系进行聚合酶链式反应(polymerase chain reaction,PCR)、荧光定量PCR(fluorescence quantitative PCR,FQ-PCR)(绝对定量)、Bt毒蛋白及抗虫性检测,以鉴定外源基因是否整合至烟草基因组及外源基因的表达情况,同时对两种载体转化的转基因株系及非转基因植株进行形态及生理生化指标测定,观测转基因植株的生长情况。结果表明,通过PCR检测,获得转单Cry1Ac基因株系9个,转双Cry1Ac基因株系8个,目的基因均整合至烟草基因组;荧光定量PCR及毒蛋白检测表明,转双Cry1Ac基因株系Cry1Ac基因的转录丰度约为转单Cry1Ac株系的2.6倍,Cry1Ac毒蛋白含量约为转单Cry1Ac基因株系的10倍,转双Cry1Ac基因株系的两个数值都显著高于转单Cry1Ac基因株系;室内饲虫实验结果表明,转两种载体株系对棉铃虫(Helicoverpa armigera)一龄幼虫和二龄幼虫的致死率均为100%,转双Cry1Ac基因株系的致死时间较短;形态及生理生化指标测定结果表明,大部分转基因株系的生长指标与对照无显著差异,部分转双Cry1Ac基因株系表现出矮化现象。总体来说,本研究中转双Cry1Ac基因株系较转单基因株系在外源基因表达量上得到提高,而在植株生长等方面未受到明显影响。本研究为探索提高外源基因表达量的方法以及转化其他植物提供基础理论基础。Plant transgenic studies have found that multiple copies insertion often lead to decreased expression of exogenous genes. However, when constructing the vector, the same exogenous gene is constructed on the same vector with series connection. Whether it will improve the expression of exogenous genes after transformation of plants has not been reported. In order to improve the expression of exogenous gene in transgenic plants and explore the effect of superposition of the same target gene on gene expressionand transgenic plant growth, in this study, the plant transformation vectors carrying single and double Bacillusthuringiensis（Bt） gene Cry1 Ac respectively were transformed into tobacco（Nicotiana tabacum） tissue culture seedlings by Agrobacterium（Agrobacterium tumefaciens）-mediated leaf disc method. Then, the transgenic lines were detected by polymerase chain reaction（PCR）, fluorescence quantitative PCR（FQ-PCR）（Absolute quantitative） and Bt toxin and insect resistance tests to identify whether or not the exogenous gene was integrated into the tobacco genome and the expression were carried out. At the same time, morphological indexes and physiological and biochemical indexes of the transgenic plants transformed with the two vectors and non-transgenic plants were observed to research the growth of the transgenic plants. The results showed that nine transgenic single Cry1 Ac gene lines and 8 transgenic dual Cry1 Ac gene lines were obtained by PCR detection, and the target genes were integrated into the tobacco genome. FQ-PCR and toxin detections showed that the Cry1 Ac gene transcriptional abundance of the transgenic dual Cry1 Ac gene lines was about 2.6 times higher than that of transgenic single Cry1 Ac gene lines, the Cry1 Ac toxin content of the transgenic dual Cry1 Ac gene lines was about 10 times higher than that of transgenic single Cry1 Ac gene lines, and the two types of values of the transgenic dual Cry1 Ac gene lines were significantly higher than that of transgenic single Cry1 Ac

关 键 词：CRY1AC基因 烟草 遗传转化 外源基因表达 生理生化指标 

分 类 号：S188[农业科学—农业基础科学]