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2025年4月6日 星期日
农业生物技术学报  2017, Vol. 25 Issue (12): 1903-1917    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
转Bt基因烟草中Cry1Ac基因的叠加效应研究
任亚超,董研,张文林,杨敏生
河北农业大学
Superposition Effect of Cry1Ac Gene in Transgenic Bt Tobacco (Nicotiana tabacum)
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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。
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任亚超 董研 张文林 杨敏生
关键词 BtCry1Ac基因烟草遗传转化外源基因表达生理生化指标    
Abstract: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 expression and transgenic plant growth, in this study, the plant transformation vectors carrying single and double Bacillus thuringiensis(Bt) gene Cry1Ac 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 Cry1Ac gene lines and 8 transgenic dual Cry1Ac 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 Cry1Ac gene transcriptional abundance of the transgenic dual Cry1Ac gene lines was about 2.6 times higher than that of transgenic single Cry1Ac gene lines, the Cry1Ac toxin content of the transgenic dual Cry1Ac gene lines was about 10 times higher than that of transgenic single Cry1Ac gene lines, and the two types of values of the transgenic dual Cry1Ac gene lines were significantly higher than that of transgenic single Cry1Ac gene lines. The result of indoor insect feeding test showed that with the increase of feeding days, the mortality of cotton bollworm (Helicoverpa armigera) fed with transgenic lines leaves increased gradually. There were some differences among different transgenic lines. The lethal rates of the two kinds of transgenic lines against the first instar larvae of Helicoverpa armigera were 100%. When feeding for three days, the larva mortality rate of transgenic dual Cry1Ac gene lines B1, B4, B6, B7 and transgenic single Cry1Ac gene lines A2, A7 reached 100%. Feeding for four days, the larva mortality rate of B3, B5, B8, A1, A3, A4, A5, and A9 reached 100%. At five days, the larva mortality rate of all transgenic lines reached 100%, while the rate of non-transgenic tobacco was less than 10%. Moreover, the lethal rates of the two kinds of transgenic lines against the second instar larvae of Helicoverpa armigera were also 100%. The larva mortality rate of B7 and B6 reached 100% at feeding for five days and six days respectively, while A2 and A5 reached 100% at feeding for eight days. Overall, the lethal time of the transgenic dual Cry1Ac gene lines was shorter than transgenic single Cry1Ac gene lines, and transgenic dual Cry1Ac gene lines had high insect resistance. The morphological and physiological and biochemical indexes detections showed that there were no significant differences in the parameters of ground diameter, net photosynthetic rate (Pn), stomatal conductance (Cond), intercellular CO2 concentration (Ci), transpiration rate (Tr), maximum fluorescence (Fm), chlorophyll a (Chla), and total chlorophyll content (CT) among transgenic dual Cry1Ac gene lines and CK and transgenic single Cry1Ac gene lines. While there were some differences in other parameters. On the whole, the growth of most transgenic lines was not significantly different from that of the control group, and some of the transgenic dual Cry1Ac gene lines showed dwarfing phenomenon. In general, in this study, compared with transgenic single Cry1Ac gene lines, the exogenous gene expression level of transgenic dual Cry1Ac gene lines was improved, and the plant growth and other aspects were not significantly affected. This study will lay the foundation for exploring ways to improve the expression of exogenous genes and to transform other plants.
Key wordsBtCry1Ac gene    Tobacco    Genetic transformation    Exogenous gene expression    Physiological and biochemical indexes
收稿日期: 2017-05-04      出版日期: 2017-12-10
基金资助:国家自然科学基金项目;国家自然科学基金项目;河北省研究生创新资助项目
通讯作者: 杨敏生     E-mail: yangms100@126.com
引用本文:   
任亚超 董研 张文林 杨敏生. 转Bt基因烟草中Cry1Ac基因的叠加效应研究[J]. 农业生物技术学报, 2017, 25(12): 1903-1917.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I12/1903
 
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