转Bt基因烟草中Cry1Ac基因的叠加效应

发布时间：2018-03-24 02:19

本文选题：CryAc基因　切入点：烟草　出处：《农业生物技术学报》2017年12期

【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。
[Abstract]:Plant transgenic studies have found that multiple copy insertions often lead to a decrease in the amount of foreign gene expression, but the same foreign gene is constructed in tandem with the same vector when the vector is constructed. In order to increase the expression of exogenous genes in transgenic plants, the effects of superposition of the same target genes on gene expression and growth of transgenic plants were explored. In this study, Agrobacterium tumefaciens-mediated leaf disk method was used to transform the plant expression vector carrying single and double Bacillus thuringiensis BT gene Cry1Ac into Nicotiana tabacum. The transgenic lines were tested by polymerase chain reaction, fluorescence quantitative PCR(fluorescence quantitative PCR, FQ-PCRG (absolute quantification of BT toxin protein) and insecticidal resistance to identify whether the foreign gene was integrated into the tobacco genome and the expression of exogenous gene. At the same time, the morphological, physiological and biochemical indexes of transgenic lines and non-transgenic plants transformed by two vectors were determined, and the growth of transgenic plants was observed. The results showed that 9 transgenic Cry1Ac gene lines were obtained by PCR detection. All the target genes were integrated into tobacco genome, and fluorescence quantitative PCR and toxin protein detection showed that, The transcriptional abundance of the Cry1Ac gene in the transgenic double Cry1Ac line was about 2.6-fold of that of the single Cry1Ac strain, and the content of Cry1Ac toxin protein was about 10 times higher than that of the single Cry1Ac gene line, and the two values of the transgenic double Cry1Ac gene line were significantly higher than that of the single Cry1Ac gene line. The results of indoor feeding experiment showed that the mortality of the two vector lines against the first and second instar larvae of Helicoverpa armigera was 100, and the lethal time of transgenic double Cry1Ac gene lines was shorter, and the morphological, physiological and biochemical indexes were determined. There was no significant difference in growth indexes between most transgenic lines and the control, and some of the transgenic double Cry1Ac lines showed dwarfing phenomenon. In general, the expression of exogenous genes in the transgenic double Cry1Ac lines was higher than that in the single transgenic lines. This study provides a basic theoretical basis for exploring ways to increase the expression of exogenous genes and transforming other plants.
【作者单位】：河北农业大学林学院;河北省林木种质资源与森林保护重点实验室;
【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)
【分类号】：Q943.2

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