农杆菌介导抗虫基因PPA的旱稻转化

发布时间：2018-03-02 06:14

本文关键词： 旱稻农杆菌介导法除草剂 PCR Southern杂交　出处：《河北科技大学》2016年硕士论文　论文类型：学位论文

【摘要】：由于水资源短缺,导致水稻的生产受到限制,旱稻作为一个需水量少耐干旱的品种,具有良好的发展及应用前景。由于虫害是导致水稻减产的一个重要因素,利用转基因培育具有抗虫性的旱稻品种,具有经济效益和生态效益。PPA基因具有杀虫的生理活性,在抗虫转基因作物育种方面应用前景广阔。本研究选用“旱稻2号”作为实验材料,以Bar基因作为筛选标记基因,通过农杆菌介导法将抗虫基因PPA导入旱稻的基因组中,对转化体系进行中的各种因素进行了优化,培育出了有抗除草剂、抗虫活性的转基因旱稻植株。通过PCR对转化旱稻植株进行了鉴定,PCR结果证明PPA基因已经成功整合到旱稻2号基因组中。大田喷洒0.5 mg/mL的草铵膦,筛选得到有除草剂抗性的T_1代植株。在实验室对T_2代转化株系进行纯合体筛选,对全部携带除草剂抗性的株系进行PCR鉴定,筛选纯合株系。本实验利用地高辛标记的Southern杂交技术对筛选得到的转PPA的旱稻2号株系进行检测。Southern杂交有许多影响因素,例如:酶切体系、样品DNA的量、探针浓度、杂交条件等。实验结果表明:在60μL的酶切体系中适宜加入15μg的DNA,90U的内切酶,酶切15-16 h。在杂交体系中5 mL杂交液中适宜加入的探针的量为150 ng。Southern杂交结果表明:PPA基因以多拷贝的形式整合到旱稻2号基因组中。
[Abstract]:Due to the shortage of water resources, rice production is restricted. As a variety with less water requirement and drought tolerance, Upland rice has a good prospect of development and application. Transgenic upland rice varieties with insecticidal resistance have economic and ecological benefits. PPA gene has insecticidal physiological activity. In this study, "Upland Rice 2" was selected as experimental material and Bar gene as screening marker gene. The insect-resistant gene PPA was introduced into the genome of Upland rice by Agrobacterium tumefaciens mediated method. The factors in the transformation system were optimized, and the herbicide resistance was developed. Transgenic upland rice plants with insecticidal activity. The transgenic upland rice plants were identified by PCR. The results showed that the PPA gene had been successfully integrated into the genome of Upland Rice 2. The plants with herbicide resistance were screened by homozygous screening in the laboratory, and all the lines with herbicide resistance were identified by PCR. In this experiment, digoxigenin labeled Southern hybridization was used to detect the PPA transgenic Upland Rice strain 2. Southern blot analysis showed that there were many factors affecting the selection, such as enzyme digestion system, sample DNA quantity and probe concentration. The results showed that 15 渭 g of DNA-90U endonuclease could be added into 60 渭 L enzyme system. The suitable amount of probes in 5 mL hybridization solution was 150 ng.Southern hybridization. The results showed that the gene was integrated into the genome of Upland Rice 2 in the form of multiple copies.
【学位授予单位】：河北科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511.6;Q943.2

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