烟草orf25基因RNAi表达载体的构建及遗传转化

发布时间：2018-03-28 05:13

  本文选题：烟草　切入点：orf基因　出处：《分子植物育种》2017年05期

【摘要】：杂种优势的利用可以有效地实现烟草高产、高抗、优质的育种目标,目前,培育雄性不育系是获取杂种烟草最有效的途径。研究发现orf25基因位于线粒体基因组中,与烟草雄性不育密切相关。本研究以烟草雄性不育相关基因orf25为研究对象,根据RNA干扰载体构建原则,将长度为606 bp的orf25基因干扰片段分别以正向、反向连接到大小为190 bp的linker连接片段的两侧,构成发夹结构的RNA干扰载体片段。将RNA干扰载体片段插入到中间载体pET30a上,最后克隆到植物表达载体pCAMBIA1301中,成功构建orf25基因的RNAi表达载体。利用农杆菌介导法将pCAMBIA1301-orf25-RNAi转入到烟草保持系中烟90中,经抗性筛选以及分子检测最终获得35株转pCAMBIA1301-orf25-RNAi烟草植株,其中阳性烟草有18株,转化率为51.4%。结果表明pCAMBIA1301-orf25-RNAi干扰载体已成功转入到烟草中烟90中。
[Abstract]:The utilization of heterosis can effectively achieve the goal of tobacco breeding with high yield, high resistance and good quality. At present, breeding male sterile lines is the most effective way to obtain hybrid tobacco. It is found that orf25 gene is located in the mitochondrial genome. In this study, the male sterility related gene (orf25) of tobacco was studied. According to the principle of RNA interference vector construction, the interference fragments of 606 BP orf25 gene were divided into positive direction. The RNA interference vector fragment with hairpin structure was constructed. The RNA interference vector fragment was inserted into the intermediate vector pET30a and cloned into the plant expression vector pCAMBIA1301. The RNAi expression vector of orf25 gene was successfully constructed. The pCAMBIA1301-orf25-RNAi was transferred into tobacco maintainer line Yan90 by Agrobacterium tumefaciens. 35 transgenic pCAMBIA1301-orf25-RNAi tobacco plants were obtained by resistance screening and molecular analysis, among which 18 were positive. The results showed that the pCAMBIA1301-orf25-RNAi interference vector had been successfully transferred into tobacco smoke 90.
【作者单位】： 江西农业大学农学院作物生理生态与遗传育种教育部重点实验室江西省作物生理生态与遗传育种重点实验室;南昌职业学院;湖南农业大学植物保护学院植物病虫害生物学与防控湖南省重点实验室;江西农业大学园林与艺术学院;
【基金】：国家自然科学基金项目(31260350;31301388) 中国博士后科学基金(2015T8087;2014M562109) 江西省教育厅科技计划项目(GJJ13275)共同资助
【分类号】：Q943.2
，

本文编号：1674900