利用RNAi技术抗玉米矮花叶病效果研究.pdfVIP

河南农业科学，2014，43(4)：69—74 Journal of Henan Agricultural Sciences 利用RNAi技术抗玉米矮花叶病效果研究 张莹莹，吴连成，库利霞，张赛赛，安允权，崔新建，陈彦惠 (河南农业大学农学院／河南粮食作物协同创新中心，河南郑州450002) 摘要：以采用RNAi技术获得的HiⅡ、HiⅡA、HiⅡB和H99抗玉米矮花叶病转基因T。和T2代 为材料，通过病毒接种、草丁膦涂抹、目的基因和标记基因PCR扩增，研究了利用RNAi原理构建 的目的基因对玉米矮花叶病的抗性效果以及玉米转基因后代群体鉴定筛选技术。结果表明， 90．00％的转基因株系抗病性超过非转基因株系，30．00％的转基因株系抗病株率超过了抗病对照 黄早4，转基因株系的抗病性明显提高，说明利用RNAi技术选育抗矮花叶病转基因玉米株系是可 行的。4种筛选鉴定方法比较结果表明，目的基因PCR和病毒接种鉴定方法最可靠，在转基因后 代群体鉴定过程中，可在苗期用200 mg／L的草丁膦筛选阳性植株基础上，再采用目的基因PCR 鉴定，筛选出抗性转基因植株或株系，不仅可以减少鉴定的工作量，而且可以提高鉴定的准确性。 关键词：RNAi技术；玉米；矮花叶病；转基因；鉴定方法 中图分类号：$513 文献标志码：A 文章编号：1004—3268(2014)04—0069一06 Effect of Maize Dwarf Mosaic Disease Resistance by RNAi Technology ZHANG Ying-ying，WU Lian-cheng，KU Li—xia，ZHANG Sai—sai，AN Yun—quan， CUI Xin-jian，CHEN Yan—hui。 (Collaborative Innovation Center of Henan Grain Crops，Agronomy College of Henan Agricultural University，Zhengzhou 450002，China) Abstract：In the paper，four T1 or T2 generation materials of transgenic maize resistant to dwarf mosaic disease were obtained with RNAi technology，which were used to study the resistant effect of target gene constructed by RNAi principle to maize dwarf mosaic disease and the techniques to identify and screen transgenic progeny population through inoculating virus。daubing PPT and PCR amplification of the target gene and marker gene．The results showed that 90．00％of transgenic plants had higher resistance than non—transgenic plants，and 30．00％of transgenic plants were even better than CK(Huangzao 4)．The increase of disease resistance in transgenic lines indicated that it was feasible to breed the transgenic plants with resistance to dwarf mosaic disease by RNAi technique．The comparison among the four identifying and screening methods showed that PCR of target gene and virus inoculation were the most reliable．During the identifying and screening procedure of transgenic progenies，the positive plants should be first selected at seedling stage with 200 mg／L of PPT。from which the resistant transgenic plants or line