棉花中草甘膦诱导型启动子ag2诱导元件的鉴定与功能分析

Currently, constitutive promoters were used in almost all reported glyphosate-resistant transgenic plants. However, due to their constitutive expression pattern, extraneous genes can express in all tissues of plants at any development stage, which would lead to great waste of both materials and energy and increase negative influences in metabolism for host plants.The most dramatically high-level expression gene was screened by mRNA differential display reverse transcription PCR and RT-PCR. It was named EST ag2 amplified from glyphosate sprayed cotton. Northern blot analysis indicated that ag2 gene was glyphosate-induced highly expression gene.We cloned gene and promoter of cotton ag2 by use RACE, hemispecific PCR and Cassette ligation PCR methods. Based on the results mentioned above, we are planning to extend this research to identify cis-elements and investigate further into the role of ag2 promoter. And we plan to analyse the growth of transgenic plant after fused this promoter with glyphosate-resistant gene and applied in field planting.In this study, a specific glyphosate-induced promoter from cotton will be identified, which can drive the extraneous glyphosate-resistant gene to just highly express after spraying glyphosate. The studies will provide scientific basis for molecular design of glyphosate-resistant transgenic plants. It has practical significance to reduce side effects and improve output of glyphosate-resistant transgenic corps.

目前报道的抗草甘膦转基因作物中，驱动抗性基因的都是强表达的组成型启动子，使抗草甘膦基因几乎在转基因作物的所有部位和所有发育阶段都高效表达，这无疑增加了植株的代谢负担，造成物质和能源上的巨大浪费。通过对棉花喷施草甘膦，利用DDRT-PCR和RT-PCR方法，我们筛选出草甘膦诱导棉花高表达EST序列ag2，Northern Blot结果证实ag2为草甘膦诱导后高表达基因。分别利用RACE方法和基因侧翼未知序列PCR方法，我们克隆了ag2基因及其启动子，并对其功能进行了初步分析。本项目拟在此基础上，对启动子进行深入的功能研究，明确其精确功能；并将该启动子与抗草甘膦基因融合，检测对转基因作物生长发育的影响。通过本项目研究，可获得一个棉花中草甘膦诱导型启动子。相关研究可为抗草甘膦转基因作物的分子设计提供依据，对于减少转基因产物对受体作物和环境产生副作用，进一步提高转基因作物产量具有重要现实意义。

目前报道的抗草甘膦转基因作物中，驱动抗性基因的都是强表达的组成型启动子，使抗草甘膦基因几乎在转基因作物的所有部位和所有发育阶段都高效表达，这无疑增加了植株的代谢负担，造成物质和能源上的巨大浪费。本项目利用草甘膦喷施棉花植株，筛选出草甘膦诱导棉花高表达ag2基因及其启动子。通过分析启动子序列结构，确定了不同反应元件作用区，并验证了转录起始位点。在构建启动子不同缺失突变体的基础上，分析了启动子诱导的时效性、诱导反应应答区及诱导表达调控的核心元件序列。并构建不同启动子驱动抗草甘膦基因表达，了解外源基因持续表达对转基因作物生长发育的影响。通过本项目研究，获得了一个棉花中草甘膦诱导型启动子。本研究可为抗草甘膦转基因作物的分子设计提供依据，对于减少转基因产物对受体作物和环境产生副作用，进一步提高转基因作物产量具有重要现实意义。