百脉根和烟草光呼吸途径改造的分析-analysis on the transformation of light breathing pathway in the root of lotus and tobacco.docx

摘要摘要高等植物的光呼吸作用是由Rubisco参与催化的，是在光照和高氧、低 浓度二氧化碳情况下，绿色细胞吸收02释放C02的过程，主要在叶绿体、 线粒体和过氧化物酶体三个细胞器中进行。高等植物的光呼吸反应对植物生长不利的一面是很明显的，它消耗了光 合作用固定的C02和能量，进入光呼吸途径的碳元素中大约25％被以C02 的形式从线粒体中释放。本研究设计了两条思路来改造C3植物的光呼吸途径，一种方法是将大肠 杆菌光呼吸途径中的三个关键基因引入到百脉根的叶绿体中；另一种方法是 将拟南芥线粒体中编码乙醇酸脱氢酶(Arabidopsis tumefaciens glycolate dehydrogenase。AtGDH)的基因AtGDH和南瓜中编码的苹果酸合酶(Malate synthase，MS)的基因MS引入到百脉根和烟草的叶绿体中，最终目的都是将光 呼吸中乙醇酸的代谢控制在叶绿体中，减少C3植物光呼吸代谢中物质与能量 的损失，达到增产的目的。在实验室前期载体构建工作的基础上，本人完成了带有叶绿体定位信号 肽编码序列的植物表达载体p1301．3MS和p1301．MS的构建工作的全过程。 通过农杆菌介导的植物转化，在优化了百脉根转化体系的基础上，经抗生素 筛选后，获得了25个转GDH百脉根抗性株系，PCR鉴定后得到了2个阳性 株系；同时进行了十几次农杆菌转化野生型烟草，目前共获得了9株转单基 因GDH的烟草与2株转双基因GDH和MS的烟草株系。关键词：光呼吸百脉根烟草遗传改造AbstractAbstractPhotorespiration in higher plants is a process that assimilates oxygen and releases carbon dioxide and is catalyzed by Rubisco under high oxygen and low carbon dioxide conditions．The reactions of this process OCCUr mainly in threeorganelles：chloroplast,mitochondfia and peroxisome．It’S very obvious that photorespiration has adverse sides for plant growth： C02 fixed by photosynthesis and ATP is consumed through photorespiration， and approximately 25％C02 is run off through the process．We used two strategies to modify the Cs plant photorespiration．One stategyis that three key genes in the pathway of E coli glycolate metabolism ate introduted into Lotus corniculatus chloroplast and the other is that AtGDH of Arabidopsis thaliana mitochondria which code glycolate dehydrogenase and MSof pumpkin which code Malate synthase ale introduced into Nicotiana tabacumand L．cornicu肠tus．The purpose of the modification iS to limit thephotorespiration reactions only within the chloroplast,in order to decrease the waste of material and energy in Cs plant photorespiration and achieve the goal ofincreasing crop yield．In this study,I completed the whole work of construction of plant expressionvectors p1301-MS and p1301-3MS．Agrobacterium-mediated transformation were carrfied out．On the basis of optimization of the system of L．corniculatus tra