根瘤菌接种提高苜蓿抗旱性相关基因功能与调控机理研究

Alfalfa (Medicago sativa L.) is a key forage crop of the world, and the important forage in China. It distributed in the area which precipitation is lower than 800mm,and is tolerant to drought.From the previous NSFC progject(30901050),we found nodulation can greatly improve the alfalfa tolorence to drought,and 12 highly expressed genes were selected by microarray and bioinformatics for further research. This research is to select 3～5 genes under different drought condition tissue and cultivars in Medicago truncatula and alfalfa seedlings by Real-Time PCR. These genes will be cloned by RACE and sequenced. The genes will be inserted into Agrobactium GV3101 by electroporation after overexpression carrier and RNAi carrier constructed. Gene modified alfalfa and Medicago truncatula will be evaluated under drought with and without nodulation by relative water content (RWC) and death rate. The research will explain the gene function and their drought resistant mechanism, and how these genes expressed under different conditions. Our research will give good gene resources and theoretical guide in alfalfa drought resistant breeding.

在前期青年基金项目（30901050）资助的基础上，证明了接种根瘤菌提高苜蓿抗旱性，利用基因芯片技术，筛选出了12个与苜蓿根瘤菌共生抗旱相关高表达基因。本项目拟以紫花苜蓿和蒺藜苜蓿为材料，开展根瘤菌接种提高苜蓿抗旱性相关基因功能与调控机理研究。借助Real-Time PCR手段,在不同干旱程度、不同组织、不同品种上进行干旱候选基因表达水平差异研究，进一步筛选出3～5个目的基因。采用RACE 法得到基因全长，构建超表达载体和RNAi 载体。应用电击法将目的基因导入农杆菌GV3101中，通过叶盘法在紫花苜蓿和蒺藜苜蓿中转化，得到转基因植株。在不接种及接种根瘤菌条件下，以相对含水量（RWC）和死亡率为指标，评价其抗旱性和目的基因的效应大小。经主要抗旱生理指标测定，结合表达水平差异研究，阐明目的基因功能，揭示其抗旱机制。为苜蓿抗旱分子机理研究提供理论指导，为苜蓿抗旱分子育种提供优异基因资源。

以紫花苜蓿幼嫩叶片cDNA为模板，克隆基因中间片段，测序。然后根据中间片段设计引物，采用RACE法获得3′末端和5′末端片段，经测序得到3′末端片段长度为953 bp，5′末端片段长度为625 bp，利用DNAMAN6.0软件拼接，得到全长cDNA为2521bp，该序列在GenBank中登录，命名为MsZEP，登录号为KM044311，初步分析与ABA合成相关。并构建了载体，已转入到紫花苜蓿，等待进行功能验证。毕业博士1人，在读硕士2人。已获发明专利1项。