根瘤提高苜蓿气孔对ABA敏感性的抗旱分子机制研究

Alfalfa is an important legume forage crop because of its high yield and high nutrition value to feeding animals. Despite its relatively high tolerance to drought, alfalfa often suffers great loss in production due to drought stress. Our recent studies showed that root nodulation enhances alfalfa survival under drought. This is, at least partly, achieved by increasing stomatal sensitivity to ABA, a key stress hormone in plants, so that water loss through transpiration is reduced. Plants transpire 90% of water they uptake from soil through stomatal. The objective of this proposal is to address how nodulation impacts stomatal sensitivity to ABA in alfalfa. Our ultimate goal is to improve drought tolerance and water use efficiency in this important crop..To understand how alfalfa root nodules increase sensitivity to ABA in guard cells, we will identify genes/proteins/pathways in leaves that are differentially affected by nodulation by comparing nodulated and non-nodulated plants when they are exposed to dehydration stress or ABA treatment. Changes in transcript and protein levels will be examined using high-throughput RNA-seq and iTRAQ, respectively. In parallel, physiological and biochemical changes will also be examined. ABA content in leaves will be monitored after leaves were treated with ABA or dehydration stress using High Performance Liquid Chromatography. Stomatal aperture will be measured under a scanning electron microscope. Bioinformatic tools will be used to integrate the gene/protein data and to establish a working model for the molecular processes involved in change in stomatal sensitivity to ABA associated with nodulation. The study will provide novel insight into the regulation of ABA signaling pathway in a nitrogen-fixing crop and may significantly benefit alfalfa breeding and management to improve its production under drought conditions.

紫花苜蓿是最重要的抗旱优质牧草，在我国草食畜牧业中具有重要地位，但干旱依然会给苜蓿生产带来严重损失。植物吸收的水分，90%以上通过气孔蒸腾散失。脱落酸（ABA）被认为是应答干旱控制气孔的重要激素。我们研究证明根瘤可提高苜蓿抗旱性，是由于根瘤增加了气孔对ABA的敏感性，但其机制尚不明晰。. 本研究拟以根瘤固氮苜蓿功能叶片为材料，通过干旱和外源ABA处理，利用RNA-seq高通量测序和iTRAQ获取其转录组和蛋白组数据并进行生物信息学分析、扫描电镜观察气孔变化、高效液相色谱法测定ABA含量，并检测生理指标变化，进行关联分析，进一步实时定量PCR进行验证，从而构建新的应答模型，揭示干旱胁迫下根瘤固氮苜蓿提高气孔对ABA敏感性的分子机制，丰富ABA途径应答干旱理论，为苜蓿抗旱栽培管理及品种选育提供理论依据和候选基因。

紫花苜蓿是重要的抗旱牧草，本研究前期以根瘤固氮苜蓿功能叶片为材料，通过干旱和外源ABA处理，利用扫描电镜观察气孔变化、高效液相色谱法测定ABA含量，并检测生理指标变化，发现结瘤能够提高苜蓿的木质素含量，降低牧草的饲用品质及消化率，提高苜蓿的耐寒性。进而利用RNA-seq高通量测序，对蒺藜苜蓿的ABA激活信号通路的PYL基因家族进行全基因组鉴定、表达和功能分析，iTRAQ获取其转录组和蛋白组数据并进行生物信息学分析、进行关联分析，进一步实时定量PCR进行验证，从而构建ABA应答模型，选取ABA相关调控途径的关键基因MsDUF进行转基因验证其功能，将MsDUF基因转入到烟草中发现过表达植株中ABA的含量和ABA合成的几个关键基因的转录水平增加。此外，还发现了一种新的水通道蛋白基因MsPIP2;2,将MsPIP2;2转入拟南芥中发现MsPIP2; 2通过调节抗氧化剂防御系统介导的ROS清除，K + / Na+离子稳态和胁迫响应基因表达来赋予拟南芥耐盐性。本研究已发表相关论文11篇，其中SCI 论文8篇，中文论文3篇，申报并授权发明专利2个。培养博硕士研究生11人，已经培养毕业博士3人，硕士6人，在读博士2人。超额完成了目标任务。本研究初步揭示了干旱胁迫下根瘤固氮苜蓿提高气孔对ABA敏感性的分子机制，丰富ABA途径应答干旱理论，为苜蓿抗旱栽培管理及品种选育提供理论依据和候选基因。