绿豆干旱胁迫应答基因及调控元件的鉴定与分析

Mung bean (Vigna radiata) is mostly grown in infertile land in arid and semi-arid areas in China, has drought stress resistance capability, especially the wild type. These properties make mung bean an ideal system to study the mechanism of drought stress responses. In this project, cultivated and wild mung bean seedlings are used as material, to identify genes and regulatory elements responding to drought stress. Previous work has determined the extent and period of seedlings exposing to drought stress through marker gene in mung bean, the expression and responses of candidate gene VrDREB2A was analyzed through qRT-PCR. The preliminary results indicated the relative expression of VrDREB2A in shoot apical meristem was observed significantly higher after drought treatment in both cultivated and wild mung bean seedlings, and the wild type showed higher expression level. Therefore, shoot apical meristem in this stage will be used as study material. This project will identify the key genes responding to drought stress in cultivated and wild mung bean, predict regulatory elements such as promoters and enhancers, and reveal the potential genetic and epigenetic variations related to drought stress responses during the process of domestication, through combining transcriptome, methylome and DNaseI hypersensitive sites data. This study will provide fundamentions for identification and functional analysis of drought stress resistance genes, and the evaluation and improvement of drought-resistant germplasms.

我国绿豆多数种植在干旱、半干旱的贫瘠土地上，具有一定的抗旱性，是研究干旱胁迫应答分子机制的理想材料。本项目以栽培型和野生型绿豆为研究对象，在全基因组水平鉴定绿豆干旱胁迫应答基因及调控元件。前期通过标记基因已确定适宜绿豆干旱胁迫的条件和时间，通过qRT-PCR分析了绿豆干旱应答响应因子候选基因VrDREB2A在叶、茎尖和根中的表达及其对干旱胁迫的响应。初步结果表明该基因只在栽培型和野生型苗期茎尖中表现出对干旱胁迫的响应而表达量显著性增加，且野生型具有较高的表达量，因而选取该发育时期茎尖作为研究材料。本项目拟通过转录组、DNA甲基化组和DNaseI超敏感位点组学数据的产生与整合，鉴定栽培型和野生型绿豆响应干旱胁迫的关键基因并初步预测启动子和增强子等调控元件，揭示在驯化过程中形成的影响干旱胁迫响应机制的潜在遗传和表观遗传变异。该研究将为绿豆抗旱基因的鉴定和功能分析及抗旱种质的评价改良奠定基础。

我国绿豆多数种植在干旱、半干旱的山坡地和贫瘠的土地上，苗期干旱严重制约着绿豆的产量。野生型绿豆和栽培型相比，具有很强的环境适应性和对逆境的抵抗性，在遗传改良中具有重要的作用。本研究以野生型（61号）和栽培型（70号）绿豆为材料，进行干旱胁迫处理，通过生理指标测定发现干旱胁迫后61号与70号相比SOD、POD和CAT含量显著性增加，而MDA的含量显著降低，表明野生型材料具有较强的抗氧化保护系统和细胞膜稳定性。转录组研究发现在野生型和栽培型绿豆中，共有2859个差异性表达基因检测出，在干旱胁迫后差异性表达基因数目增加到3121，表明其遗传背景对干旱胁迫的响应的较大差异。另外，在野生型绿豆中，干旱胁迫后有更多的差异性表达基因、转录因子和与植物激素相关的基因被检测出，其中下调的差异性表达基因数目要多于上调的差异性表达基因数目，与之相对应，干旱胁迫后高甲基化的差异性甲基化区域数目要多于低甲基化的差异性甲基化区域数目，这与栽培型种质干旱胁迫后变化趋势恰好相反。进一步分析发现，野生型和栽培型材料甲基化差异主要集中在启动子CHH位点上，表明在三种DNA甲基化形式CG, CHG, CHH中，CHH甲基化比较容易受基因型和干旱胁迫的影响。对差异性甲基化区域和差异性表达基因的相关性分析发现，干旱胁迫后野生型和栽培型绿豆与其对照相比，gene body的差异性甲基化区域与差异性表达基因均呈负相关趋势。这些发现将为绿豆抗旱基因的鉴定和功能分析及抗旱种质的评价改良奠定基础，该研究对野生种质在提升作物的抗旱性及抗旱育种中的应用提供了理论依据。