NCED基因表达及ABA积累在小麦耐倒春寒中的作用机理研究

Spring frost is one of the major causes of yield reduction in common wheat (Triticum aestivum L.). It has important significance to identify the responsive genes to spring frost and study the molecular mechanism of frost resistance. In our previous study, we performed both RNA-Seq and digital gene expression analysis with samples under normal conditions and low temperature stress in wheat reproduction stage (simulated spring frost). Using digital gene expression tag profiling, we constructed an expression profile to identify genes related to spring frost resistance. The NCED gene was found significantly up-regulated, which was the key enzyme in the ABA biosynthesis pathway. According to the EST database information, we cloned two members of the NCED gene family, designated as TaNCED1 and TaNCED2. In this research, we want to investigate the relationship between the NCED gene expression, ABA level and the wheat spring frost resistance. We plan to analyze the NCED gene expression profile and their promoter activities. Construct plant expression vectors and analyze the effects of NCED gene on the gene expression and stress resistance of transgenic wheat plants. These results may provide valuable information for us to understand the function of NCED family and the ABA response system in the spring frost responses in wheat，thereby providing a theoretical basis of candidate genes for spring frost tolerance in wheat breeding.

倒春寒（晚春霜冻）是造成小麦产量损失的主要灾害之一，分析倒春寒胁迫响应基因，研究小麦耐倒春寒的分子机理具有重要的意义。我们在前期工作中，对处于生殖发育阶段的小麦在正常条件和低温胁迫（模拟倒春寒）条件下进行了转录组和数字表达谱测序。对差异表达基因进行分析时，发现ABA合成途径的关键限制酶基因NCED基因上调表达显著。根据数据库中EST信息，我们克隆得到了两个NCED基因，分别命名为TaNCED1和TaNCED2。本项目选择小麦NCED基因表达、ABA水平、小麦耐倒春寒抗性三者之间的关系进行研究：在分析NCED基因表达调控模式和启动子元件的基础上，利用转基因技术来深入研究小麦NCED基因的表达以及ABA响应系统与小麦生殖阶段耐寒性的关系，进一步解析小麦耐倒春寒的分子机制，为耐倒春寒小麦新品种选育提供理论依据。

本研究分析比较了17个抗旱性不同的小麦品种的TaNCED1基因序列等位变异，通过序列比对发现在B基因组上存在4个SNP位点的变异，针对这些变异开发了KASP分子标记。通过分析干旱胁迫和复水过程中不同小麦品种间TaNCED1基因表达和ABA积累的变化规律，发现不同品种间TaNCED1基因对水分变化的响应快慢程度及其表达水平存在显著差异。构建TaNCED1基因的过表达、RNAi及基因编辑载体，通过农杆菌介导的小麦遗传转化得到了转TaNCED1基因的小麦株系。利用转基因植株分别在苗期进行干旱胁迫处理和在雌雄蕊分化期模拟倒春寒处理，对各项生理指标进行测定，并且分别进行了叶片和幼穗的转录组测序。过表达TaNCED1基因小麦植株表现出较强的抗逆性。通过过表达NCED基因增加植物体内的ABA含量和降低NCED表达降低体内ABA水平，来研究NCED基因表达、ABA含量和小麦抗逆性之间的关系，阐明NCED基因如何通过ABA信号途径发挥功能，进一步解析小麦抗逆过程调控途径的分子机制，为小麦抗逆育种提供候选基因和理论依据。