乙醛脱氢酶ADH1相关天然反义lncRNA在菜用大豆低温胁迫应答中的功能及作用机制研究

Low temperature stress in the early spring is one of the major environmental factors that affect the production of vegetable soybean. Recently, long non-coding RNA (lncRNA) has been shown to play critical role in plant stresses response. However, the lncRNA and their regulatory roles in response to cold stress remain largely unknown. Study of lncRNAs in vegetable soybean remains in its infancy. In our previous study, we identified total of 1,254 lncRNAs in vegetables soybean and found that 73 lncRNAS showed significantly different expression under colds stress treatment. Among them, we identified one antisense lncRNA, named ADH-AS1 (ADH-antisense1), which was associated with sense alcohol dehydrogenase 1 (ADH1) gene. In addition, we found that the expression of ADH-AS1 was significantly up-regulated in response to cold stress with its sense gene ADH1. Based on these previous studies, in order to understand the molecular function and regulatory role of ADH-AS1 in cold stress tolerance, this study will investigate the biological function of ADH-AS1 in vegetable soybean cold stress response and the regulatory role of ADH-AS1 on ADH1 mRNA expression. Moreover, we will determine whether the ADH-AS1 regulated ADH1 expression by mediating interaction with RNA binding protein, regulation chromatin modification or formation of RNA-RNA complex with ADH1. The aim of our project is to reveal ADH-AS1 mediated regulatory network involved in the response to cold stress in vegetable soybean, which will supply theoretical bases and novel candidates for breeding cold stress resistant varieties of vegetable soybean.

早春季节的低温寒潮是制约菜用大豆生产最主要的环境因子之一。最新研究发现长链非编码RNA(lncRNA)在植物抗逆反应中发挥重要作用，但目前对lncRNA介导的植物低温胁迫应答机制研究较少。本项目前期在菜用大豆全基因组水平鉴定到1254个lncRNA，发现其中73个lncRNA受低温胁迫显著诱导。从中筛选到一个低温诱导lncRNA，该lncRNA位于低温响应关键基因ADH1的反义链上，命名为ADH-AS1。前期研究发现ADH-AS1与ADH1在低温胁迫下协同上调表达，但具体作用机理还不清楚。为此，本项目拟在蛋白互作、染色质的表观遗传修饰及RNA互作等层面深入解析ADH-AS1与ADH1的互作模式，明确其对ADH1的表达调控模式及作用方式，从而探明ADH-AS1在菜用大豆低温胁迫应答中的生物学功能，揭示ADH-AS1介导的低温胁迫应答机制及调控网络，为菜用大豆低温抗性育种提供理论基础和新思路。

菜用大豆是一种重要的豆类蔬菜，我国是世界上最大的菜用大豆生产国和速冻加工出口国。早春季节设施及露地栽培的菜用大豆极易遭受寒潮和倒春寒引起的低温冷害影响，已成为当前制约菜用大豆安全、优质生产最主要的因素之一。研究菜用大豆低温胁迫应答的分子机制，挖掘关键调控因子具有重要意义。本项目针对乙醛脱氢酶ADH1及其天然反义lncRNA在菜用大豆低温胁迫应答中的调控机制开展了研究。针对菜用大豆与粮（油）用大豆不同性状上存在的长期人为选择和自然选择，以项目组自主育成的主栽品种‘浙农6号’为材料，获得了首个高质量菜用大豆基因组，群体遗传学分析发现，菜用大豆与粮（油）用大豆明显划分为两个不同的亚群，为lncRNA更加精准鉴定奠定基础。转录水平分析发现ADH1及ADH-AS1低温胁迫下显著上调表达，二者呈现协同表达模式。菜用大豆转基因生物学功能验证表明过表达植株抗低温性显著增强。进一步调控机制研究发现ADH1通过MAPK3、MYC、ERF109、JAZ1等途径形成复杂的激酶-转录因子-激素互作网络，从而调控菜用大豆低温胁迫应答。研究结果为菜用大豆低温抗性育种提供新的分子靶标和理论基础。