CsARN6.1调控淹涝胁迫下黄瓜不定根形成的机制解析

Cucumber is one of the major vegetable crops cultivated in China that is generally considered as waterlogging sensitive, due to its shallow root system and strict oxygen requirement. Adventitious root (AR) formation at the soil surface is considered a key step in adaptation to waterlogging because ARs can obtain oxygen directly from the air. A major-effect QTL on chromosome 6, ARN6.1, that was responsible for waterlogging tolerance due to increased AR formation in the cucumber line Zaoer-N, were identified in our previous study. Through combined use of SNP marker design and genotyping of the recombinants, we narrow down the QTL to a 36.1kb region. Through multiple lines of evidence, we show that CsARN6.1 is the most possible candidate for ARN6.1 which encodes an AAA ATPase. In current study, we aimed to clone the CsARN6.1 through fine mapping in large segregating population and regional association study in natural cucumber populations. The underlying molecular mechanisms of CsARN6.1 in regulating of cucumber AR formation will be revealed by yeast-two hybrid technique and GST pull-down assay, which will help us to understand the fundamental mechanism of CsARN6.1 under waterlogging stress and breed of waterlogging tolerant varieties.

黄瓜是我国栽培的主要蔬菜之一，其根系入土浅、通气组织不发达、极易受到淹涝胁迫危害，造成产量损失和品质下降。不定根是植物对淹涝胁迫最重要的适应能力之一，它代替了由于淹涝导致的初生根死亡，有利于受涝器官获得氧气。本实验室前期在黄瓜Chr6上定位到1个淹涝胁迫下不定根数的主效QTL ARN6.1，结合SNP标记开发和重组株鉴定将其定位至~36.1kb区域内，其中一个编码AAA ATPase蛋白的CsARN6.1推测为黄瓜耐淹涝候选基因。本项目旨在进一步通过扩大分离群体完成精细定位，结合区域关联分析，克隆CsARN6.1；通过基因组编辑技术及构建近等基因系法验证其功能；通过构建酵母文库并利用酵母双杂交技术与GST pull-down技术，结合质谱鉴定，构建蛋白互作网络，最终解析CsARN6.1调控淹涝胁迫下黄瓜不定根形成的分子机制，对今后创新黄瓜耐涝种质，选育耐涝黄瓜新品种具有重要意义和应用前景。

黄瓜是我国栽培的主要蔬菜之一，具有须根系、好气性强、易受涝危害等特点。涝胁迫后下胚轴产生的不定根数是黄瓜耐涝能力强弱的特征性表型。前期以耐涝品系Zaoer-N和敏涝品系Pepino构建的F2为定位群体，在黄瓜Chr6上定位到一个控制涝胁迫下不定根数的耐涝主效QTLARN6.1。通过精细定位，基因表达及黄瓜转基因分析初步确定一个编码AAA ATPase蛋白的CsARN6.1推测为黄瓜耐淹涝候选。本项目通过对12000株超大F2分离群体重组株鉴定及多态性分子标记开发，最终将主效QTLARN6.1精细定位至8.1kb区间，仅含有2个候选基因，均编码AAA ATPase蛋白。基因克隆及序列发现，仅CsARN6.1在双亲间存在序列差异。基于黄瓜花叶病毒体系的VIGS实验发现，沉默CsARN6.1的黄瓜涝胁迫后不定根形成数量显著降低。依据350份黄瓜自然群体的单倍型分析发现，CsARN6.1存在三个单倍型，且5’UTR的InDel（TC/T）和第二外显子上的InDel（A/ACGTGGG）变异对CsARN6.1调控涝胁迫下黄瓜不定根形成的功能起关键作用。运用酵母单杂、EMSA、双荧光素酶等技术方法发现，乙烯信号通过CsEIL3蛋白与CsARN6.1基因启动子上的ATGTATCT元件特异性结合，从而转录激活CsARN6.1基因的表达。运用pull-down结合质谱鉴定、酵母双杂、双分子荧光互补等技术方法发现，CsARN6.1蛋白通过第三家族过氧化物酶Csa6G495000互作，从而诱导黄瓜下胚轴中H2O2积累，从而激活下游一系列与不定根形成相关的基因表达，以促进不定根原基形成与伸长。上述研究结果不仅揭示了AAA ATPase蛋白调控黄瓜耐涝性的新机制，也为深入开展黄瓜耐涝性分子遗传改良提供新的基因资源和理论基础。