黄瓜涝胁迫下不定根数基因精细定位及功能鉴定

Cucumber (Cucumis sativus L.) is easily damaged by waterlogging because of the absence of developed taproot,which might inhibit the oxygen assimilation. Isolation and identification of waterlogging tolerant genes, genetic germplasm resources innovation based on molecular biology are effective ways to improve the waterlogging tolerance, and finally get high yield and good quality of cucumber. Adventitious root formation is one of the most important adapted morphology to waterlogging stress, and plays a central role in waterlogging resistance of cucumber. Previously, we mapped QTLs of the adventitious root number(ARN)of cucumber by using ISSR and SRAP marker. However, as the publication of cucumber genome sequencing, the SSR markers which allocated in the cucumber chromosome were identified. In present research, the gene related to ARN will be fine mapped based on F2 and F2:3 populations by using SSR markers and SNP and inDel which would be developed by genome resequencing of parental lines. By combination of digital gene expression profile and candidate gene methods, the candidate genes of ARN will be predicted. To verify the function of candidate genes, RNAi combined transient expression methods will be applied. The research results will help us to understand the fundamental mechanism of waterlogging resistance in cucumber, and ultimately improve the cucumber waterlogging resistance by genetic manipulation and breeding.

黄瓜主根不发达，好气性强，极易受到淹涝胁迫危害。分离鉴定耐涝基因，利用现代分子生物技术手段创新种质，选育耐涝品种是克服黄瓜淹涝危害、生产优质高产黄瓜的有效途经。黄瓜淹涝胁迫下不定根形成能力决定其耐涝性强弱。本实验室前期已利用ISSR和SRAP标记对该性状进行了初步定位，随着黄瓜基因组测序的完成及大量SSR分子标记的公布，本项目拟以耐涝品系和敏涝品系构建的F2和F2:3群体为基础，对淹涝后不定根数进行基于SSR、SNP和inDel标记的精细定位。预期结果将结合黄瓜基因组数据库信息和前期高通量数字基因表达谱预测目标区段内的候选基因，通过RT-PCR获得基因表达的初步证据，进一步构建候选基因过量表达和RNAi载体，利用农杆菌介导法进行遗传转化确证基因功能。研究结果不仅对阐明黄瓜耐涝机制具有重要意义，也将为黄瓜耐涝性种质创新和新品种选育奠定重要基础。

黄瓜为我国露地和设施栽培的主要蔬菜作物之一。随着全球气候变暖和雨涝灾害的频繁发生，涝已成为危害作物生长的重要制约因子。黄瓜根系具有入土浅、通气组织不发达、吸收力弱、再生能力差等特点，极易受到涝胁迫危害，造成产量损失和品质下降。因此，探明黄瓜对涝胁迫的遗传与适应机制，分离鉴定耐涝关键基因，创新耐涝种质和选育耐涝品种，对生产优质高产黄瓜具有重要理论意义和应用价值。选择精确可信且能够稳定遗传的表型是定位耐涝基因及解析耐涝机制的关键。本课题组前期通过对大量不同生态类型的黄瓜遗传资源耐涝性鉴定，筛选出耐涝品系Zaoer-N（华南型）和不耐涝品系Pepino（美国露地型）。对这两个品系连续多年淹涝观察均发现Zaoer-N的下胚轴形成大量不定根，而Pepino下胚轴上只有极少量的不定根的形成。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原生根死亡，有利于受涝器官获得氧气。本基金以Zaoer-N和Pepino为材料，利用RNA-seq和iTRAQ比较了两者下胚轴淹涝48h后全基因组在基因转录水平和翻译后蛋白水平上的差异；以不定根数（Adventitious Root Numbers，ARN）为表型，对黄瓜耐涝性进行了遗传分析及QTL初步定位。在此基础上，通过双亲重测序开发KASP/dCAPs标记鉴定大群体F2中的重组株，精细定位了主效QTLARN6.1并对目标基因进行了图位克隆及功能鉴定，最终获得一个编码AAA-ATPase（ATPase associated with various cellular activities）的关键候选基因，命名为CsARN6.1。在黄瓜耐淹涝品系Zaoer-N中，CsARN6.1基因通过增加不定根的产生来促进黄瓜对淹涝的适应。这种不定根增加的效应可能是Zaoer-N中CsARN6.1蛋白的coiled-coil 结构域的一个非同义SNP突变导致的。CsARN6.1基因调控不定根数依赖于其ATP酶的活性。在拟南芥中异源表达CsARN6.1基因导致种子萌发后主根生长速度显著快于对照株系，且侧根形成时间更早。将来源于Zaoer-N中的CsARN6.1基因转入与不耐涝亲本Pepino等位基因遗传背景一致的品系后，其在淹涝状态下比对照表现出显著的不定根数量增加。