黄瓜/南瓜嫁接亲和性基因的筛选与鉴定

Grafting is widely practiced for improving tolerance of cucumber to biotic and abiotic stresses. Whereas the role of graft compatibility is not well understood, and it has been seriously constraint to develop a rapid screening of high affinity cucumber rootstock technology by molecular identification means. So the identification of gene controlling graft compatibility is top priority. In this study, cucumber is grafted on rootstock of compatible and incompatible pumpkin. We have identified series of histological changes during graft union formation. Through Laser Capture Microdissection and RNA sequencing technique, we will analyse gene changes respectively in vascular bundles and parenchyma cell adjacent to the graft junction. The proposal will identify the potential of related genes involved in vascular reconnection. In addition, we will explore the genetic effect characteristics of graft compatibility in genetic populations and locate compatibility gene precisely through high-throughput sequencing-based bulked segregant analysis (BSA-seq) and conventional genetic method. Combining the identified novel gene to interface healing with compatible gene location, to identify the key gene and substantiate their function through transgenic technique. The proposal will elucidate initially the mechanism of cucumber/pumpkin heterograft compatibility, and found theoretical bases for establishing a novel technique for rapid screening of high affinity cucumber rootstocks by molecular identification.

嫁接可以提高黄瓜对生物和非生物胁迫的抵抗能力，在设施黄瓜生产中已大面积应用。但嫁接亲和性分子机制尚不明确，制约了快速筛选高亲和性黄瓜砧木分子鉴定技术的开发。因此，探明黄瓜嫁接亲和性基因成为解析其亲和性机制这一科学问题的首要策略。本项目以黄瓜(接穗)和与其亲和性高低的两个南瓜品种(砧木)为试材，在已明确划分黄瓜/南瓜嫁接接口愈合时期的基础上，采用激光显微切割(LCM)技术，对亲和性高低的嫁接组合接口愈合各时期的砧木和接穗，进行组织水平转录组分析，明晰主要代谢通路，确定候选的亲和性基因。同时，构建遗传分离群体，采用BSA-seq和传统遗传学手段对亲和性基因进行精细定位，并结合接口愈合候选基因，通过转基因功能验证，确定影响嫁接亲和性的关键基因。项目可初步阐明黄瓜/南瓜嫁接亲和性的分子机制，为开发快速筛选高亲和性黄瓜砧木的分子鉴定技术奠定基础。

嫁接亲和性分子机制尚不明确，制约了快速筛选高亲和性黄瓜砧木分子鉴定技术的开发。嫁接接口愈合是决定嫁接亲和性的关键事件。本项目以黄瓜为接穗，以南瓜为砧木，系统的研究了嫁接方法对黄瓜/南瓜嫁接接口愈合生理特征影响、黄瓜/南瓜嫁接接口愈合的生理特征及转录组分析、糖及CmANT1.2在嫁接接口愈合中的作用机制。发现相比较于插接法和贴接法，靠接法在嫁接接口愈合期具有较高的日生长速率及较早的维管束重连，可能与其维持较高的抗氧化酶活性及较低的木质素含量有关；黄瓜-南瓜嫁接接口愈合过程中，愈伤组织优先在接穗一侧出现，韧皮部重连早于木质部重连2d，发生在嫁接后3d；转录组分析表明，接穗和砧木在嫁接接口愈合中转录响应水平不同，特别是糖代谢、分生组织发育及植物激素相关基因；相比于WT嫁接体，含糖量低的黄化苗嫁接体接口愈合中的韧皮部和木质部重连均延迟，嫁接成活率降低，生物积累量降低，而喷施外源糖(5%葡萄糖)对此有所缓解，即促进了木质部重连、嫁接成活率和嫁接体植株的生长，且外源糖显著促进了WT嫁接体生物量积累；TOR抑制剂AZD-8055对嫁接体成活率无显著影响，但使嫁接接口处维持较低的能荷水平，延缓了韧皮部和木质部重连，外源糖的喷施对此有所缓解，表明Glc-TOR信号可能参与嫁接接口愈合；异源过表达CmoANT1.2可增大拟南芥器官大小，特别是果实长度；异源过表达CmoANT1.2可通过加速嫁接接口愈合来促进嫁接体生长，这种促进作用在砧木一侧表现显著。项目初步阐明黄瓜-南瓜嫁接接口愈的分子机制，丰富了嫁接亲和性机制研究，为开发快速筛选高亲和性黄瓜砧木的分子鉴定技术奠定基础。培养硕士研究生1人，博士研究生1人，博士后1人，发表7篇，其中SCI论文5篇，中文核心期刊论文2篇。圆满完成了计划任务和考核指标。