薄壳山核桃CiARF5基因调控嫁接体砧穗愈合的分子机理研究

Pecan, as a member of the Juglandaceae family, has low rates of graft success. Low rates of graft success is an important factor that hampers the industrial process of pecan. In former researches, we found that the upregulation of pecan ARF5 gene on the stage of vascular formation between scion and rootstock was involved in successful grafting. However, potential regulation roles of ARF5 underlying graft union healing process are still unclear. In this research, full length cDNA of CiARF5 will be cloned through RACE, based on our former RNA-seq data. Overexpressing CiARF5 vectors will be constructed and transformed into Arabidopsis thaliana to identify its functions. The potential down-stream regulators of CiARF5 will be screen from our previous RNA-seq dates, and then be confirmed by co-expression analysis and yeast one-hybrid assay, so as to reveal the biological processes in which CiARF5 is involved. Meanwhile, the promoter + GUS vectors of CiARF5 will be transformed into Arabidopsis thaliana, and the cis-elements of CiARF5 would be evaluated following grafting. These results will reveal the regulation mechanism of CiARF5 on graft union healing, and provide scientific reference for grafting improvement of pecan, which has wide prospects of application.

薄壳山核桃是一种嫁接难成活的胡桃科树种，嫁接成活率低是限制该树种产业化发展的一个重要因素。申请人在前期的研究中发现，薄壳山核桃ARF5基因（CiARF5）在砧穗间维管组织形成期的上调表达与嫁接成活相关。但是，ARF5在嫁接体砧穗愈合过程中的调控机理仍不清楚。本申请拟以前期构建的RNA-seq测序数据为基础，利用RACE技术克隆CiARF5基因的cDNA全长；构建CiARF5超表达载体转化拟南芥，分析其功能；在转录组数据中，挖掘出受CiARF5调控的潜在下游基因，并利用实时荧光定量、酵母单杂技术进行验证，以揭示CiARF5在植株体内参与的生物学过程；构建启动子融合GUS基因的载体在拟南芥中转化，进行嫁接后，分析CiARF5基因的响应元件。研究结果将揭示薄壳山核桃CiARF5基因在嫁接体砧穗愈合中的调控机理，并为嫁接技术的改进提供科学依据，应用前景广阔。

嫁接是进行薄壳山核桃扩繁的重要手段，生长素在嫁接愈合过程中起主导作用。生长素响应因子（Auxin response factor，ARF）在生长素信号传导过程中处于中心位置，为生长素信号传导途径中的关键转录因子。本研究对ARF其中的一个成员CiARF5调控薄壳山核桃嫁接愈合的分子机理进行系统研究。首先，为筛选适合薄壳山核桃嫁接愈合分析的内参基因，选择了17个已报道的内参，使用实时荧光定量PCR（RT-qRCR）技术对它们在薄壳山核桃中的表达稳定性进行评价，结果发现2个内参基因组合26S proteaseregulatory subunit 7A (PR26S)与Serine/threonine-protein phosphatase-1 (PP1)在嫁接愈合中的稳定性最高。在此基础上，使用RT-qPCR检测薄壳山核桃ARF（CiARF）家族基因在砧穗愈合过程中的表达模式，结果显示CiARF5基因仅在接穗中持续上调表达，表明该基因参与接穗的愈合反应。为探索接穗在嫁接愈合过程中主要参与哪些愈合反应，从解剖、生理生化、转录表达方面分析了接穗在嫁接愈合中的变化，结果显示接穗主要参与低氧、高温、病菌入侵的逆境响应，推测在接穗中高表达的CiARF5基因功能是抵御嫁接引起的逆境胁迫。对CiARF5基因的启动子进行克隆和元件预测，发现存在低氧诱导的作用元件。为验证CiARF5基因是否参与抵御嫁接引起的低氧胁迫，克隆CiARF5基因的ORF全长，构建超表达载体转化烟草，结果发现在低氧胁迫（水涝）处理下，转基因烟草中的PDC和ADH酶活性显著高于野生型。经加权基因共表达网络分析（WGCNA），预测CiARF5互作的基因有homeobox-leucine zipper protein (HD-ZIP，同源异型-亮氨酸拉链蛋白)、pathogenesis-related protein (PR，病程相关蛋白)、heat shock factor protein (热激因子蛋白)、1-aminocyclopropane-1-carboxylate oxidase (ACO，1-氨基环丙烷羧酸氧化酶)等，CiARF5可能调控抗性相关的生物学通路。综合以上结果表明，CiARF5基因在薄壳山核桃嫁接愈合过程中的功能可能是抵御接穗遭受的低氧胁迫。