核桃复幼提高生根能力的多基因作用机制解析

Walnut (Juglans L.) propagation by cutting is difficult to realized, especially for the mature cultivars. Rooting of softwood cutting from mature walnut cultivars was successfully achieved in our research with the rejuvenation treatments of buried stem etc., the rooting rate was over 95% (Invention patent), but its molecular basis remains largely unknown. To address this question, we examined the transcriptome of softwood cuttings of rootstock cultivar ‘Zhongningsheng’ treated with or without rejuvenation at the critical period of adventitious rooting respectively. Moreover, transcript assembly and differential expression analysis were performed preliminarily. In this study, a strategy of multi-genes and systems biology will be adopted, further differential expression analysis will be conducted, approaches of gene network analysis such as the WGCNA method will be used to construct the co-expression network, functional module segmentation will be performed, the co-expression network orientation of functional modules will be identified with the methods such as D-NetWeaver, GO (Gene Ontology) and pathway enrichment of the modules will be performed to make functional classification and interpretation, the main regulator or functional modules of the network will be determined through the differential network analysis in the process of adventitious rooting and in mature and rejuvenated softwood cuttings, and functional verifications of the main regulator genes are conducted with the in vitro rooting system of walnut cotyledon and other rooting systems. The aim of this research is to answer the question of how dose the transcriptional pattern change and which important regulatory factors and functional gene modules are involved in the rooting promoting by the rejuvenation treatment. The result of this study is of great significance to enrich the theory of rejuvenation and adventitious rooting of woody plants and to guide practice of cutting propagation.

核桃（Juglans L.）扦插繁殖困难，成龄核桃品种则更难。我们通过埋干等复幼处理，成功地诱导成龄核桃品种生根，扦插生根率可达到95%以上（发明专利），然而对其分子机制还缺乏了解。为此，我们选取核桃砧木品种‘中宁盛’的成龄和复幼嫩枝插穗，分别获得其不定根发生关键时期的组织样品，进行了转录组深度测序，开展了转录本组装和初步的差异表达分析。本研究拟采用多基因的系统分析策略，进一步筛选差异表达基因；利用WGCNA等方法构建共表达网络并进行功能模块切分；通过D-NetWeaver等方法对功能模块的共表达网络进行定向，对功能模块进行GO (Gene Ontology)功能富集和代谢通路分析；通过不同状态下调控网络的差异分析确定主调控因子；利用已有的核桃子叶不定根发生等体系，对主调控基因进行功能验证，以期回答埋干等复幼处理究竟改变了那些重要调控因子及基因功能群进而提高了不定根发生能力的科学问题。

优良砧木无性繁育体系的建立，是实现经济林产业整株无性系化的关键。然而，核桃等经济林树种很难产生不定根，特别是随着树龄的增长，其不定根发生能力显著降低甚至完全丧失，严重制约了优良砧木良种的推广应用。前期，本课题组创新性地通过连续嫁接和埋干等复幼技术，成功地诱导了成龄核桃品种的不定根发生，扦插生根率可达到95%以上（发明专利授权号：ZL200410092014），但是对于其中的分子仍缺乏了解。为了系统、全面地探索埋干等复幼提高不定根发生能力的分子机制，我们以核桃砧木品种‘中宁盛’的成龄和复幼插穗为试材，从多个层面开展相关研究，并取得如下主要结果：①通过连续切片观察确定了复幼插穗的不定根原基起源于髓射线正对的形成层和韧皮部交叉区域，明确了核桃不定根发生的关键时期，利用原位免疫胶体金技术分析了IAA、GA3、ABA和ZR在不定根发生过程中的时空分布，并提出了复幼促进核桃不定根发生的多激素作用模型；②通过RNA-Seq技术捕获复幼和成龄插穗不定根诱导过程中的基因时序表达数据，利用高纬微分方程和变量选择相结合的方法，在功能模块和基因两个层次构建了基因调控网络，挖掘出复幼促进核桃不定根发生过程的5个功能模块和35个核心基因，包括参与生长素代谢和信号转导调控的基因BGL和AGD3，参与细胞扩张、形态发生和发育的基因WAK2，参与水杨酸和茉莉酸信号途径的基因GRXC1等；③ 建立了稳定、较高效的核桃体胚发生和遗传转化体系，体胚诱导率和转株率分别达到86%和53%以上，转化效率达到20%以上；④ 在全基因组范围内鉴定出12 个WOX家族基因，在‘84K’杨中过表达JrWOX4、JrWOX5和JrWOX11基因，转基因植株均表现出不定根发生能力增强的表型。本项目研究结果不仅可以回答复幼处理究竟改变了那些重要调控因子及基因功能群进而提高了不定根发生能力的科学问题，而且对于丰富和发展树木复幼、不定根发生理论及指导实践均具有重要意义。