microRNA调控红树植物无瓣海桑抗盐作用和机制研究

It is a prerequisite to illuminate the salt-tolerance mechanism of plants in resistance breeding and alleviation of soil salinization. As a typical salt-producing plant, mangrove is often used as a typical woody material for plant salt tolerance research. The salt-tolerant mechanism of mangrove is particularly complex, and how the related genes are regulated remained to be further explored. microRNA (miRNA) has been demonstrated to be the important element in the regulation of gene expression of the plant stress response. In order to deeply analyze the regulation mechanism of miRNA in the salt tolerance of mangrove, the southern coastal preponderant mangrove, Sonneratia apetala Buch.-Ham, is chose as the research object. Firstly, the high-throughput small RNA sequencing technology and bioinformatics methods are used to the miRNAs involved in the regulation of salt tolerance, and then constructs the genetic regulatory network between miRNA and target genes under the salt stress. Then, the regulation of key candidate miRNAs on the salt tolerance for Sonneratia apetala Buch.-Ham are verified by using stem-loop PCR, overexpression and RNAi technology. This project can not only improve the salt-tolerant molecular regulation mechanism of higher woody plants, but also lay a foundation for forest salt-tolerant breeding and provide powerfully technical support and theoretical guidance for the ecological construction in coastal saline-alkali areas.

阐明植物的抗盐性机制是进行抗性育种、缓解土壤盐渍化问题的前提。红树作为典型的盐生高等植物，是植物抗盐性研究的典型木本材料。红树植物抗盐性机制尤其复杂，相关基因如何被调控，还有待探索和揭示。microRNA（miRNA）已被证明是植物逆境胁迫应答中调控基因表达的重要元素。为了深入剖析miRNA在红树植物抗盐性中的调控机制，本项目以南部沿海优势红树植物无瓣海桑（Sonneratia apetala Buch.-Ham.）为材料，首先利用高通量小RNA测序技术和生物信息学方法，鉴定参与调控无瓣海桑耐盐性的miRNA并构建盐逆境胁迫中miRNA与靶基因的遗传调控网络。然后利用stem-loop PCR、过表达及RNAi技术，验证关键候选miRNA对无瓣海桑抗盐性的调控作用。该研究不仅能完善高等木本植物抗盐性分子调控机制，为林木抗盐育种奠定基础，还能为滨海盐碱地区生态建设提供有力理论指导和技术支撑。

红树作为典型的盐生高等植物，是植物耐盐响应与适应机制研究的理想材料。探究红树植物的耐盐性调控机制，对红树抗盐型品种选育及沿海滩涂植被恢复具有重要意义。miRNA被证实在植物逆境胁迫应答中具有重要的调节作用。为揭示miRNA在红树植物中的抗盐作用及调控机制，本项目以无瓣海桑为材料进行盐胁迫响应特性研究，结果显示，无瓣海桑生长速度、净光合速率及抗氧化酶活性等在胁迫处理后均发生了明显变化。在此基础上，首先利用PacBio三代测序技术获得平均长度为1,418 bp的295,501个无瓣海桑全长转录本序列，进而借助小RNA测序技术鉴定获得261个miRNA，包含197个已知miRNA和64个新miRNA。特征分析显示，无瓣海桑miRNA长度分布在18~24 nt之间，且保守miRNA表达水平相对新发现miRNA较高。通过比较分析盐胁迫处理前后miRNA的表达特性，发现共有113个miRNA表达量发生显著差异。进一步对差异表达miRNA的靶基因进行预测，共获得631对miRNA-靶基因调控关系，对应105个miRNA和573个靶基因。功能注释分析显示这些靶基因能够参与调控植物信号转导、离子平衡、解毒、渗透保护等胁迫应答反应过程。通过对miRNA在物种间的差异性进行分析，共获得62个在无瓣海桑响应盐胁迫过程中具有物种特异性的miRNA，包括45个已知miRNA和17个新miRNAs。结合miRNA和靶基因转录表达数据，构建完成34个miRNA和131个靶基因的共表达调控网络，涉及140个miRNA-靶基因作用对，其中13个miRNA的18个靶基因被注释到与环境信息处理相关的代谢通路。最后利用RT-qPCR技术，对miRNA和miRNA-靶基因作用对在表达水平上进行真实性和调控关系验证。本项目研究结果对完善木本植物抗盐机理具有重要的学术价值，同时为红树植物抗盐遗传改良及滨海盐碱地区生态建设提供有力指导。