miR159和miR169在甘蓝型油菜干旱应答中的功能分析

Drought is a major environmental factor that restricts the production and quality of Brassica napus. As a group of crucial non-coding small RNA molecules, miRNAs play a vital role in the regulation of gene expression in plant response to abiotic stress and the process of growth and development. Although many plant miRNAs have been discovered in recent years, their biological functions and mechanisms need further investigation. In our previous study, we constructed small RNA libraries of B. napus under normal and drought stress conditions, and preliminarily identified 50 drought-responsive miRNAs using high-throughput sequencing analysis. Based on these identified miRNAs, we choose miR159 and miR169 as our candidates in the current study. We will predict and verify the target genes of miR159 and miR169 using bioinformatics approach and RLM-RACE technology, and establish the expression pattern of the candidate miRNAs and their targets by qPCR. We will also perform functional verification by genetic transformation to illustrate their function through various aspects,such as phenotyping under drought stress, physiological response, and molecular regulation. The project will decipher the molecular regulatory mechanism of candidate miRNAs and their targets in response to drought stress in B. napus, and lay the theoretical foundation of drought-resistance breeding of B. napus.

干旱胁迫是制约甘蓝型油菜产量和品质的重要环境因子。miRNA作为一种关键的非编码小 RNA分子，在植物的逆境应答和生长发育相关的基因表达调控中发挥着至关重要的作用。虽然近年来已有许多植物miRNA被发现，但是它们的生物学功能和作用机制还需要更深入的研究。在前期研究中，我们构建了甘蓝型油菜在正常和干旱胁迫条件下的small RNA文库，并对其进行了高通量测序分析，初步鉴定出50个受干旱胁迫诱导表达的miRNA。在此基础上，本项目拟将miR159和miR169作为重点候选研究对象，利用生物信息学和RLM-RACE手段鉴定和验证它们的靶基因，通过qPCR方法分析候选miRNA和靶基因的表达模式，通过转基因技术对它们进行功能验证，从表型、生理反应以及分子调控水平对它们进行深入研究，探索候选miRNA及靶基因在甘蓝型油菜对干旱胁迫响应过程中的分子调控机制，为甘蓝型油菜的抗旱育种提供理论依据。

干旱胁迫严重影响甘蓝型油菜的正常生长发育和产量，危害油菜生产。miRNA在调控植物对干旱的应答方面具有重要调控作用。本项目以利用small RNA-Seq技术鉴定出的在正常和干旱胁迫条件下显著差异表达的miR159和miR169作为研究对象，结合qPCR、转基因功能验证、酵母生化分析等手段,对miR159和miR169在甘蓝型油菜干旱应答中的生物学功能进行了分析。利用生物信息学方法和降解组数据分析鉴定得到了miR159的靶基因BnbHLH62、BnMYB65和BnMYB81，以及miR169的靶基因BnNF-YA22、BnNF-YA23、BnNF-YA27。利用转录组分析和qPCR技术检测了miR159和miR169及其靶基因在逆境胁迫条件下的表达模式。研究显示，过表达miR159a和miR159c能够增强转基因植株对渗透胁迫的抗性，其靶基因编码蛋白BnbHLH62、BnMYB65和BnMYB81均定位于细胞核中，BnMYB65全长蛋白没有转录激活活性，BnbHLH62和BnMYB81全长蛋白具有转录激活活性，BnMYB81转录激活活性位于蛋白的C端，而BnbHLH62蛋白的N端和C端序列均对其转录激活活性的维持具有作用。过表达miR169g能够增强转基因植株对ABA的敏感性和对渗透胁迫的抗性，BnNF-YA22、BnNF-YA23、BnNF-YA27定位于细胞核中，全长蛋白均不具有转录激活活性，BnNF-YA23蛋白可能通过与EIN2和PAP6互作参与逆境应答。本研究初步阐明了miR159和miR169在甘蓝型油菜干旱应答中的生物学功能，为甘蓝型油菜抗旱性改良的分子遗传育种提供了新的途径。