大豆高pH盐碱胁迫下MicroRNA1510的鉴定、表达分析及其调控作用机制研究

Soybean [Glycine max (L.) Merrill], one of the most important oil and grain crops in China, plays an important role in agriculture industrial structure and people's lives. However, natural calamities, especially the high pH alkalinity limit soybean growth and productivity dramatically. Therefore, an increase in high pH saline-alkaline tolerance of soybean is desirable and has been a major goal of variety breeding. MicroRNAs (miRNAs) are a class of single-stranded small RNAs, involved in regulation or changing of the endogenous target gene expression via post-transcriptional degradation or post-translational suppression mechannism, which are extensively focused by scientists in the world. Previously, we identified miRNA1510 in soybean which was differentially expressed under the saline-alkali stress. In this project, in order to further clarify the role of miRNA1510 involved in the regulation mechanism of high pH saline-alkaline tolerance of soybean, target gene of miRNA1510 was aimed to be predicted and verified firstly. Then，to further study the interaction of miRNA1507 and its target gene, the transcripts of both miRNA1510 and its target gene in roots and leaves during a time course under saline-alkali stress are to be assayed. Furthermore, overexpression and reverse genetic technology are to be carried out to further ascertain the important role of miRNA1510 involved in the regulation of saline-alkaline tolerance of soybean. This study can serve as a basis for further study of the molecular mechanism of saline-alkaline tolerance in soybean; on the other hand, it will provide a starting point to establish molecular tool of miRNA to increase saline-alkaline tolerance of soybean by genetic engineering.

大豆是我国重要的粮油作物，在农业产业结构和人民生活中占有重要地位。但由于自然灾害，尤其是我国东北地区西部高pH盐碱的危害，其产量受到极大影响。因此，提高大豆耐高pH盐碱的能力，培育耐盐碱大豆新品系已迫在眉睫。MicroRNA是植物中一种单链小分子RNA，通过在转录后水平介导靶mRNA降解或翻译后抑制来调节或改变内源基因表达，是目前分子生物学研究的热点之一。本项目在鉴定大豆高pH盐碱胁迫下差异表达miRNA1510基础上，预测并验证其靶基因，深入研究它们在高pH盐碱胁迫下的时空表达情况,探明它们之间的相互作用；同时利用超表达和抑制miRNA1510表达等反向遗传学方法进一步分析miRNA1510的功能，从而明确miRNA1510在调控大豆耐高pH盐碱分子机制中的作用。该研究一方面为深入探索大豆耐高pH盐碱分子机制奠定基础；另一方面为利用基因工程手段培育耐盐碱大豆新品系提供候选miRNA基因。

植物microRNA（miRNA）是植物中广泛存在的一类非编码的小RNA分子，它不仅对植物的生长和发育有巨大的调控作用，而且能促使植物对多种生物和非生物胁迫处理产生应答。本项目在获得干旱等逆境胁迫下miRNA 转录组测序数据和全面分析基础上，选择在处理前后表达量变化相对明显的miR1510a基因家族作为研究对象进行分析。. miR1510a是目前仅在某些豆科植物中发现的一个非保守的miRNA，但其在大豆响应逆境胁迫中的作用仍然未知。本项目利用在线分析软件预测到gma-miR1510a-5p的靶基因为PPF-1，gma-miR1510a-3p的靶基因有24个；利用qRT-PCR、改良的5′RACE技术和原生质体瞬时转化鉴定出gma-miR1510a与靶基因确有互作关系；对转gma-miR1510a前体基因拟南芥表型分析发现，150 mM NaCl和300 mM甘露醇处理后，转miR1510a基因拟南芥的发芽率及长势均优于野生型植株。而烟草花叶病毒侵染后，野生型拟南芥生长状况要优于转基因型。这说明miR1510a可以正调控植物对盐及干旱胁迫的耐受，而在病原菌侵染过程中则起着负调控作用。另外，利用农杆菌介导的子叶节遗传转化法将miR1510a转化至大豆中，并获得14株T1代转基因大豆植株，这为miR1510a在大豆中进一步的分子机制研究奠定了基础。. 通过该项目的实施，共发表标注有该项目资助的SCI论文8篇；授权发明专利1项；获得吉林省科技进步奖一等奖等3项；在此基础上获得国家转基因生物新品种培育重大专项子课题1项；培养博、硕士研究生共9名，超额完成了合同规定的任务指标。