蒺藜苜蓿miR2687调控细胞壁代谢酶基因XTH及其响应重金属汞的机理分析

Functional analysis have demonstrated that miRNA plays an important role in plant resistance to abiotic as well as biotic stresses. Recently, we identified a species-specific miR2687 and its target xyloglucan endotransglucosylase/hydrolase (XTH), a cell wall metabolism enzyme，from M. truncatula. Our previous experiments have demonstrated that miR2687 was down-regulated while its target XTH was differentially up-regulated in expression under Hg stress in M. truncatula, suggesting that miR2687 likely negatively regulates XTH. In this study, we will construct overexpression vectors of the miR2687 and XTH with specific site mutation and transform them into M. truncatula, respectively. By the methods of northern blot and in situ hybridization etc., the expressions of miR2687 and XTH, the characteristics of phenotype and mechanisms of physiology and biochemistry will be studied in the control and transgenic plants under heavy metal Hg stress. The results will facilitate our better understanding the regulatory function of miR2687 and the role of XTH in cell wall metabolism under the control and Hg stress. The study also may provide the evidences in investigation of regulatory mechanisms for plant tolerance to stresses.

国内外研究表明miRNA在植物抗逆中具有重要的调控作用。最近，申请人鉴定了蒺藜苜蓿特异的miR2687及其靶基因- - 木葡聚糖内糖苷转移/水解酶XTH(-种细胞壁代谢酶)，初步研究表明，汞胁迫下miR2687表达下调、XTH表达上调，呈现负相关。本项目拟通过构建miR2687和抗miR2687-XTH（定点突变）两个过表达载体，转化获得蒺藜苜蓿转基因植株，结合northern blot、原位杂交等手段探索miR2687和XTH的时空表达模式、转基因植株的表型变化及其在重金属汞胁迫下的生理生化反应。项目研究结果可以阐明miR2687调控靶基因XTH的机制和XTH在细胞壁代谢中的功能，并可以揭示miR2687和XTH在植物耐受重金属汞胁迫中所发挥的作用，为植物抗逆机制研究提供理论依据。

已有研究表明，miRNA可以通过切割其靶标mRNA或者抑制其靶标蛋白翻译调控植物生长发育或响应各种生物或非生物胁迫。miRNA及靶标的鉴定成为首要任务，我们综合公布的46个sRNA和5个降解组文库，从蒺藜苜蓿鉴定到了24个新miRNA和133个I型编码基因靶标及91个I型转座子靶标，分析了它们在Hg胁迫下的表达差异。木葡聚糖内糖苷转移/水解酶（XTH) 是一类细胞壁代谢酶，其影响细胞分化、扩展和细胞特化等多个生物过程和对多种胁迫的响应。我们在蒺藜苜蓿全基因组水平上鉴定了44个编码XTH蛋白的基因，并发现多数响应高盐、干旱和Hg等胁迫。重点研究了miR2687预测的靶标——MtXTH3，组织表达分析表明其在蒺藜苜蓿成苗成熟叶片和节间表达强烈，这与我们采用启动子和GUS融合技术进行的组织定位结果一致。Hg胁迫下该基因表达上调。35S::MtXTH3过表达材料叶形发生了变化，对盐的敏感性增强，但Hg胁迫下表型没有发生明显变化。通过本项目的研究，丰富了蒺藜苜蓿miRNA及其靶标和XTH鉴定结果，初步阐明MtXTH3的功能，揭示了它们对重金属汞等胁迫的响应，为揭示植物抗逆机制提供了理论依据。