不同磷效率杉木基因型响应低磷胁迫的miRNA及其多肽调控机制

The situation of plantations of Chinese fir facing with effect of low phosphate in soil is increasingly severe recently. Insight into the adaptive strategy and regulatory mechanism during low-phosphate stress is important to improve the phosphorus-use efficiency in Chinese fir. Early research has shown that 6 miRNAs (Cln-miR166, Cln-miR827 and et al.), a kind of important genes of negative regulatory function, were significantly responsive to low-phosphate stress in Chinese fir. However, little is known about how they response and effect during low-phosphate stress in Chinese fir. In this research project, two low-phosphate-resistant genotypes (genotype that initiative to capture the soil phosphorus & genotype that passive to endure low-phosphate) annual seedlings of Chinese fir were used. The annul seedlings grow in water culture solution with control and low-phosphate treatment. Expression pattern analysis of similarities and differences between different genotypes and treatments were conducted for miRNAs, miRNA precursors, and target genes. It is reported that the miRNA peptide could induce the expression of downstream miRNA. Then the annual seedlings were treated with both low-phosphate condition and miRNA peptide solution. The expression pattern and regulatory mechanism of induced miRNAs and their targets were identified. The targets would be further proved by 5’ RACE. Through implementation of this project, it will increase our knowledge about response process of miRNA by low-phosphate stress, establish a method to prove the function of miRNA using miRNA peptide, and lay the foundation for the understanding of adaptation strategy and regulation mechanism to tolerance to low-phosphate stress in Chinese fir.

近年来杉木林土壤有效磷缺乏日益严重，解析杉木适应低磷胁迫的策略和调控机制对提高杉木的磷利用效率极为关键。课题组前期研究表明：作为一类重要的负调控基因，Cln-miR166、Cln-miR827等6个miRNA在杉木适应低磷胁迫中存在显著响应表达的现象。但目前对这些关键miRNA是如何参与应对低磷胁迫的调控机制还不清楚。本项目以“主动捕获土壤磷”和“被动忍受低磷”的杉木基因型为研究材料，开展低磷胁迫条件下不同磷效率基因型对低磷胁迫响应的关键miRNA、miRNA前体和靶基因表达模式的差异研究。利用miRNA多肽在杉木中诱导关键miRNA，研究其靶基因表达，并通过5‘RACE技术验证，探讨耐低磷杉木体内miRNA的响应和调节过程，完善以miRNA多肽技术为基础的杉木miRNA功能研究方法，揭示不同磷效率杉木响应低磷胁迫的多肽调控机制，为阐明耐低磷杉木基因型高效利用土壤磷的内在机制提供科学依据。

中国南方人工林以为杉木种植为主，但经过多代连栽后出现产量下降的现象。其土壤有效磷的缺乏日益严重是导致这一现象的重要原因之一。而miRNA在植物体内磷平衡和低磷响应过程起着重要的作用。本项目从杉木自身响应miRNA基因的调控应对土壤环境缺磷的分子机制方面着手研究。利用沙培的控制元素供给的方法模拟土壤缺磷的条件，筛选杉木中低磷胁迫响应的miRNA基因并探究其前体和靶基因，及其前体和靶基因在低磷胁迫下的响应表达模式，最终探究miRNA调控通路在低磷胁迫中的分子机制。本研究筛选出杉木5个miRNA（miR399d、miR166e、miR169a、miR1、和miR2）在低磷胁迫下上调表达；鉴定了它们的前体序列并探究其前体在低磷胁迫下的响应表达情况，其中miR166e前体在低磷胁迫下下调表达而miR2前体在低磷胁迫下上调表达；通过miR166e前体序列中设计miRPEP166e多肽浇灌杉木幼苗发现其对miR166e成熟体表达无显著影响。最后利用杉木降解组数据鉴定它们的靶基因，其中miR166e的3个HD-ZIP靶基因（1B166、2B166、4B166）和miR169a的2个NF-YA靶基因（1B169、4B169）是最有可能的靶基因。其中1B166、2B166、4B169靶基因在低磷胁迫中上调表达。这些靶基因都是杉木中重要转录因子，因此低磷胁迫响应的miR166e和miR169a可能通过其靶基因参与低磷胁迫调控的通路。该研究有助于了解杉木响应低磷胁迫的分子机制为后期深入研究提供一定的依据。