microRNA399调控番茄缺磷胁迫响应的机理研究

Phosphorus (P)-deficiency is a major stress factor that affects the yield and quality of tomato. Tomato microRNA399 (Sly-miR399) is induced by low-phosphorus signal and plays potentially important roles in P-deficiency responses in tomato, therefore, investigation of the biological functions of Sly-miR399 will help elucidate the mechanisms that control the developmental and physiological responses of tomato plants under P-deficiency stress. However, our current understanding of the target genes and regulatory mechanisms of Sly-miR399 is still limited. Previous research in our group found that the sequence of Sly-miR399 is different from its homologues in the model species Arabidopsis thaliana. Furthermore, overexpression of Sly-miR399 in tomato also results in phenotypes that haven’t been reported in the model systems, which suggests that Sly-miR399 might have novel target genes and regulatory mechanisms in controlling P-deficiency responses in tomato. In this study, we plan to first identify the major target genes of Sly-miR399 using degradome sequencing combined with other approaches, and characterize the biological functions of these Sly-miR399 targets by phenotypic analysis of their gain and loss of function mutants. We will also examine the expression patterns of these target genes during plant development, as well as their transcript levels in Sly-miR399 overexpressing (Sly-miR399-OE) and knock-out (Sly-miR399-KO) tomato lines compared to wild type in different P-concentration treatments to further elucidate how these genes function in the Sly-miR399-mediated P-deficiency responses in tomato. Moreover, we will analyze the gene expression profiles of wild type, Sly-miR399-OE and Sly-miR399-KO in different P-concentration conditions using whole transcriptome RNA-sequencing, and also screen the interacting proteins of Sly-miR399 target gene products. Results from these studies will help construct the downstream gene network of Sly-miR399 in the regulation of P-deficiency responses in tomato. We hope our work in this project will further our understanding of the molecular mechanisms that Sly-miR399 utilizes to regulate the developmental and physiological responses of tomato to P-deficiency stress, and also provide novel insights in generating new tools for breeding low-P tolerant tomato lines in the future.

缺磷是影响番茄产量和品质的主要胁迫之一。番茄microRNA399 (Sly-miR399) 受低磷诱导，在调节缺磷响应中发挥潜在作用，分析其功能对阐明番茄响应缺磷胁迫的过程及机理有重要意义，但现有研究对其靶基因及调控机制的解析还几乎空白。申请人前期发现Sly-miR399在序列及超表达植物表型上与模式系统拟南芥的miR399有明显差异，推测其可能存在不同的靶基因及作用机制。因此，本项目将通过降解组测序等方法鉴定Sly-miR399的靶基因，并结合表型及基因表达分析阐释靶基因的功能，尤其在缺磷响应中的作用。本项目还将利用转录组测序分析过表达及敲除Sly-miR399的番茄在不同磷浓度下的基因表达情况，同时结合筛选其靶基因的互作蛋白，深入分析Sly-miR399如何通过调节靶基因及下游遗传网络来参与番茄响应缺磷胁迫的生理及发育变化过程，同时希望上述研究结果为培育磷高效的番茄品种提供理论帮助。

缺磷是番茄生长中面临的主要逆境胁迫之一，对番茄响应缺磷胁迫机理的深入认识可为培育高效利用磷的番茄品种提供理论依据，然而相关的研究还很不系统。番茄microRNA399 (Sly-miR399)在调节缺磷响应中发挥潜在功能，但对其作用机制还缺乏深入探究。本课题基于以上问题，对番茄响应缺磷胁迫的调节通路，以及Sly-miR399在其中所起作用进行了详细研究。我们通过对Sly-miR399过表达株系进行表型检测，鉴定了Sly-miR399在磷利用及信号调节中的重要功能。我们还通过多组学分析探究了野生型番茄在磷缺乏条件下基因及小RNA表达、基因选择剪切和DNA甲基化变化，从中总结番茄缺磷响应的分子机制。并通过将Sly-miR399过表达苗中的基因表达情况与野生型对比，分析Sly-miR399在番茄缺磷响应中的作用机理。此外，我们还利用分子及生化手段研究了磷缺乏对番茄果实品质的影响及调节机理，并通过组学方法比较Sly-miR399过表达果实与野生型果实在磷缺乏条件下的基因表达及代谢产物差异，从中解析Sly-miR399在缺磷胁迫下对果实品质的具体调控作用。在上述组学数据分析基础上，我们还鉴定了Sly-miR399的主要靶基因SlPHO2，并通过对CRISPR/Cas9敲除突变体表型分析发现其在番茄磷吸收中的潜在作用。我们还挖掘出多个SlPHO2可能的互作蛋白编码基因，为后续研究SlPHO2及其互作蛋白的功能奠定了基础。我们希望上述研究为深入认识番茄缺磷响应的调控机制提供帮助，我们还希望后续结果能有助于开发改良番茄性状的分子工具，为番茄育种做出贡献。