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。

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.