DELLA蛋白PRO调控番茄响应低磷胁迫的机制研究

Phosphorus deficiency in soil has become an important factor to limit crop yield and agricultural development. To solve this problem, it may be an effective way to exploit and take advantage of the abilities of plants in response to low phosphorus stress. As an important vegetable crop grown extensively in the world, the ability of tomato to adapt to low phosphorus stress has been paid more and more attention by researchers. In this study, a gibberellin signaling repressor in tomato, DELLA protein PRO, is focused, and its subcellular localization and spatiotemporal expression pattern are examined. The mutants and overexpression lines of PRO are obtained and further analyzed to test their responsive abilities to low phosphorus stress with the help of phenotypic analysis and physiological index. RNA-seq and iTRAQ techniques are used to identify the genes and proteins related to the low phosphorus stress responsive abilities regulated by the PRO at the transcriptome and proteome levels, respectively. The interactive proteins of PRO are screened by Y2H and confirmed by Pull-down, BiFC, Co-IP and LC. Subsequently, the transient transcriptional expression analysis and stable genetic analysis are performed to reveal the network mechanism of PRO regulation of low phosphorus stress response. The results of this study can provide theoretical basis and technical support for the breeding and green cultivation of anti-low-phosphorus tomato varieties, so it is of great significance.

土壤有效磷缺乏已成为限制作物产量和农业发展的重要因素，挖掘植物自身应答低磷胁迫的能力是解决此问题的有效手段之一。作为世界上广泛种植的一种重要蔬菜作物，番茄响应低磷胁迫的能力逐步受到研究者的关注。本研究围绕番茄的赤霉素信号负调控因子DELLA蛋白PRO，分析其亚细胞定位和时空表达模式；获得其突变体和超表达体并结合表型分析和生理指标测定明确PRO参与的低磷胁迫应答过程；借助RNA-seq和iTRAQ技术分别在转录组和蛋白质组水平揭示PRO调控的低磷胁迫应答相关的基因和蛋白质；利用Y2H技术筛选PRO的互作蛋白，并通过Pull-down、BiFC、Co-IP和LC等技术进行验证，再采用瞬时转录表达分析和稳定遗传分析最终阐明PRO调控低磷胁迫应答的网络作用机制。本研究的结果能为抗低磷番茄品种的选育和绿色栽培提供理论依据和技术储备，因此具有重要的意义。

赤霉素（GA）参与植物生长发育的多个生理过程，但是它在植物应答低磷胁迫中的功能还知之甚少。土壤中无机磷（Pi）缺乏是影响植物生长发育的重要限制因素之一，植物具有多条途径应对这一非生物胁迫。低磷响应相关的研究已经在拟南芥和水稻中大量开展，而番茄应答低磷胁迫的信号转导以及赤霉素在其中是否扮演一定的作用还尚不清楚。.我们首先探索了GA在番茄低磷胁迫应答中的潜在功能。研究发现低磷胁迫诱导花青素积累、增强酸性磷酸酶活性，促进主根生长、上调高亲和磷酸根转运体编码基因（SlPT）的表达等。外源施加GA3会抑制低磷诱导的花青素合成；而gib3同野生型相比，低磷胁迫诱导的主根生长、SlPT的表达以及体内Pi含量都被降低，这表明GA在番茄响应低磷胁迫中发挥双重作用：正向调控地上部而负向调控根部。进一步发现低磷处理降低地上部而增加根部内源GA4的含量。此外，pro同野生型相比，低磷诱导的花青素合成被削弱，而低磷诱导的主根生长、SlPT的表达以及体内Pi含量却被增强，这表明GA调控番茄应答低磷胁迫可能通过PRO发挥作用。.我们随后从番茄中分离到一个PRO的潜在互作蛋白SlPHL1。它包含典型的MYB和CC保守域，定位于细胞核，具有转录激活活性。在拟南芥和番茄中过表达SlPHL1增强多个低磷响应途径，包括根毛生长、酸性磷酸酶活性、无机磷吸收、SlPT的表达等；而且过表达SlPHL1能够回补AtPHR1突变导致的低磷响应减弱。SlPHL1能与包含P1BS的DNA序列结合，且能够结合于番茄低磷响应基因（PSI）启动子的P1BS基序。总之，SlPHL1是一个新颖的MYB-CC转录因子，它通过直接调控PSI的转录表达来正向调控植物的低磷应答过程。.这些实验结果为揭示番茄的低磷信号转导和GA在其中的潜在作用机制奠定了基础，也为将来培育磷素高效利用的农作物提供了一定的理论支撑。