SlWRKY33参与外源Spd调控番茄抗盐碱性的功能解析

Soil salinization is one of the main factors limiting the stable and high tomato production in northwest district. Application of exogenous spermidine (Spd) can effectively improve the saline-alkali stress resistance of tomato, and WRKY transcription factors are closely related to the stress responses in plants. In the preliminary study, SlWRKY33 was strongly induced by saline-alkali stress, while exogenous Spd could decrease this trend. However the function and mechanism of SlWRKY33 in enhancing tomato salt-alkaline tolerance by Spd has not been fully elucidated. In this study, tomato cultivar ‘Jinpengchaoguan’ will be used as experimental material in this project. By means of transgenic and Real-tmie RCR technolgy, SlWRKY33 genes will be performed the sequence characteristics. We will characterize the molecular mechanisms of SlWRKY33 by loss-of-function and gain-of-function analysis, identifying its down-stream targets by ChIP-Seq and interacting partners by IP-MASS, BiFC, and Co-IP analysis, and examining the biological functions of its targets and interaction partners under saline-alkali stress plus Spd condition. The present study will clarify the response mechanisms of SlWRKY33 transcription factors in the Spd-mediated resistance of tomato to saline-alkali stress. All those will help to provide new gene for the breeding of saline-alkali stress resistant tomato cultivars, and explorea new way based on Spd in the anti-salinity-alkalinity and high-efficiency tomato cultivation.

土壤盐碱化是番茄高产优产的主要限制因素，外源Spd可有效提高番茄抗盐碱性，WRKY转录因子与植物胁迫应答密切相关。项目组前期研究发现SlWRKY33受盐碱胁迫强烈诱导，外源Spd可降低此趋势，但SlWRKY33在Spd增强番茄盐碱抗性中的功能及作用机理尚不清楚，因此，本项目以“金鹏朝冠”番茄为试材，基于转基因和Real-time PCR技术分析SlWRKY33序列特性，通过探究SlWRKY33基因沉默和超表达番茄植株对盐碱胁迫及Spd的响应，明确Spd诱导番茄抗盐碱性中SlWRKY33的功能；拟结合ChIP-Seq、RNA-Seq技术确定SlWRKY33的靶基因；通过亲和纯化-质谱、双分子荧光互补技术确定SlWRKY33的互作蛋白，并研究其互作机制。本研究有望阐明SlWRKY33参与外源Spd调控番茄盐碱胁迫抗性的功能及作用机制，为番茄耐盐碱育种提供新的基因来源和抗逆栽培提供途径。

本项目通过研究外源亚精胺（Spd）和精胺（Spm）调控Na+/K+及信号响应机理及SlWRKY33基因在番茄抗盐碱性的功能及其参与外源Spd增强番茄盐碱抗性的调控机制，主要研究结果如下：.（1）预喷施Spd和Spm能够显著提高盐碱胁迫番茄幼苗的抗氧化能力，H2O2和GSH参与预喷施Spd调节番茄幼苗盐碱耐性的调控，缓解植株氧化损伤，提高盐碱胁迫下番茄幼苗的抗氧化的能力。外源Spm可增加液泡膜上Na+/H+反转运蛋白基因（NHX1和NHX2）的表达量，激活NHX的活性，减少番茄根、茎和叶中Na+的积累，促进根、茎和叶中K+的积累，提高番茄幼苗的盐碱胁迫耐性。沉默SlSPMS削弱了盐碱胁迫下番茄抗氧化能力，破坏了Na+和K+离子稳态。.（2）SlWRKY33受到盐碱胁迫、NaCl、ABA的诱导，且预喷施Spd后盐碱性胁迫对SlWRKY33的诱导出现滞后效果。SlWRKY33（Solyc09g014990）开放阅读框（ORF）全长1590bp，编码529个氨基酸。SlWRKY33B与SlWRKY33A和SlWRKY26的同源性最高，而且与CaWRKY1、CaWRKY26、AtWRKY33也有很高的同源性。SlWRKY33蛋白定位在细胞核中，说明SlWRKY33在细胞核中发挥转录调控作用。.（3）SlWRKY33高表达于番茄的根和叶组织中，且能够被盐碱胁迫逆境诱导表达，外源Spd能够降低盐碱胁迫下SlWRKY33的表达。.（4）转录激活试验表明SlWRKY33的转录激活域位于SlWRKY33序列的N端（P1段），并通过酵母双杂交实验，得到2个与SlWRKY33互作的蛋白，分别为Solyc03g121290（F-box家族蛋白）、Solyc02g036370（Myb家族转录因子REVEILLE1）。