SbWRKY55调节甜高粱根系拒盐的分子机理研究

Sweet sorghum [Sorghum bicolor (L.) Moench] is considered as energy and forage crop of high-yield and salt-resistant, which is most suited to grow in the saline-alkali land of Yellow River Delta. Sweet sorghum has a high ability to exclude Na+. However, the mechanism of salt exclusion in sweet sorghum is still unknown. A sweet sorghum gene WRKY55 (Sb02g011050) has been identified by high-throughput sequencing in the presence of NaCl by compering a salt-resistant line M-81E and a salt-sensitive line Roma. The analyses of RT-PCR show that the expression level of SbWRKY55 decreases with increasing NaCl concentration. In addition, overexpression of SbWRKY55 in Arabidopsis leads to increased salt sensitivity, suggesting that SbWRKY55 is a negative regulating factor during salt response. In this project, we will knock out SbWRKY55 by CRISPR/Cas9 system and overexpress SbWRKY55 in sweet sorghum to investigate the salt exclusion ability of these transgenic plants, to identify the target gene regulated by SbWRKY55, and to analyze the biochemical regulation relationship between SbWRKY55 and the target gene. The possible function of the target gene during salt exclusion in sweet sorghum will be further analyzed to determine which the salt exclusion pathway is involved in sweet sorghum by the target gene. Through these analyses, we will ultimately reveal the molecular mechanisms of SbWRKY55 regulating salt exclusion of roots in sweet sorghum. The results in this project will provide the important information to understand the mechanisms of salt exclusion and molecular breeding in sweet sorghum for the utilization of saline-alkali soil.

甜高粱为C4作物，生物量高，耐逆性强，是最适合在黄河三角洲盐碱地上生长的高产耐盐饲用及能源作物。甜高粱抗盐能力较强，但甜高粱拒Na+机理尚不清楚。我们前期通过高通量转录组测序，鉴定到一个在耐盐自交系M-81E中差异表达的WRKY55基因（Sb02g011050）。RT-PCR结果表明随着盐浓度的增加，SbWRKY55的表达量逐渐降低；将SbWRKY55在拟南芥中过表达，转基因植株对盐的敏感性增加，表明该基因可能是植物耐盐途径的一个负调节因子。本项目拟在前期工作的基础上，利用CRISPR/Cas9系统在甜高粱中敲除SbWRKY55，并过表达该基因，研究转基因植株的拒盐能力，鉴定SbWRKY55调控的靶基因，分析SbWRKY55与靶基因之间的生化调节关系以及靶基因在甜高粱拒盐中的作用，揭示SbWRKY55调控甜高粱根系拒盐的分子机制，研究结果将为理解甜高粱拒盐机理及分子育种提供重要的理论信息。

植物的耐盐性是由多种环境胁迫因素和内源性激素信号触发的。大量研究表明，WRKY转录因子参与调节植物盐耐受性。然而，WRKY转录因子调控盐应激反应和信号转导途径的潜在机制在很大程度上仍然未知。我们的研究表明，SbWRKY55转录因子过表达后能够显著降低高粱和拟南芥的抗盐性和ABA含量。SbWRKY55的同源基因AtWRKY55在拟南芥中突变后，突变体的抗盐能力显著提高，而且SbWRKY55能够回补拟南芥的突变表型。在SbWRKY55过表达的转基因高粱中，参与脱落酸（ABA）途径的基因表达水平发生改变，内源性ABA含量降低。酵母单杂交和双荧光素酶报告实验表明，SbWRKY55直接与SbBGLU22的启动子结合并抑制其表达。此外，体内和体外生化分析均表明，SbWRKY55与FYVE锌指蛋白SbFYVE1相互作用，阻断ABA信号通路。这可能是平衡SbWRKY55介导的盐应激反应的重要反馈调节途径。综上所述，本研究结果提供了令人信服的证据，证明SbWRKY55是ABA介导的信号通路的关键组成部分，突出了SbWRKY55在ABA信号传导中的双重作用。该研究还表明，SbWRKY55可以负调节高粱的耐盐性。