GhbZIPX及其靶基因调控棉花盐胁迫应答及叶片衰老的分子机制

Salt stress is a main limiting factor for cotton growth and productivity. Plants have well developed sophisticated signaling pathways i.e. receptors, secondary messengers, phytohormones, and signal transducers for their survival in salt stress. The response of plants against salt stress also required differential gene expression mainly regulated by transcription factors (TFs). The bZIP TFs are key components in controlling transcription initiation rates to modulate salt stress-associated genes expression for the salinity signal cascade transmission. However, its regulatory mechanism is poorly understood. Using RNA-sequencing analysis, large-scale gene expression and transgenic plant analyses, we obtained a novel member of cotton bZIP TF, named GhbZIPX. Overexpression of GhbZIPX significantly enhanced salt tolerance and produced a marked delay of salt-induced leaf senescence, whereas knockdown of GhbZIPX increased salt sensitivity and promoted senescence proceeding, confirming the role of GhbZIPX TF in the salt responsive of cotton. We further performed RNA-sequencing analysis on GhbZIPX-overexpression cottons and wild type. Strikingly, among 659 up-regulated genes with alteration of 2-fold or more, GhZTG1-GhZTG4 genes were up-regulated in ABA and salt treatment. Moreover, activated and repressed expressions of GhZTG1-GhZTG4 were observed in GhbZIPX overexpression and RNAi cottons, respectively. According to the AthaMap, the 2,000-bp GhZTG1-GhZTG4 promoter regions contain several A-box, C-box, or G-box motifs. Consistently, a yeast one-hybrid assay also showed that the GAL4 transcriptional activation domain-GhbZIPX (AD-GhbZIPX) fusion protein activated the LacZ reporter gene driven by the promoters of GhZTG1-GhZTG4. Taken together, we reasonably speculate that GhbZIPX act as a transcription factor to regulate the expression levels of GhZTG1-GhZTG4 by directly binding to their promoter region in vivo. In this proposed study, we would like to investigate the mechanism of GhbZTG1-GhZTG4 involved in salt stress response and leaf senescence by using genetics, molecular biology, and biochemistry means, and also explore its possible application in cotton salt tolerance improvement.

盐胁迫严重影响棉花的产量和品质，深入研究棉花盐害响应对于培育耐盐棉花具有重要意义。bZIP类转录因子在植物耐逆中起关键的作用，但其调控机制却知之甚少。GhbZIPX转录因子是我们通过转录组和大规模转基因实验获得的响应盐胁迫蛋白，过表达棉花耐盐性显著提高，盐害诱发早衰有效延缓；相反，干涉棉花盐敏感增强、衰老加速。进一步研究鉴定到4个GhbZIPX候选靶基因：GhZTG1-GhZTG4。此4个基因编码蛋白属于同一家族，表达均在GhbZIPX过表达棉花中上调，干涉棉花中下降。GhZTG启动子区均含有bZIP类蛋白结合元件，酵母实验中GhbZIPX可直接结合GhZTG启动子调控其表达，说明GhZTG可能是GhbZIPX直接调控的靶基因。本项目在此基础上，进一步在体内和体外确定GhbZIPX对GhZTG表达的直接调控，研究他们在盐胁迫响应和叶片衰老中的分子遗传机理，探索在提高棉花耐盐中的应用潜力。

棉花是我国重要的经济作物，提质和增效是棉花研究的两大主要目标。粮棉争地的矛盾，决定了棉花种植不断向新疆地区集中和东部滨海盐碱地延伸。这些地区共同的特征是土壤含盐量高，盐渍化严重。因此，培育耐盐棉花且有效防止盐害诱发的叶片早衰，对棉花增产和稳产据有重要的意义。项目主持人研究发现bZIP类型转录因子GhABF2是ABA介导逆境响应信号通路的核心元件，其所指导的转录调控过程是棉花逆境信号通路的核心。本项目围绕GhABF2进一步开展研究，利用生物信息学、ChIP-PCR、qRT-PCR和酵母等实验，证明同一亚家族4个GhZTG基因GhZTG1-4是GhABF2直接调控的下游候选靶基因，其拟南芥中同源基因与逆境响应密切相关。转基因实验结果表明，无论在过表达GhABF2棉花还是野生型棉花背景下，利用VIGS (Virus-Induced Gene Silencing)干涉GhZTG1-4基因表达棉花耐盐性显著降低，而过表达GhZTG1基因棉花耐盐性明显提升，盐害诱导的叶片衰老和细胞死亡水平显著下降。GhABF2-GhZTG信号途径是ABA介导的盐胁迫及其诱发的叶片早衰的重要调控路径，通过降低高盐胁迫下植物体内的氧化逆境，降低细胞氧化损伤，从而提升植物耐盐胁迫以及减缓盐胁迫下的叶片衰老。同时，本项目还鉴定到棉花逆境下ABA信号途径的关键受体蛋白GhPYL9-11A，进一步完善了棉花响应高盐逆境胁迫下的信号通路。而且通过精准调控AtCMO等关键逆境基因在棉花中的表达可显著提升棉花抗旱、耐盐和低温胁迫能力。此外，建立了农杆菌介导的棉花CRISPR/Cas9基因编辑系统，是最早报道利用CRSPR/Cas9技术成功编辑棉花基因的四篇研究论文之一。共同搭建了棉花基因组分析平台CottonFGD，目前网站已被国内外多家棉花实验室使用，受到广泛好评。项目执行期间，发表学术论文7篇，其中SCI论文6篇，申请专利1项，开展学术交流9次，在国内外学术会议上做报告2次，参与组织2017年和2019年“中-巴棉花生物技术与创新国际研讨会”，获得中巴双方科研人员高度评价。共同组织在生命科学主流科普期刊《生命世界》撰写3期转基因科普专题（22篇），取得了良好的学术效应和社会效应。总之，在项目主持人和参加人共同努力下，圆满完成申请书中设定的研究目标。