超量表达PhZFP1转基因矮牵牛提高转基因植物的耐寒性

PhZFP1结合PhGolS1-1启动子并激活其表达

PhZFP1调控矮牵牛抗寒性的作用机制

南湖新闻网讯（通讯员 张慧琳）低温限制植物地理分布和生长季节，制约作物产量，是影响植物生长发育的关键环境因子。在胁迫环境下，植物会通过代谢调控积累一定的渗透调节物来维持细胞及细胞膜的稳定性。其中由GolS（Galactinol synthase，GolS，EC 2.4.1.123）介导的棉子糖积累在逆境应答中发挥重要作用。目前关于逆境条件下调控GolS转录水平的调控因子鲜有报道。

近日，我校园林植物遗传育种团队在Journal of Experimental Botany发表题为The C2H2-type zinc finger protein PhZFP1 regulates cold stress tolerance by modulating galactinol synthesis in Petunia hybrid的研究论文。该研究揭示了矮牵牛锌指蛋白PhZFP1通过调控肌醇半乳糖苷代谢途径，从而应答低温胁迫的分子机制。

矮牵牛（Petunia hybrida）是重要的园林观赏植物，被喻为“世界花坛花卉之王”。然而，矮牵牛不耐低温，晚春或晚霜冻害对其伤害很大，尤其在北方冬季极端低温的条件下难以露地越冬，即使顺利越冬其开花数量和质量也会受到严重影响。课题组前期通过分析低温胁迫矮牵牛基因表达谱，结合多个候选基因在不同非生物逆境胁迫下的表达模式，筛选出一个C2H2型锌指蛋白基因PhZFP1（Li et al., 2015 Front. Plant Sci.）。该基因显著且特异地受到低温强烈诱导，但此前研究对其响应低温的分子机制并不了解。

该研究通过遗传转化证据表明，超量表达PhZFP1提高了转基因矮牵牛株系的耐寒性，而PhZFP1干涉植株对低温胁迫的敏感性增加。低温胁迫下转基因株系中肌醇半乳糖苷合酶基因PhGolS1-1和棉子糖合酶基因PhRS表达也发生了变化，并且这一过程与肌醇半乳糖苷及棉子糖的积累密切相关。研究人员进一步通过Y1H、EMSA和ChIP-qPCR技术，以及双荧光素酶报告基因检测系统证实PhZFP1能够直接作用于PhGolS1-1 启动子并激活其表达，进而促进肌醇半乳糖苷和棉子糖的生物合成，最终提高矮牵牛植株的抗寒性。

此外，PhZFP1还能通过ROS的清除以抵御外界低温胁迫。值得一提的是，超量表达PhZFP1矮牵牛植株并未出现矮小、早衰或叶片畸形等症状，且异源表达该基因还能提高其他转基因植物的抗寒性。综上，该研究揭示了一个新的冷响应锌指蛋白PhZFP1调控矮牵牛抗寒性的分子机理和生理机制，为植物的逆境适应提供了新的理论依据，同时为植物抗寒基因工程提供了具有良好应用前景的候选基因。

我校园艺林学学院博士研究生张慧琳为论文第一作者，园艺植物生物学教育部重点实验室包满珠教授和张蔚副教授为论文共同通讯作者。博士研究生孙政、在站博士后冯珊以及张俊卫副教授、张帆教授、王文恩研究员和胡惠蓉副教授等也参与了该项研究。研究得到了国家自然科学基金和中央高校基本科研业务费专项基金的资助。

审核人：张蔚

【英文摘要】

The C2H2 zinc finger proteins (ZFPs) play essential roles in regulating cold stress responses. Similarly, raffinose accumulation contributes to freezing stress tolerance. However, the relationship between C2H2 functions and raffinose synthesis in cold tolerance remain uncertain. Here, we report the characterization of the cold-induced C2H2-type zinc finger protein PhZFP1 in Petunia hybrida. PhZFP1 was found to be predominantly localized in the nucleus. Overexpression of PhZFP1 conferred enhanced cold tolerance in transgenic petunia lines. In contrast, RNAi mediated suppression of PhZFP1 led to increased cold susceptibility. PhZFP1 regulated the expression of a range of abiotic stress responsive-genes including genes encoding proteins involved in reactive oxygen species (ROS) scavenging and raffinose metabolism. The accumulation of galactinol and raffinose, and the levels of PhGolS1-1 transcripts were significantly increased in PhZFP1-overexpressing plants and decreased in PhZFP1- RNAi plants under cold stress. Moreover, the galactinol synthase (GolS)-encoding gene, PhGolS1-1 was identified as a direct target of PhZFP1. Taken together, these results demonstrate that PhZFP1 functions in cold stress tolerance by modulation of galactinol synthesis via regulation of PhGolS1-1. This study also provides new insights into the mechanisms underlying C2H2 zinc finger protein-mediated cold stress tolerance, and has identified a candidate gene for improving cold stress tolerance.

论文链接：https://academic.oup.com/jxb/advance-article-abstract/doi/10.1093/jxb/erac274/6611932