木豆激酶CIPK14介导的微丝蛋白ADF2促进根毛生长抗旱分子机制研究

As an important stress-responsive kinase, CIPK can improve plant drought resistance by promoting polar growth of root hairs in pigeon pea, but its mechanism is still unclear.Previous studies have shown that CIPK14, the most capable of responding to drought stress, has been screened from 29 homologous genes of CIPK family in Pigeonpea, and the drought-resistant function of CIPK14 has been preliminarily verified by hairy-root transgenic system.CIPK14 was used as bait to screen the root cDNA library to obtain the interaction protein, and we found that ADF2 interacted with CIPK14 strongly and frequently. By analyzing the expression pattern of ADF2, we found that ADF2 was highly expressed in root hairs, and possessed actin severing activity. In view of this, this study first further verified the co-localization and interaction between CIPK14 and ADF2 in vivo. Secondly, the phosphorylation site of ADF2 was determined by mass spectrometry and point mutation.Further analysis of CIPK14 phosphorylated ADF2 regulates the activity of cutting microfilaments by F-actin immunofluorescence assay to analyze how to promotes the polar growth of root hairs, thereby improving the utilization of water by root hairs. Finally, hairy-root rapid transgenic system and CRISPR/Cas9 fixed-point knockout were used to verify their involvement in drought resistance in vivo.Ultimately, the molecular mechanism of drought-resistant root hair growth promoted by microfilament protein ADF2 mediated by CIPK14 in response to drought was clarified.

植物CIPK是重要的干旱响应因子，但其在林木中如何通过调控微丝骨架促进根毛极性生长提高林木抗旱性的分子机制尚不明确。在前期研究中，申请者利用生物信息学等方法从29个木豆CIPK家族基因中筛选获得影响干旱的CIPK14，hairy-root转基因分析初步确认了CIPK14的抗旱功能。为解析其分子信号通路，申请者以CIPK14为诱饵筛选酵母文库获得微丝蛋白ADF2，初步验证其在根毛中表达较高且具有微丝切割活性，可能参与CIPK14调控的根毛微丝生长与解聚过程。鉴于此，首先确定CIPK14与ADF2在体内共定位和互作关系；其次，通过质谱检测及点突变分析确定ADF2的磷酸化位点；微丝免疫荧光实验验证CIPK14能够磷酸化ADF2调控其切割微丝活性；最后，利用根部转基因和CRISPR验证CIPK14和ADF2在体内参与抗旱反应。最终阐明CIPK14磷酸化ADF2促进根毛生长提高其抗旱的分子机制。

植物CIPK是重要的干旱响应因子，但其在林木中如何通过调控微丝骨架促进根毛极性生长提高林木抗旱性的分子机制尚不明确。本项目严格按照任务书计划执行，并根据实验进展情况进行了拓展和延伸，相关研究成果发表在Plant Physiology、Plant Journal、Journal of Experimental Botany等植物学重要期刊上，其中第一标注2篇；授权国家发明专利1件；培养硕士研究生2人；本人完成副教授职称晋升和遴选为硕士生、博士生导师，顺利完成任务书各项指标。项目获得了三部分主要成果：第一，探究了微丝骨架蛋白在植物抗逆过程中的作用，在前期通过 CIPK14 筛选到ADF蛋白的基础之上，对ADF基因家族进行了系统分析。同时，通过改变不同浓度ADF以及外源施加不同浓度的次生代谢物金丝桃苷，探究了切割微丝的效率；第二，项目研究了外源性ABA影响木豆抗旱性的分子机制。在干旱条件下，木豆叶卷曲程度（LRI）显著增加到80%，施加ABA后，叶枯萎程度下降，存活率提高到约70%，增强了木豆的抗旱能力。同时，外源ABA可以促进气孔闭合，诱导转录因子表达水平提高，调节多个黄酮合成途径上关键基因的表达，最终引起代谢物的积累，从而产生抗旱性；第三，项目解析了CIPK14调节类黄酮生物合成提高植物耐旱的分子机制，筛选到CcCIPK13和CcCIPK14可能是木豆抗旱过程中的关键基因。通过酵母双杂交筛库，Y2H和BiFC实验验证，证明了CcCIPK14在体外表现出自磷酸化以及对下游互作蛋白的磷酸化。此外，进一步获得了CcCBL1的RNAi和过表达株系，证明了CcCIPK14-CcCBL通过促进类黄酮生物合成增强木豆的抗旱性，为加快木本植物抗旱基因功能研究和抗性新品种培育进程提供了理论支持。