类钙调素ZmCML21介导玉米抵御低磷胁迫的功能及机制研究

Phosphate (Pi) is a macronutrient that is essential for plant growth and development. However, low Pi availability is a major constraint on maize production. Therefore, it is important to gain insight into the mechanism underlying low Pi tolerance in maize. Calmodulin-like proteins, CMLs, are known to be involved in abiotic and biotic stress responses. However, the role of CML in low Pi adaptive responses remains largely unknown. Our preliminary results showed that miR1432 played a role in improving low Pi tolerance in maize, and that a CML coding gene, ZmCML21, is one of target genes regulated by miR1432, suggesting the possible involvement of ZmCML21 in regulating low Pi adaptive responses of maize. Moreover, we found that the expression of ZmCML21 in maize was regulated by low Pi stress, and over-expression of ZmCML21 in Arabidopsis enhanced low Pi tolerance in transgenic Arabidopsis. These results further suggested that ZmCML21 might play an important role in regulating low Pi tolerance in maize and deserve further research. In this study, to investigate the role of ZmCML21 in improving low Pi tolerance of maize, we will generate ZmCML21 over-expression or gene-knockout maize plants and analyze their low Pi tolerance. To investigate the possible molecular mechanism of ZmCML21 in regulating low Pi tolerance, the interacting protein of ZmCML21 will be identified based on yeast-two-hybrid system. Subsequently, candidate interacting protein coding gene related to low Pi tolerance will be selected for further functional characterization, including subcellular localization analysis and time-space characteristics of gene expression. Moreover, preliminary functional verification of the candidate gene will be performed. This project will gain insight into the molecular mechanism underlying low Pi tolerance of maize, and will be useful for screening and breeding maize varieties with enhanced low Pi tolerance.

土壤有效磷不足是限制玉米生产的主要因素之一，深入研究玉米低磷应答调控机制对其耐低磷遗传改良具有重要意义。申请人前期研究表明miR1432参与调控玉米低磷应答过程，而类钙调素基因ZmCML21是miR1432调控的下游靶基因之一，初步分析发现ZmCML21对低磷逆境产生明显应答，而且在拟南芥中过表达该基因可增强其耐低磷能力，暗示ZmCML21可能在玉米抵御低磷过程中发挥重要功能，值得深入研究。本项目主要研究内容包括：分析ZmCML21的时空表达模式和蛋白亚细胞定位；创建ZmCML21过表达玉米和基因敲除玉米,分析其表型及低磷应答过程；通过酵母双杂交技术鉴定ZmCML21蛋白的互作靶蛋白；挑选潜在关键的候选互作蛋白，验证其与ZmCML21蛋白的互作关系；对候选基因进行表达特性分析和初步功能验证。本项目有助于深入理解玉米低磷应答调控机制，可为培育和筛选耐低磷玉米品种提供研究思路。

土壤有效磷不足是限制玉米生产的主要因素之一，深入研究玉米低磷应答调控机制对其耐 .低磷遗传改良具有重要意义。申请人前期研究表明miR1432参与调控玉米低磷应答过程，而类钙调素基因ZmCML21是miR1432调控的下游靶基因之一，初步分析发现ZmCML21对低磷逆境产生明显应答，而且在拟南芥中过表达该基因可增强其耐低磷能力，暗示ZmCML21可能在玉米抵御低磷过程中发挥重要功能，值得深入研究。经过进一步研究，本项目获得主要研究结果如下：（1）通过5’RACE实验和烟草瞬时共表达实验验证miR1432和ZmCML21的互作关系。（2）解析ZmCML21的蛋白亚细胞定位和该基因响应低磷胁迫的表达模式，发现该基因的表达水平受低磷胁迫的显著调控。（3）创建ZmCML21过表达和基因敲除玉米转基因株系，并分析其表型及低磷耐受能力，发现ZmCML21正向调控玉米耐低磷能力。（4）解析ZmCML21参与玉米低磷响应过程的机制，发现该基因可显著调控根系发育及根系有机酸分泌能力，从而有效提高玉米的耐低磷特性。（4）通过酵母双杂交技术鉴定ZmCML21蛋白的互作靶蛋白，测序成功27个互作基因，并回转验证成功。本项目有助于深入理解玉米低磷应答调控机制，可为培育和筛选耐低磷玉米品种提供研究思路。