液泡加工酶及其上游调控蛋白介导平邑甜茶根系镉胁迫应答的分子机理

Cadmium (Cd) is one of the important factors that affect fruit safety and human health, and the root is the vital organ that feeling and absorbing of cadmium and other heavy metals in soil. Vacuolar processing enzymes (VPEs), a type of the asparagine specific cysteine proteinases existing in plant vacuole, play an important role in the process of growth and development, as well as plant defense responses. Our previous work had demonstrated that MhVPEγ gene was involved in the process of cadmium stress response in the root of Malus hupehensis, however, the core cadmium responsive promoter region and the promoter binding protein are still unclear at present. In this study, Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang, which is one of the excellent apple rootstocks, was used as materials, and construction of the promoter sequence deletants was first started, then, the core cadmium responsive promoter region was identified by GUS histochemical analysis; after that, the promoter binding protein and its binding sites were identified through yeast one-hybrid and EMSA methods; finally, the function of the promoter binding protein and its relationship between MhVPEγ gene was analyzed by transforming into Arabidopsis and apple callus. The above work would eventually elucidate the molecular mechanism of vacuolar processing enzyme and its upstream regulatory protein that mediated cadmium stress response in the root of Malus hupehensis. The results would provide a theoretical basis for revealing the resistance mechanism in the root of fruit trees and guiding fruit safety production.

镉是影响果品安全和人类健康的重要因素之一，根系是感受和吸收土壤镉等重金属的重要器官。液泡加工酶（vacuolar processing enzymes, VPEs）在植物的生长发育与防御反应方面具有重要作用。我们的前期工作已证实MhVPEγ基因参与平邑甜茶根系镉胁迫应答的过程，但其启动子镉应答核心功能区域及结合蛋白尚不明确。平邑甜茶是优良的苹果砧木，本项目以平邑甜茶为试材，从构建MhVPEγ启动子序列缺失体入手，通过GUS组织化学分析鉴定启动子镉应答核心功能区域，并利用酵母单杂交和EMSA技术鉴定启动子结合蛋白及结合位点，通过转基因手段分析启动子结合蛋白基因的功能及其与MhVPEγ的关系，最终阐明液泡加工酶及其上游调控蛋白介导平邑甜茶根系镉胁迫应答的分子机理，为进一步揭示果树根系抗逆机制及指导果品安全生产提供理论依据。

镉是影响果品安全和人类健康的重要因素之一，根系是感受和吸收土壤镉等重金属的重要器官。液泡加工酶（vacuolar processing enzymes, VPEs）在植物的生长发育与防御反应方面具有重要作用。在已明确MhVPEγ基因参与平邑甜茶根系镉胁迫应答的基础上，本项目利用生物信息学手段对苹果VPE基因家族进行了全基因组鉴定和表达分析，并对MhVPEγ进行了原核表达分析、多克隆抗体制备和亚细胞定位分析；分离了MhVPEγ基因上游启动子序列，构建了启动子序列系列缺失表达载体并转化拟南芥，通过GUS组织化学分析明确了启动子100bp核心序列为镉应答功能区域；利用酵母单杂交技术和EMSA技术鉴定镉胁迫下MhVPEγ启动子结合蛋白为MhARF4，并证实了二者的体外结合；通过转基因手段初步分析了启动子结合蛋白MhARF4的功能及其与MhVPEγ的关系，初步阐明了液泡加工酶及其上游调控蛋白MhARF4协同调控平邑甜茶根系镉胁迫应答的分子机理；此外，还进行了镉胁迫下平邑甜茶根系转录组和蛋白组关联分析，为进一步揭示果树根系抗逆机制及指导果品安全生产提供理论依据。