MhMYB2-L通过MhNCED3介导的ABA合成调控平邑甜茶根系响应镉胁迫的机理

Cadmium is a highly toxic heavy metal. Many orchards have been contaminated by cadmium. Root is the very organ that feels cadmium pollution in soil and the major organ that produces the stressed hormone ABA. This study focused on the transcription factor of MhNCED3 which is the key gene mediated ABA biosynthesis in Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang. The transcription factor MhMYB2-L was isolated and identified using the yeast one-hybrid system, EMSA method and ChIP technology. The response of MhMYB2-L to cadmium stress and the regulation of MhNCED3 transcriptional activity were analyzed using the overexpressed and silenced MhMYB2-L callus. At the same time,MhMYB2-L was transformed into the apple to obtain the transgenic material that MhMYB2-L gene expression of up-regulated, down-regulated, or silenced. Using the transgenic materials to further verify the response of MhMYB2-L to cadmium stress and the regulation on MhNCED3 gene expression and ABA biosynthesis. The mechanism of MhMYB2-L gene regulation by MhNCED3 mediated ABA synthesis in root response to cadmium stress was further explored. These results would provide a deep basis for promoting the root system of fruit tree to resist heavy metal stress such as cadmium, screening specific apple rootstocks and the safety production of fruit.

镉是毒性很强的重金属，不少果园已出现土壤镉超标问题；根系是果树最先感知土壤镉污染的器官，也是合成胁迫激素ABA的主要器官。本项目以平邑甜茶ABA合成关键酶基因MhNCED3上游转录因子为对象，利用酵母单杂交、凝胶迁移实验和染色质免疫共沉淀技术分离并确定MhNCED3上游转录因子MhMYB2-L，利用获得的过表达和沉默表达MhMYB2-L的苹果愈伤分析MhMYB2-L对镉胁迫的应答及其对MhNCED3转录活性的调节；同时，将MhMYB2-L转入苹果，获得MhMYB2-L基因表达上调、下调或沉默的转基因材料，利用转基因材料进一步验证MhMYB2-L对镉胁迫的响应及其对MhNCED3基因表达和ABA合成的调节，进而探明MhMYB2-L基因通过MhNCED3介导的ABA合成调控根系应答镉胁迫的机理，为促进果树根系抵御镉等重金属胁迫、筛选特异苹果砧木及果品安全生产提供深层次依据。

镉（Cd）污染是果园管理当中一般不被考虑的污染因素，随着工业发展和三废污染物的累积，有的果园已经出现镉。镉的出现势必会造成果品质量安全问题。脱落酸（ABA）是植物的逆境激素，在多种植物抗逆响应中发挥重要作用。本研究围绕脱落酸的生物合成，以有无融合生殖特性的苹果砧木平邑甜茶为试材，研究了外源ABA对镉胁迫下根系镉吸收和细胞死亡的影响，探讨了MhNCED3介导脱落酸合成的上游调控机制及对镉引起的细胞死亡的影响。结果显示外施ABA可显著降低平邑甜茶根系镉吸收、积累，降低镉引起的根系细胞死亡；将MhNCED3在拟南芥和苹果愈伤中异位表达，可上调植株内源ABA合成，降低拟南芥根系和苹果愈伤组织的Cd吸收和积累，降低细胞死亡程度。分离出MhNCED3上游转录因子MhDIV3，经EMSA和酵母单杂交验证了其与MhNCED3启动子的结合，证实其对MhNCED3有转录激活活性；更进一步，将MhDIV3转入苹果愈伤发现，过量表达MhDIV3的苹果愈伤组织的NCED3表达和内源ABA含量更高，且在镉胁迫下的长势更好，细胞死亡率更低；但是，在外源ABA处理下，过量表达MhDIV3的苹果长势却更差，说明MhDIV3作为MhNCED3上游转录因子可正向调控镉胁迫下ABA的生成，但与ABA之间是反馈调节的关系。因此，MhDIV3—MhNCED3—ABA途径是受ABA反馈调节的逆境调控途径。这为ABA的逆境调控机制，根系镉胁迫响应机制提供了一定的理论依据。