MdFRK2和MdERD6.7转基因苹果中果糖含量稳态调控的生理与分子机制

Fructose (Fru) is the sweetest sugar, and plays an important role in controlling the flavor quality of fruit. Our study on the transgenic apples with over-expressing MdFRK2 or MdERD6.7 indicated that there is a regulating system for Fru homeostasis in apple. To know the physiological and molecular mechanism for Fru homeostasis regulation in apple, this project will intend to MdFRK2 and MdERD6.7 transgenic apple plants as research materials, combined with the physiological and transcription sequencing analysis, sugar metabolism and transport will be carried out to investigate the reason that Fru recovery to normal levels in the transgenic plants, which will help understand the regulating relationship among different carbohydrates. Meanwhile, we will screen the environment factors influencing Fru homeostasis in transgenic apple leaves. Moreover, based on the previous research results, we will screen and identify the interaction proteins regulated MdFRK2 post-translational modification, and the transcription factors regulated MdFRK5 transcription, meanwhile their role will be confirmed in sensing Fru signal and regulating Fru homeostasis and content through the transforming apple and solid molecular experiments. After studying on the steady-state regulation mechanism of Fru content in MdFRK2 and MdERD6.7 analysis of transgenic apples, the results would get the new insight in Fru signal and its regulation pathway, and obtain new genes and methods to improving Fru content, which would provide new theoretical and technical support for fruit flavor quality breeding.

果糖（Fru）是最甜的糖，对水果风味品质具有重要影响。项目组对不同环境条件下MdFRK2和MdERD6.7转基因苹果研究发现，苹果中存在Fru稳态调控系统，为探明Fru稳态调控的生理与分子机制，本项目拟以MdFRK2和MdERD6.7转基因苹果为材料，首先结合生理和转录组分析，从糖代谢和转运分析转基因苹果叶片Fru恢复稳态的生理原因及不同糖代谢之间的相互调控，筛选分析影响转基因苹果叶片Fru调控的环境因子。同时，基于前期研究结果，筛选鉴定MdFRK2翻译后调控的互作蛋白及调控MdFRK5转录的转录因子，并通过苹果转化与分子实验分析它们在感受Fru信号中的作用及与Fru含量的关系，挖掘苹果中Fru稳态调控基因。项目通过解析MdFRK2和MdERD6.7转基因苹果Fru含量的稳态调控机理，以获得Fru调控基因和途径，为水果风味品质育种提供重要的理论与技术支撑。

果糖（Fru）是苹果果实糖的主要组成，也是最甜了可溶性糖，为了探明苹果Fru含量的调控机制，在课题组前期关于MdFRK2和MdERDL6遗传转化和糖含量研究的基础上，本项目从胞质Fru代谢和液泡Fru贮存积累两个方面探索了苹果Fru含量的调控机制，获得如下结果，对MdFRK2转基因苹果中果糖含量稳态调控研究证实MdFRK2转基因苹果在大田条件下，MdFRK2蛋白发生了翻译后修饰，降低了MdFRK2的蛋白水平，恢复了果糖Fru含量，而胞质的Fru水平能调控参与蛋白质泛素化途径的MdCacyBP表达，在MdFRK2转基因苹果成熟叶片和果实中高度表达。MdCacyBP能与MdFRK2蛋白水平互做，促进MdFRK2泛素-蛋白酶体降解，调控着MdFRK2蛋白及FRK活性及Fru含量。在过表达转基因苹果成熟叶片和果实中，MdCacyBP表达的增加是FRK活性降低和果糖含量不变的主要原因。同时，发现转录因子AIB3 能通过调控SDH2表达控制苹果果糖的含量。然而，FRK1表达的降低不是MdERDL6过表达转基因苹果中Fru含量增加的主要原因。在MdERDL6过表达转基因苹果中，MdERDL6介导的液泡葡萄糖外排能诱导糖内吸TST1/2表达，进而增加了Fru等糖进入液泡储存的能力，促进了Fru向液泡中的积累能力，证实了液泡中两类糖转运蛋白可以相互调控影响果实中糖的积累能力。进一步研究证实MdERDL6过表达引起的Glc外排能够诱导SnRK2.3的表达， SnRK2.3能与AREB1.1/1.2互做，引起AREB1.1/1.2的磷酸化，激活其对TST1/2转录激活能力，建立了Glc/SnRK2.3/AREB1.2/TST调控苹果果实糖含量的分子途径，解析了果实中糖高度积累的分子调控途径。项目的相关研究结果已在PNAS、Plant Physiol、Plant J等期刊发表研究性论文8篇，培养博士生4人，硕士生1人。上述研究结果深化了果实Fru含量调控的认知，回答了果实糖积累的主要原因，挖掘了一批基因资源，为果实糖酸品质改良奠定了基础。