桃果实酸性转化酶基因家族及其抑制子在低温胁迫下的表达与互作研究

During the previous program, we focused on the relationship between soluble sugar and chilling injury in peach fruit. Results showed that sucrose level decreased sharply, which accompanied with the increasing of gene expression and activity of acid invertase (AI). The objective of the present program is to investigate the target AI gene sensitive to the chilling stress by cloning all of AI gene family members and measuring the space and time changes of genes transcript level in "Yulu" peach stored at different temperatures. Then, yeast two-hybrid cDNA library of peach will be constructed, screen and obtain the endogenous proteinaceous inhibitor (INH) interacting with target AI. After the investigation of subcellular localization of AI and INH gene, the interactive positioning in the cell could be confirmed by using bimolecular fluorescence complementation test. The gene recombinant protein of AI and INH will be expressed and purified to reveal the characterization and interactive effects of INH of AI in vitro test. Mediated transient expression system in peach will be established to confirm the post-translational regulation of AI. Finally the relationship between the AI and INH target gene and chilling injury will be investigated by the peach treated with different concentration of 1- methylcyclopropene (1-MCP). Our findings is benefit for the understanding the changes rule and interactive of AI and INH in peach under chilling stress. It also provides further evidence for the prediction of postharvest chilling and the quality improvement of fruit tree, and it has significant theoretical and practical value.

前期研究发现低温胁迫导致桃果实酸性转化酶（AI）表达量和活性上升，蔗糖分解。本项目以冷敏感“玉露”桃为研究对象，借助桃基因组数据库，获取并验证AI家族成员的基因序列；分析不同温度下AI家族基因的时空表达，筛选出低温敏感的AI目的基因。构建桃果实酵母双杂交cDNA文库，筛选与AI有互作的转化酶抑制子（INH）。测定AI、INH目标蛋白的亚细胞定位，运用双分子荧光互补定位验证二者在细胞内的互作关系。对上述目的基因进行蛋白重组表达和纯化，分析其功能特性，明确INH对AI的体外抑制作用及影响因素。建立INH在桃中的瞬时表达系统，验证INH对AI的翻译后调控。最后采用1-甲基环丙烯（1-MCP）调控桃冷害，研究目的基因、蔗糖变化与冷害的关联。研究有望揭示低温下桃AI、INH的变化规律和互作关系，更好的阐述低温下蔗糖的变化，丰富冷害相关理论；也利于今后冷害预测、品种改良，具有理论和实际意义。

本项目克隆分析了桃果实酸性转化酶（AI）家族成员——包括5条细胞壁型PpCWIN1-5和2条液泡型PpVIN1-2，明确仅PpVIN2对低温敏感。克隆分析了5条转化酶抑制子PpINH1-5，酵母双杂交（Y2H）发现仅PpINH1与PpVIN2存在互作；亚细胞定位及双分子荧光互补（BiFC）证实PpINH1与PpVIN2在烟草细胞的细胞膜上发生互作；在“玉露”桃果实体内过表达及沉默PpINH1，可以引起VIN活性的相应降低和升高，证实了PpINH1对VIN活性的抑制作用；重组蛋白体外互作研究也证实PpINH1可显著抑制PpVIN2活性。项目组进一步构建了桃果实酵母双杂交 cDNA文库，筛选得到与PpVIN2互作的蛋白22个，发现多聚半乳糖醛酸酶抑制子PpPGIP1和锌指蛋白PpZAT10在5 ℃下被显著诱导上调。PpPGIP1与PpVIN2共定位在烟草细胞膜并发生互作，瞬时过表达及沉默PpPGIP1分别提高和抑制了桃果实VIN活性，表明PpPGIP1正调控VIN酶活，加速蔗糖分解。亚细胞定位显示PpZAT10为核蛋白，BiFC证实其与PpVIN2互作，其重组蛋白可促进PpVIN2活性。1μL/L 1-甲基环丙烯和1% 海藻糖外源处理采后桃果实，可以提高PpINH1表达量，降低VIN表达量和活性，减缓蔗糖分解从而减轻冷害。总之，本项目明确了AI家族中唯一冷敏感基因是PpVIN2，明确1个反向抑制蛋白（PpINH1）、2个正向调控蛋白（PpPGIP1和PpZAT10）对PpVIN2有翻译后调控作用，相关研究揭示了低温下桃果实糖代谢变化机制与抗冷性。项目已培养硕士研究生4名，在Horticultural Research、Postharvest Biology and Technology等期刊发表SCI论文9篇，申报国家发明专利3项，参与获得教育部科技进步一等奖1项。