葡萄新型N末端截短质膜H+-ATPase活性调控及其在抗碱性盐胁迫中的作用

Cultivation of grape is restricted due to soil alkalized. So it is important for sustainable development of grape to improve alkali resistance of the grape. The regulation of plasma membrane H+-ATPases activity of roots plays a key role to sustain pH homeostasis of plant in alkali stress. In our previous study, a full length cDNA of root main PM-H+-ATPase isoform VvPMA1 were isolated from alkalinity-insensitive Crimson seedless. Yeast function complementation assay showed that the isolated VvPMA1 had powerful function. Surprisingly, the sequence analysis in grape genomic data and further assay showed that the isolated VvPMA1 might be an undiscovered splicing variant and it would generate a novel N terminus shortened PM-H+-ATPase (VvPMA1β) with higher activity, which would be helpful to alkali resistance of the grape. To verify this speculation, the expression, activity and regulation of VvPMA1β will further be studied. Firstly, it will be explicit whether VvPMA1 will produce different amount and activity of VvPMA1 splicing variant influencing the total activity of root PM-H+-ATPase. Secondly, the organic and variety specificity of VvPMA1β expression will be discussed. Thirdly, how to control the activity of VvPMA1β will be ascertained. This study aims to clarify the regulatory mechanism of PM-H+-ATPase activity of grape root in gene and protein expression level.

葡萄种植受到土壤盐碱化束缚，因此提高葡萄的抗盐碱性对产业发展非常重要。植物抗盐碱胁迫的重要部位为根，其中根质膜H+-ATPase(质子泵)的活性对植物维持盐碱环境中pH稳恒态至关重要。前期研究中，申请人从盐碱抗性较强的克瑞森无核葡萄根cDNA中克隆了质膜质子泵基因 VvPMA1，酵母功能互补已证实了其强功能，随后序列分析和试验发现克隆的VvPMA1可能是尚未报道的剪接变体，将产生具有较高活性的N末端截短的新型质膜质子泵（VvPMA1β），利于葡萄盐碱抗性。为了验证推测，本项目将对VvPMA1β表达、活性及调控进行进一步分析明确碱性盐胁迫下葡萄是否通过选择剪接产生活性和数量不同的VvPMA1剪接变体影响根质膜质子泵总活性；VvPMA1β是否具有组织、抗性品种特异性；VvPMA1β活性如何调节。希望从基因及蛋白水平了解葡萄根质膜质子泵的表达调控方式，提高盐碱胁迫下葡萄根质膜质子泵活性。

葡萄作为已知基因组全序的多年生果树，碱性盐胁迫如何对根质膜H+-ATPase进行精确调控来维持体内pH恒稳态至今无深入研究。本项目发现葡萄经碱性盐胁迫后根质膜H+-ATPase主要表达基因VvPMA1在基因表达的RNA加工过程中会发生选择性剪切，通过内含子保留机制产生剪接变体VvPMA1β。缺失酵母突变体功能互补实验发现，相对组成型剪接体VvPMA1ɑ，该变体缺失N末端35个氨基酸残基，恰好解除了质膜H+-ATPase N末端自抑制区的抑制，呈现出较高的质膜质子泵活性。在盐碱胁迫初期耐盐碱能力强的葡萄根VvPMA1发生选择性剪切的比例较高，其活性也会相应增加。因此这种葡萄根质膜H+-ATPase基因表达调控方式有利于提高盐碱胁迫下葡萄根质膜H+-ATPase活性。将这两个剪接变体转化番茄和葡萄愈伤组织，发现VvPMA1β有助于提高葡萄愈伤在盐碱条件下的生存能力，但却严重影响了转基因番茄在正常条件下的生长，而VvPMA1ɑ对转基因番茄和愈伤在盐碱条件下的生长皆起到积极作用。说明过高活性的VvPMA1β变体消耗了大量的ATP，反而影响了植株正常生长。因此植株需通过选择性剪接精准调节盐碱条件下根质膜H+-ATPase选择性剪接体的比例，适当提高总活性来达到抗盐碱目的。