海水喷施诱导葡萄果实糖积累的分子机理研究

Multiple applications of diluted seawater could induce sugar accumulation in grape found in our previous study. Two commonly cultivated grape cultivars 'Ju feng' and 'Mei ren zhi' will be used in this study. To find out the best treatment, sugar and ion concentrations in fruits are determined after treated with diluted seawater at different developmental stages. Then, gene expression pattern of grape from the best treatment are analyzed by RNA-Seq. More up-regulated or down-regulated genes from best seawater treatment and control fruits were screened by RNA-Seq analysis method according to gene expression. By screening the RNA-Seq data, candidate genes for sugar metabolism, ion absorption and transportation or Ca2+ signal transduction were selected through bioinformatics method. And, gene regulation network for sugar metabolism was constructed. Furthermore, the candidate key genes for sugar metabolism and their regulated genes were cloned and functionally analyzed by transforming into tomato with over-expression and RNAi expression vector. In a word, this study could not only clarify molecular mechanism of sugar accumulation in grape induced by seawater spray, but also find gene regulation network for sugar metabolism, which will be useful for grape quality improvement by genetic engineering.

课题组前期研究发现，多次喷施低浓度海水可促进葡萄果实的糖分积累。本项目以葡萄主栽品种'巨峰'和'美人指'为试材，不同时期叶面喷施多浓度的稀释海水，果实成熟期分析果实糖积累及矿质元素含量，筛选最佳处理方案。采用数字基因表达谱升级版测序技术（RNA-Seq）进行海水处理后葡萄果实基因表达变化分析，筛选上调或下调程度较大的基因；生物信息学方法分析、筛选候选糖代谢相关关键基因，离子吸收、转运及Ca2+参与的信号转导途径等相关基因；构建海水诱导葡萄果实糖积累的基因调控网络。依据已知葡萄全基因组序列，设计引物，克隆糖代谢关键候选基因、离子吸收、转运及Ca2+参与的信号转导途径等相关基因，构建超量表达与RNAi表达载体，转化番茄，研究它们在番茄果实糖代谢中的功能。本项目完成后不仅能阐明海水喷施诱导葡萄果实糖积累的分子机制，还可获得葡萄果实糖代谢的基因调控网络，为基因工程方法进行葡萄品质改良奠定分子基础

低浓度海水喷施可以促进葡萄果实的糖分积累，研究海水喷施诱导葡萄果实糖积累的分子机制，筛选果实糖代谢关键基因，不仅可以为提高葡萄品质提供技术支撑，还可以为基因工程方法改良葡萄品质奠定分子基础。项目实施过程中，两个葡萄品种（‘巨峰’和‘金手指’）对喷施稀释海水的反应一致，喷施海水提高了果实葡萄糖和果糖含量，但积累速率有差异；喷施海水未对植株生长造成显著抑制作用，增加了枝条可溶性糖含量，降低了淀粉含量，但游离氨基酸、相对电导率变化不大；喷施海水增加了土壤pH值、氯离子及多数阳离子浓度，但对碳酸氢根、硝态氮含量影响较小。由以上结果筛选出喷施海水最佳处理方案。采用数字基因表达谱升级版测序技术（RNA-Seq）进行喷施海水后葡萄果实基因表达变化分析，发现差异表达基因305个，其中，上调基因116个，下调基因189个，主要的差异基因集中在信号转导和蔗糖代谢途径上。克隆相关基因，转化番茄，由于时间所限，仍在进行中，以后将继续这方面的工作。在本项目的资助下，已经发表SCI收录论文3篇，中文核心期刊论文1篇，另有2篇英文稿件在投稿中。