转录因子应答ABA和糖信号调控酿酒葡萄果实糖积累关键酶基因表达的分子机制

The type and content of sugar is one of important agronomic traits to determine winemaking raw material grape berry quality. The study of the trait helps to improve the quality of the berries and wine. Berry sugar accumulation in the metabolism is a complex network system, which was effected by the interaction of sugar and ABA hormonal signal dual signal pathways. The wine grape 'Cabernet Sauvignon' as test material, transcription factors regulating sugar accumulation metabolism key enzyme genes in response to ABA regulatory pathway and glucose and sucrose regulating pathway were get via the use of transcriptome sequencing and annotation mining. The spatial and temporal expression of transcription factors were detected by using real-time quantitative PCR technology. The function of transcription factors were explored in grape berry sugar accumulation response to sugar signal pathway and ABA hormonal signals pathway. The subcellular localization of transcription factors was carried out via fluorescence positioning technique GFP. The functional validation studies of transcription factors were made by using grapes cell suspension system and transgenic Arabidopsis system. Transcription factor and sucrose synthase and acid invertase gene promoter binding rules will be revealed to regulate the acid invertase and sucrose synthase gene expression at the transcriptional level. Ultimately revealing response transcription factor signaling regulates ABA and sugar grape berry sugar accumulation key enzyme molecular mechanisms of gene expression.

酿酒葡萄果实内糖种类和含量是决定酿酒葡萄果实品质的重要农艺性状，对该性状的研究有助于提高浆果和葡萄酒的品质。浆果内糖积累代谢是一个错综复杂的网络系统，受到糖信号和ABA激素信号双重通路的交互作用。本项目以酿酒葡萄‘赤霞珠’为试材，利用转录组测序技术注释并挖掘与ABA调节通路和葡萄糖和蔗糖调节通路调控糖积累代谢关键酶基因的转录因子。利用实时荧光定量PCR技术，检测转录因子的时空表达，探究转录因子分别在糖信号和激素ABA信号调节果实糖积累通路中的功能。利用荧光定位技术对转录因子进行GFP亚细胞定位，确定转录因子在细胞中的功能部位。利用葡萄果实细胞悬浮系统和拟南芥转基因系统，进行转录因子功能验证研究，以揭示转录因子与蔗糖合酶和酸性转化酶基因启动子结合规律，并在转录水平上调节蔗糖合酶和酸性转化酶基因表达。最终揭示转录因子应答ABA和糖信号调控酿酒葡萄果实糖积累关键酶基因表达的分子机制。

葡萄浆果中含糖量决定了酿酒原料葡萄果实品质。研究转录因子对糖和ABA信号转导途径中糖代谢关键酶基因的调控，有助于提高酒葡萄浆果的品质。为了进一步探讨转录因子对酿酒葡萄‘赤霞珠’浆果糖代谢的调控机制，对酿酒葡萄浆果发育过程30天、70天和90天的葡萄果实进行了转录组测序分析。通过基因共表达网络分析，找到了能够富集糖代谢功能的转录因子模块。通过基因注释信息获得葡萄浆果中VvNF-Y、WRKY、MYB、VvbZIP、VvHD-Zip、VvNAC、VvABF和VvABI等转录因子与糖代谢有密切关系。利用荧光定位技术GFP对这些转录因子进行亚细胞定位。克隆了酿酒葡萄‘赤霞珠’浆果VvbZIP37、VvHD-Zip11/21、VvNF-Y、VvNAC8/26、VvWRKY22/23、VvMYB16/92、VvNF-YB13/C8、VvABF2和VvABI5等转录因子基因。利用克隆技术构建pCXSN-过表达转录因子和pCXSN-反转录因子载体。然后利用农杆菌介导法对这些载体进行转基因葡萄愈伤组织的构建。利用这些pCXSN载体上独特的潮霉素抗性基因进行筛选，最终获得转化的葡萄愈伤组织。利用葡萄糖、蔗糖、脱落酸以及葡萄糖和蔗糖分别与ABA结合处理野生型愈伤组织和转基因型愈伤组织，实时荧光定量PCR检测糖和脱落酸信号途径中糖代谢关键酶基因的相对表达量。综合结果分析，这些转录因子的过表达增加了植物对糖和ABA信号的敏感性，而它们的沉默则导致相反的结果，并且响应ABA和糖信号调控VvPYL6、VvPP2C2、VvSnRK1、VvABI3、VvSS、VvSPS、VvHXK、VvCWINV、VvTPS的表达。此外，利用定点突变技术对VvbZIP11和VvbZIP53的uORF进行点突变，并构建载体，用农杆菌介导法转化入葡萄愈伤组织。证实了VvbZIP11和VvbZIP53转录因子均表现出蔗糖诱导的转录表达抑制（SIRT），即uORF在高蔗糖浓度下抑制表达。最后，为阐明这些转录因子参与糖和ABA信号传导的机制，我们还利用酵母双杂交和BiFC系统研究了一些转录因子与VvSnRK1和HXK三者的蛋白质与蛋白质的相互作用。结果表明，糖信号与激素ABA信号的交叉可能是由转录因子介导的SnRK1和HXK的蛋白质互作完成的。