大蒜AsWOX9调控鳞芽休眠的功能分析

Garlic (Allium sativum L.) is one of the most widely cultivated Allium vegetables throughout the world. The sprouting of garlic bulbs during storage is a major factor limiting storage life, as it leads to a reduction of vegetable quality, loss of dry matter, and onset of disease. However, the molecular mechanism of dormancy maintenance and sprouting in Allium crops is poorly studied and remains largely unknown. WOX（WUSCHEL-related homeobox）is an important transcription factor in plants and plays an important role in the process of organ formation and development. In this investigation, the full-length AsWOX9 cDNA has been subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense and antisense constructs. The 35S-CaMV AsWOX9 constructs has been introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method. The transgenic plants has been selected by 5‰ kanamycin. The kanamycin-resistant transgenic plants will be further verified by Northern and Western hybridization. A recombinant of prokaryotic expression vector pET-LeVDE will be constructed and transformed to E.Coli BL21 to express. The strong induced fusion protein bands will be collected into PBS solution and used to immunize white mice to obtain antiserum. ABA and GA levels will be measured in freshly-harvested bulbs to determine whether changes in ABA and GA could explain the differences in dormancy between WT and transgenic plants and the changes observed in the examined transcripts during sprouting. Genes involved in ABA and GA signalling and metabolism（CYP707A1, CYP707A2, NCED9, GA3OX1, GA3OX2, ABI3, ABI4, ABI5）were analysed to determine whether there was altered expression of these genes in the AsWOX9 mutant. We tried to find out the interaction proteins with AsWOX9 using the yeast two-hybrid system. The functional analysis of AsWOX9 will provide a better understanding of the mechanisms regulating dormancy maintenance and release in garlic.

鳞芽的休眠特性对大蒜生产及产品贮藏有重要影响。大蒜休眠的机制有待从分子水平深入研究，以便合理调控。WOX是植物所特有的转录因子，在器官形成和发育过程中发挥重要调控作用。本项目在已克隆大蒜AsWOX9基因，获得过表达及抑制表达AsWOX9转基因大蒜的基础上，探讨AsWOX9参与调控大蒜鳞芽休眠的功能和分子机理。研究拟通过检测转基因大蒜鳞芽中赤霉素和脱落酸的含量及相关代谢基因的表达，分析AsWOX9对赤霉素和脱落酸信号途径的影响；通过测定转基因大蒜鳞芽中可溶性糖等的含量，蔗糖合成酶等酶的活性，明确AsWOX9对碳水化合物代谢的影响；通过测定转基因大蒜鳞芽中氨基酸、蛋白质含量，谷丙酰胺合成酶等酶的活性，分析AsWOX9对蛋白质代谢的影响，从而探讨AsWOX9调控鳞芽休眠的生理生化机制。并通过酵母双杂交寻找AsWOX9的互作蛋白，深入分析AsWOX9调控鳞芽休眠的分子机制。

鳞芽的休眠特性对大蒜生产及产品贮藏有重要影响。大蒜休眠的机制有待从分子水平深入研究，以便合理调控。WOX是植物所特有的转录因子，在器官形成和发育过程中发挥重要调控 作用。本项目克隆了大蒜AsWOX9基因，获得了过表达及抑制表达AsWOX9转基因大蒜， 探讨了AsWOX9参与调控大蒜鳞芽休眠的功能和分子机理。通过检测转基因大蒜鳞芽中赤霉 素和脱落酸的含量及相关代谢基因的表达，分析了AsWOX9对赤霉素和脱落酸信号途径的影响；通 过测定转基因大蒜鳞芽中可溶性糖等的含量，蔗糖合成酶等酶的活性，明确了AsWOX9对碳水化合 物代谢的影响；通过测定转基因大蒜鳞芽中氨基酸、蛋白质含量，谷丙酰胺合成酶等酶的活性，分析了AsWOX9对蛋白质代谢的影响，从而探讨AsWOX9调控鳞芽休眠的生理生化机制。