大蒜HAP3基因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. In this investigation, the full-length AsHAP3 cDNA has been subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense and antisense constructs. The 35S-CaMV AsHAP3 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, RGL2）were analysed to determine whether there was altered expression of these genes in the AsHAP3 mutant. We tried to find out the interaction proteins with AsHAP3 using the yeast two-hybrid system. The functional analysis of AsHAP3 will provide a better understanding of the mechanisms regulating dormancy maintenance and release in garlic.

大蒜鳞芽的休眠特性对大蒜生产及产品贮藏有重要影响。处于休眠期的鳞芽播种后不能萌发。关于大蒜休眠的机制有待从分子水平深入研究，以便合理调控。本项目在已克隆大蒜HAP3基因（AsHAP3），获得含AsHAP3正义、反义基因的转基因大蒜植株的基础上，构建AsHAP3的原核表达载体，体外表达HAP3蛋白，制备抗体；Northern和Western杂交检测转基因植株，并分析休眠期不同植株AsHAP3的表达；测定大蒜对照植株和转基因植株鳞芽中赤霉素和脱落酸的含量；测定大蒜鳞芽中脱落酸、赤霉素信号转导及代谢相关基因ABI3、ABI4、ABI5、CYP707A1、CYP707A2、NCED9、RGL2、GA3OX1、GA3的表达。酵母双杂交技术寻找HAP3蛋白的互作蛋白。通过本研究进一步证明AsHAP3对大蒜鳞芽休眠的调控作用，探讨其对大蒜鳞芽休眠的调控途径，将为促进或阻止大蒜休眠提供理论依据

大蒜鳞芽的休眠特性对大蒜生产及产品贮藏有重要影响。处于休眠期的鳞芽播种后不能萌发。关于大蒜休眠的机制有待从分子水平深入研究，以便合理调控。本项目在已克隆大蒜AsHAP3（AsNF-YB3）基因，获得含AsHAP3正义、反义基因的转基因大蒜植株的基础上，构建了AsHAP3的原核表达载体，体外表达HAP3蛋白，制备了抗体；Northern和Western杂交检测了转基因植株，并分析了休眠期不同植株AsHAP3的表达；AsHAP3在MeJA、GA3处理后基因上调表达明显，在IAA、ABA 、ET处理后基因下调表达明显， AsNF-YB3能参与响应多种激素信号调控途径。测定了大蒜对照植株和转基因植株鳞芽中赤霉素和脱落酸的含量；提取转基因大蒜吸水后6 h, 12 h, 24 h, 48 h后大蒜嫩芽激素，采用酶联免疫吸附（ELISA）测定活性ABA含量。大蒜吸水后，ABA含量变化不明显。大蒜吸水6 h, 12 h时，GA3和GA4含量变化不明显，但是在24 h以及48 h，GA3和GA4含量都表现出明显增长。测定大蒜鳞芽中脱落酸、赤霉素信号转导及代谢相关基因ABI3、ABI4、ABI5、CYP707A1、CYP707A2、NCED9、RGL2、GA3OX1、GA3的表达。利用荧光定量PCR对脱落酸信号转导及代谢相关基因ABI3, ABI4, ABI5、CYP707A1, CYP707A2, NCED9的表达情况进行了检测，发现ABI3, ABI4, ABI5在转基因鳞芽中的的表达受到了抑制；而CYP707A1, CYP707A2, NCED9的表达在吸水后的表达变化不明显。对GA分解代谢途径的关键基因GA2ox3和GA20ox1的表达情况进行了检测，发现GA3ox1的表达在吸水后的6 h, 12 h, 24 h, 48 h等不同时段都表现出了不同程度的增长；而GA20ox1的表达在吸水后的6 h, 12 h, 24 h, 48 h虽然也有增长，但是增长幅度不如GA3ox1大。对转基因大蒜进行了转录组测序。在转基因植株中上调表达的有锌指蛋白（Zinc finger，RING/FYVE/PHD-type），GH3生长素应答启动子（GH3 auxin-responsive promoter），伴侣蛋白Cpn60 / TCP-1， GRAS转录因子（GRAS Transcriptio