细胞壁蔗糖转化酶基因（AoCWIN1）调控绿芦笋采后品质变化的分子机理

The process of quality deterioration of postharvest green asparagus was closely tracked with the spatial-temporal dynamic changes of sugars composition and content. Cell wall invertase can regulate the form and flow of sugars. However, the molecular mechanism of on regulating the quality changes of postharvest green asparagus has not been reported. We previously screened one cell wall invertase candidate gene AoCWIN1 and one cell wall invertase inhibitor gene AoINVHIN1 which may be involved in regulating the quality of postharvest green asparagus. In this project, full-length of above two candidate genes will be cloned. The function and mechanisms of these candidate genes involved in regulating the texture ageing process of immature stem will be investigated through constructing the overexpression vectors of two candidate genes and transforming these genes to Arabidopsis thaliana. Subsequently, the promoter of AoCWIN1 gene will be cloned. Then, the upstream and downstream genes regulated by AoCWIN1 and involved in regulating the texture ageing process of immature stem will be isolated using yeast one-hybrid and RNA sequence technologies. Finally, the spatio-temporal expression patterns of these genes in those immature stems of green asparagus at different development stages and at different storage stages of different post-harvest treatments will be studied by real-time quantitative PCR. The results of this project will not only help us to understand the molecular mechanism of AoCWIN1 on regulating the quality changes of postharvest green asparagus and enrich the biological theory of postharvest quality deteriorates, but also can find out more functional genes which can be used to enhance the storage quality of postharvest green asparagus by genetic engineering.

绿芦笋采后品质劣变进程与糖的组分及其含量的时空动态变化密切相关。CWIN能够调节糖的形态与流向。但目前有关CWIN参与调控绿芦笋采后品质变化的分子机制未见报道。本项目拟对申请者前期从采后绿芦笋中筛选出的候选AoCWIN1基因及其抑制蛋白AoINVHIN1基因序列进行克隆；通过构建上述基因的超量表达载体转化拟南芥对候选基因参与调控嫩茎质地老化的功能进行验证；随后克隆AoCWIN1基因启动子序列，并通过酵母单杂交、转录组测序等技术筛选AoCWIN1调控的上下游基因。最后利用实时定量PCR分析2个候选基因以及AoCWIN1调控的上下游基因在绿芦笋嫩茎不同发育时期以及采后不同处理贮藏期间的时空表达模式。研究结果将帮助我们阐明AoCWIN1基因调控绿芦笋采后品质变化的分子机制，并顺着AoCWIN1调控的上下游基因，找到更多的采后品质调控基因，为绿芦笋采后品质调控基因工程提供新的基因资源和理论基础。

绿芦笋采后品质劣变进程与糖的组分及其含量的时空动态变化密切相关。本项目针对一个绿芦笋贮藏前后表达差异倍数超高的糖代谢相关基因（立项时命名为AoCWIN1，现更名为Aoeh4a）的结构、生物学功能及其参与调控绿芦笋采后品质变化的作用机制展开研究。研究结果如下：Aoeh4a的cDNA长1689 bp，其编码蛋白属于糖苷水解酶32家族，亚细胞定位于细胞壁上，具有59个磷酸化位点；系统进化分析表明Aoeh4a与已报道的果聚糖6G&1-外切水解酶蛋白eh4a（BBE49465.1）亲缘关系最近；获得了AoEH4a基因起始密码子上游1856bp长的启动子序列，该启动子上存在多个ABA、JA、SA、ETH以及光响应因子等顺式元件；GUS染色分析发现，Aoeh4a启动子的核心区域主要在基因上游的988bp内，ABA和MeJA对启动子活性有诱导效应而SA和蔗糖则起抑制作用；绿芦笋Aoeh4a的表达水平具有品种、器官、组织部位差异性，与品质和产量形成密切相关；证实了Aoeh4a表达水平与绿芦笋糖代谢水平以及采后品质变化进程呈正相关，同时Aoeh4a表达可响应多种植物激素及糖信号，但头部和基部组织的响应模式不同；通过构建Aoeh4a过表达载体并稳定转化烟草和番茄植株研究Aoeh4a功能，发现转基因烟草及番茄植株的株型较野生型小，结实率大大降低，但烟草茎以及番茄果实中的果聚糖含量增加，转基因番茄果实的耐贮性更强；通过酵母单杂文库筛选到25个可能与Aoeh4a启动子结合的上游调控基因，其中包括14个转录因子和2个激酶；通过酵母双杂文库筛选出了315个可能与Aoeh4a互作的基因，这些候选基因中有多个基因在绿芦笋贮藏过程中被强烈诱导表达。酵母双杂及双荧光素酶实验表明转录因子myb30与Aoeh4a存在互作，并通过瞬时过表达技术分析了myb30与Aoeh4a参与调控绿芦笋采后糖代谢变化的分子机制。本研究结果明确了Aoeh4a参与了调控绿芦笋采后品质变化进程，初步阐明了其作用的生理生化及分子机制，丰富了绿芦笋采后品质调控理论。并可以顺着Aoeh4a调控的上下游基因，找到了更多的采后品质调控的候选基因，为绿芦笋采后品质调控基因工程提供新的基因资源。