木薯细胞壁转化酶基因家族启动子的克隆及功能研究

Cassava is a very important energy source, which is a starch storage root crop. It is reported that the amount of photosynthate produced by leaves transfers to the roots for strach accumulation is less than 1/5. So it is meanful to improve the energy transferring rate from leaves to roots. Sucrose cell wall invertase， is the key enzyme of the sucrose-metabolizing enzymes which transfers photosynthate from leaves to roots. The project will focus on investigation of the promoters of cassava sucrose cell wall invertase gene family. Firstly, isolation of the promoters by chromosome walking method; Construction of a set of the 5' deletion promoter vectors; then transformation of them to Arabidopsis and cassava seedings to identify the core promoters. Online analyses of the promoter sequences to find the light response and adversity cis acting elements binding sites. Gel mobility shifting assays，yeast one-hybrid system and lack of cis element analysis　to identify the binding sites in the promoters. Under various treatment such as light and stresses to test the interactional regulation of the transcription factors and promoters using Real-time PCR, GUS and invertase activities anlysis.Then the temporal and spatial rules of gene expression regulated by the promoters will be identified. This results will be scientific fundation for researching on the role of cassava sucrose invertase in cassava starch accumulation from leaves to roots, and provide a transgenic breeding basis for improving the starch content in cassava.

木薯是以块根淀粉为主要储能物质的新型能源、工业原料和粮食作物。木薯块根中储藏同化物的量不足叶片中合成同化物量的1/5，提高光合同化物从叶（源）到块根（库）的转化效率对木薯淀粉积累意义重大。细胞壁转化酶是负责此过程的关键酶，在调节韧皮部糖的卸载、控制贮藏器官中糖的组成等方面具有重要作用。本研究利用染色体步移法扩增获得木薯细胞壁转化酶基因家族6个成员的启动子序列，用生物信息学在线分析和5'缺失法确定各启动子的结构组成；利用凝胶迁移滞后实验、酵母单杂交技术、顺式元件缺失分析，研究确定其上的核心调控元件；不同外界诱导条件下，采用Real-time PCR、GUS 活性及酸性转化酶活性检测确定各启动子对目的基因的调控作用，鉴定其调控基因表达的时空特征，筛选出能提高木薯淀粉积累的组织特异型启动子和诱导型启动子。为进一步研究细胞壁转化酶基因功能提供依据，并为高淀粉木薯品质改良分子育种提供基础。

细胞壁转化酶在调节韧皮部糖的卸载、控制贮藏器官中糖的组成和参与植物逆境胁迫等方面具有重要作用。本课题扩增获得了6个木薯细胞壁转化酶基因启动子；对克隆获得启动子序列进行比对和调控元件的生物信息学分析，确定了木薯细胞壁转化酶基因家族各启动子的结构组成,并分析了各基因启动子的结构差异和其上所存在的顺式作用元件的异同；GUS组织化学染色及荧光定量分析确定启动子活性；顺式元件缺失分析、凝胶迁移滞后实验和酵母单杂交实验确定转录因子MeAHL和MeABI5与木薯细胞壁转化酶基因启动子的相互作用；不同外界诱导条件下，检测细胞壁转化酶基因及其启动子活性变化，分析了分析了转录因子MeAHL和MeABI5的表达模式。为进一步研究细胞壁转化酶基因功能提供了依据，并为高淀粉木薯品质改良分子育种提供了基础。项目共发表标注发表期刊论文11篇（其中SCI论文4篇，EI论文3篇，中文核心期刊论文4篇），会议论文2篇（EI收录），培养硕士研究生4名。