调控小麦TaAGPL1基因表达的转录因子的分离及其功能研究

ADP-glucose pyrophosphorylase (AGPase) plays the key role in plant starch synthesis. AGPase in higher plants is a heterotetrameric enzyme composed of two large subunits and two small subunits, and has plastidial and cytosolic isoforms. Accordingly, there are four types of AGPase subunits (cytosolic small subunit, AGPS1, plastidial small subunit, AGPS2; cytosolic large subunit, AGPL1; and plastidial subunit, AGPL2) in higher plants. In our previous studies, the comprehensive transcriptional levels of 26 starch synthesis-related genes during the grain filling were measured using qRT-PCR and the relations between the expression levels of these genes and the rates of grain starch accumulation were further calculated. The results indicated that, during the grain filling periods, thirteen genes (TaAGPS1-a, TaAGPL1, TaGBSSI, TaSSI, TaSSIIa, TaSSIIIa, TaSSIV, TaBEI, TaBEIIa, TaBEIIb, TaPUL, TaPHOL and TaDPE1) were richly expressed during the middle and late stages of grain development, their transcription levels were positively related to the rates of starch accumulation, and these genes are presumed to play important roles in starch synthesis. Among these genes, TaAGPL1 gene had the highest positive relation to the rates of starch accumulation,and was presumed to play the most important role in starch synthesis among thirteen genes. Overexpression of TaAGPL1 gene has significantly increased the activities of AGPase and the rates of starch accumulation in grains. These results have verified the crucial role of TaAGPL1 gene in both AGPase enzyme and starch synthesis in wheat grains. Accordingly, the promoter of TaAGPL1 has been isolated (GenBank number accession: JQ346192) in our lab.. To further explore the molecular mechanism of TaAGPL1 gene, in the present study, the endosperm-specific elements of TaAGPL1 promoter will be isolated using fragment-deletions method. The transcription factors, which combine with the endosperm-specific elements of TaAGPL1 gene, are futher identified using yeast one-hybrid method. The overexpression and RNAi interference vectors of the isolated transcription factors will be constructed and transformed into wheat plants. In two types of the transgenic wheat plants, the trascription levels of TaAGPL1 gene and other starch synthesis-related genes, the activities of the AGPase, and the rates of starch accumulation in grains will be measured in their grains in order to explore the function of the isolated transcription factors in starch synthesis. These results will help to further explore the molecular mechanism of TaAGPL1 gene on starch synthesis in important crops.

AGPase是调控作物淀粉合成的关键酶，申请人前期研究发现它的胞质型大亚基（TaAGPL1）基因表达量的高低与小麦籽粒的淀粉积累速率呈显著正相关，推测它在AGPase活性和淀粉合成中发挥着重要作用；进一步研究发现，TaAGPL1基因过表达后，小麦籽粒内AGPase活性与淀粉积累速率均显著提高，从而验证了上述推测。为进一步探讨调控TaAGPL1基因表达的作用机制，申请人分离了它的启动子序列。在此基础上，本项目拟采用内部缺失法，鉴定出此启动子的胚乳特异性响应元件；利用酵母单杂交法，筛选出调控TaAGPL1表达的转录因子；进而获得转录因子的过表达与抑制表达的转基因植株，通过测定两类转基因小麦植株籽粒内，TaAGPL1基因、AGPase活性与淀粉积累速率等的变化，来验证分离的转录因子是否调控TaAGPL1基因的表达，从而进一步解析调控TaAGPL1基因表达的作用机制。

AGPase是调控作物淀粉合成的关键酶，项目负责人前期研究发现它的胞质型大亚基（TaAGPL1）基因表达量的高低与小麦籽粒的淀粉积累速率呈显著正相关，推测它在AGPase活性和淀粉合成中发挥着重要作用；进一步发现，过表达TaAGPL1基因后，小麦籽粒内AGPase活性与淀粉积累速率均显著提高，从而验证了上述推测。为进一步探讨调控TaAGPL1基因表达的作用机制，项目负责人分离了它的启动子序列。在此基础上，本项目采用内部缺失法，鉴定出此启动子的胚乳特异性响应元件；利用酵母单杂交法，筛选出调控TaAGPL1表达的转录因子-bHLH39和分子伴侣-PDIL1-2；又采用酵母单杂交和LUC试验，发现bHLH39和PDIL1-2分别能与TaAGPL1启动子之间存在着互作关系；进而利用BSMV-VIGS沉默法，分别获得了bHLH39或PDIL1-2短暂沉默的小麦植株，发现bHLH39沉默小麦植株籽粒内TaAGPL1基因、淀粉含量、粒重等均显著降低，而PDIL1-2短暂沉默的小麦植株上述指标则呈现出相反的趋势，表明bHLH39转录因子与PDIL1-2分子伴侣分别通过正向与负向调控TaAGPL1基因的表达，从而参与了小麦淀粉的合成。