小麦淀粉合成关键酶TaSSU II基因上游转录因子的辨析

ADP-glucose pyrophosphorylase (AGPase) is the key enzyme involved in crop starch synthesis. In higher plants, AGPase is a heterotetrameric enzyme composed of two large subunits and two small subunits, and has plastidial and cytosolic isoforms. In rice, maize and other cereals, the majority (more than 85%) of total AGPase activities are from cytosolic isoforms, and plastidial isoforms merely account for a small proportion (5%~15%). Different from plastidial AGPase in rice and maize, wheat plastidial AGPase accounts for about 36% of total AGPase activities. In our previous study, a cDNA sequence encoding the small subunit (TaSSU II) of plastidial AGPase in bread wheat was isolated and its precursor protein subunit was characterized with a truncated transit peptide (38 amino acids shorter than those of other plant SSU IIs). Using subcellular location assay, however, our experimental results showed that TaSSU II protein was still localized into plastids by targeting with this shorten transit peptide. Further, overexpression of TaSSU II gene has significantly increased the activities of plastidial AGPase and the rates of starch accumulation in transgenic wheat plants. These results have further verified the important role of TaSSU II gene in both AGPase enzyme and starch synthesis in wheat. . To explore the molecular regulatory mechanism of TaSSU II gene, in the present study, the TaSSU II promoter will be hybrided with cDNA library using yeast one-hybrid method and the transcription factors, which combine with the promoter of TaSSU II gene, are identified. The overexpression and RNAi interference vectors of the isolated transcription factors will be further constructed and transformed into wheat plants by genetic transformation. And the function of the isolated transcription factors in starch synthesis will be further explored by measuring the transcription levels of TaSSU II gene and other starch synthesis-related genes, the contents and activities of the plastidial AGPase, and the rates and characteristics of storage starch in grains in two types of the transgenic wheat plants. These results will help deeply explore the molecular regulatory mechanism of TaSSU II gene in starch synthesis and develop wheat germplasms with desirable agronomic traits.

申请人前期研究从小麦中克隆出淀粉合成关键酶—AGPase一个新的质体型小亚基基因（TaSSUII），发现其5´端缺失114bp一段序列，导致其蛋白质N端转移肽缺失了38个氨基酸，但亚细胞定位与转基因试验结果分别表明TaSSUII前体蛋白转移肽的转运功能与成熟蛋白的催化功能未发生改变，且过表达转基因小麦植株AGPase活性与淀粉合成能力均显著提高，表明TaSSUII在小麦淀粉合成中发挥了重要功能。为探究调控TaSSUII基因表达的作用机制，本项目利用酵母单杂交筛选出与该基因启动子相结合的上游转录因子，进而获得转录因子的过表达与抑制表达两类转基因小麦植株，通过测定两类转基因植株籽粒内TaSSUII基因表达水平、AGPase酶活性与淀粉含量等的变化，来验证分离的转录因子是否调控了TaSSUII基因表达及淀粉合成，从而辨析出调控TaSSUII基因表达的转录因子并有助于创制出优异的小麦种质资源。

淀粉是小麦籽粒最主要的组分（65%-80%），因此，提高小麦粒重主要在于增强淀粉的合成能力。腺苷二磷酸葡萄糖焦磷酸化酶（ADP-glucose pyrophosphorylase, AGPase）是作物淀粉合成的关键酶，有关其深入研究对增强小麦淀粉的合成能力具有重要意义。申请人前期研究发现质体型AGPase在小麦籽粒淀粉合成中发挥了重要功能，但有关小麦质体型AGPase的调控机制尚未报道。本项目通过酵母单杂交分离出小麦质体型AGPase亚基基因TaSSU II（TaAGPS1）上游的转录因子，并进行了功能验证。在本项目中，负责人将TaAGPS1基因启动子（promoter）与小麦籽粒cDNA文库进行酵母单杂交，初步筛选出一批调控因子，包括TaJAZ1、TabHLH39、TaEFRL1a等多个转录因子与TaPDIL1分子伴侣等；进一步，将上述调控因子与TaAGPS1 promoter进行了一对一的酵母单杂交和LUC实验，证明了它们均能与TaAGPS1基因启动子相结合，从而调控后者的表达；最后，利用瞬时沉默和基因编辑获得了TaJAZ1基因表达沉默的两类实验材料，结果发现两类沉默材料小麦籽粒内TaAGPS1基因表达量显著降低、淀粉合成显著受阻，表明TaJAZ1基因通过调控TaAGPS1基因的表达，在小麦淀粉合成中发挥了重要功能。上述研究结果有助于揭示小麦淀粉合成的分子调控机制。