BACKGROUND ADP-glucose pyrophosphorylase (AGPase), the key enzyme in plant starch biosynthesis, is a heterotetramer composed of two identical large subunits and two identical small subunits. AGPase has plastidial and cytosolic isoforms in higher plants, whereas it is mainly detected in the cytosol of grain endosperms in cereal crops. Our previous results have shown that the expression of the TaAGPL1 gene, encoding the cytosolic large subunit of wheat AGPase, temporally coincides with the rate of starch accumulation and that its overexpression dramatically increases wheat AGPase activity and the rate of starch accumulation, suggesting an important role. METHODS In this study, we performed yeast one-hybrid screening using the promoter of the TaAGPL1 gene as bait and a wheat grain cDNA library as prey to screen out the upstream regulators of TaAGPL1 gene. And the barley stripe mosaic virus-induced gene-silencing (BSMV-VIGS) method was used to verify the functional characterization of the identified regulators in starch biosynthesis. RESULTS Disulfide isomerase 1-2 protein (TaPDIL1-2) was screened out, and its binding to the TaAGPL1-1D promoter was further verified using another yeast one-hybrid screen. Transiently silenced wheat plants of the TaPDIL1-2 gene were obtained by using BSMV-VIGS method under field conditions. In grains of BSMV-VIGS-TaPDIL1-2-silenced wheat plants, the TaAGPL1 gene transcription levels, grain starch contents, and 1000-kernel weight also significantly increased. CONCLUSIONS As important chaperones involved in oxidative protein folding, PDIL proteins have been reported to form hetero-dimers with some transcription factors, and thus, our results suggested that TaPDIL1-2 protein could indirectly and negatively regulate the expression of the TaAGPL1 gene and function in starch biosynthesis.

中文翻译：

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PDIL1-2可以间接和负调控面包小麦中AGPL1基因的表达。

背景技术ADP-葡萄糖焦磷酸化酶（AGPase）是植物淀粉生物合成中的关键酶，是由两个相同的大亚基和两个相同的小亚基组成的异四聚体。AGPase在高等植物中具有质体和胞质亚型，而AGPase主要在谷类作物的谷物胚乳的胞质溶胶中检测到。我们以前的研究结果表明，编码小麦AGPase胞质大亚基的TaAGPL1基因的表达在时间上与淀粉积累速率一致，并且它的过表达显着提高了小麦AGPase活性和淀粉积累速率，表明了重要作用。方法在这项研究中 我们用TaAGPL1基因的启动子作为诱饵，并用小麦籽粒cDNA文库作为猎物进行了酵母一杂交筛选，以筛选出TaAGPL1基因的上游调节子。并利用大麦条纹花叶病毒诱导的基因沉默（BSMV-VIGS）方法来验证已鉴定的调节剂在淀粉生物合成中的功能特征。结果筛选出二硫键异构酶1-2蛋白（TaPDIL1-2），并使用另一种酵母单杂交筛选进一步证实了其与TaAGPL1-1D启动子的结合。在田间条件下，使用BSMV-VIGS方法获得了TaPDIL1-2基因的瞬时沉默小麦植株。在沉默了BSMV-VIGS-TaPDIL1-2的小麦植株中，TaAGPL1基因的转录水平，籽粒淀粉含量和1000粒重也显着增加。