Functional alteration of enzymes for starch biosynthesis to advance plant productivity

植物ADP葡萄糖焦磷酸化酶（AGPglucose pyrophosphorylase，AGP 1）催化淀粉合成的唯一底物ADP葡萄糖的供应。AGG是由两个大亚基和两个小亚基组成的异源四聚体酶，并受3-磷酸甘油酸和Pi的变构调节。本研究的目的是通过改善AGPase的变构特性来定量改变淀粉含量。1）从表达拟南芥AGPase天然小亚基cDNA和突变大亚基cDNA的大肠杆菌中制备对变构效应物低敏感性的突变AGPase。结果表明，ApL 1基因由14个内含子和15个外显子组成。3）根据从大肠杆菌细胞中制备的突变型AGPases的结果，通过定点突变将相同的突变引入ApL 1基因。将突变体基因连接到自身启动子下。4）在转别构突变体ApL 1基因的拟南芥TL 46植株中，淀粉的最大积累量是TL 46植株的4倍多，是野生型植株的1.2倍左右，说明AGL 1的别构特性可以改变淀粉的合成速率。

Plant ADPglucose pyrophosphorylase (AGPase) catalyzes the supply of ADPglucose that is a sole substrate of starch biosynthesis. AGPase is a heterotetrameric enzyme composed of two large and two small subunits, and allosterically regulated by 3-phosphoglycerate and Pi. The aim in this study was a quantitative alteration of starch contents by improvements of allosterical properties of AGPase.1) Mutant AGPases with low sensitivities to allosteric effectors were prepared from E.coli expressing the native samll subunit cDNA and the mutated large subunit cDNA of Arabidopsis AGPase.2) The ApL1 gene conaining the promoter region was isolated from an Arabidopsis genomic library. The sequence revealed that the gene consists of 14 introns and 15 exons.3) Based on the results of mutant AGPases prepared from E.coli cells, the same mutations were introduced in the ApL1 gene by a site-directed mutagenesis. The mutant genes were ligated under the self promoter. Arabidopsis TL46 plants, that are deficient in ApL 1 protein in leaves, were transformed with the constructed mutant genes.4) In leaves of transgenic plants expressing allosteric mutant ApL1, the maximum starch accumulation was more than 4-fold of TL46 plants and about 1.2-fold of wild type plants.These results suggest that the allosteric properties of AGPase can alter the rate of starch biosynthesis.