Functional alteration of cereal starches using the genes encoding starch-synthesizing enzymes

为了研究编码淀粉合成酶的基因是否能够利用改变谷物淀粉的特性，我们利用这些基因进行了生化实验，得到了以下结果：1)从水稻发育种子粗提取物中纯化出淀粉分支酶的三个同工型，分别为RBE1、RBE3和RBE4。根据纯化蛋白的氨基末端氨基酸序列，我们鉴定并鉴定了编码RBE1、RBE3和RBE4的cDNA克隆。利用RBE1、RBE3和RBE4的cDNA片段，建立了在大肠杆菌细胞可溶性提取物中生产重组酶的表达体系。纯化后的重组RBE1比重组RBE3和RBE4具有更快的直链淀粉和支链淀粉分支的能力。重组酶制备的支链α-葡聚糖表现出不同的低聚糖链转移模式；RBE1主要转移超过10个葡萄糖单位的长链，而RBE3倾向于转移6和7个葡萄糖单位的短链。RBE4在低聚糖链的可转移性方面具有RBE1和RBE3的特点。3) Northern blot分析表明，RBE4基因在叶片和发育中的种子中均有表达。RBE4基因与RBE1和RBE3基因是通过在种子发育早期的表达来区分的。因此，RBE4可能在支链淀粉合成的早期阶段发挥重要作用。

To examine whether the genes encoding starch-synthesizing enzymes are able to utilize alteration of the properties of cereal starches, we have carried out biochemical experiments using these genes, and have obtained the following results :1) Three isoforms of starch branching enzyme, termed RBE1, RBE3, and RBE4, were purified from crude extracts of rice developing seeds. On the basis of the amino-terminal amino acid sequences of the purified proteins, we have identified and characterized cDNA clones encoding each of RBE1, RBE3, and RBE4. An expression system for production of recombinant enzymes in soluble extracts of Escherichia coli cells has been established using the cDNA fragments of RBE1, RBE3, and RBE4.2) Purified recombinant RBE1 was capable of branching amylose and amylopectin more rapidly than recombinant RBE3 and RBE4. The branched α-glucans produced by the recombinant enzymes from potato amylose revealed the different patterns of oligosaccharide chain transfer ; RBE1 predominantly transferred longer chains of more than 10 glucose units, whereas RBE3 preferred to transfer short chains of 6 and 7 glucose units. RBE4 showed both characteristics of RBE1 and RBE3 in the transferability of oligosaccharide chains.3) Northern blot analysis demonstrated that the RBE4 gene is expressed in both leaves and developing seeds. The RBE4 gene was distinguished from the RBE1 and RBE3 genes by expression at the earlier stages of seed development. It is thus possible that RBE4 may play an important role in the early stages of amylopectin synthesis.