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谷物淀粉性质的改变，我们利用这些基因进行了生化实验，并获得了以下结果：1）从水稻发育种子的粗提物中纯化了三种淀粉分支酶同工酶，命名为RBE 1，RBE 3和RBE 4。纯化的蛋白质的氨基末端氨基酸序列的基础上，我们已经确定和表征的cDNA克隆编码每个RBE 1，RBE 3，和RBE 4。利用RBE 1、RBE 3和RBE 4的cDNA片段建立了大肠杆菌可溶性提取物中重组酶的表达系统。2）纯化的重组RBE 1能够比重组RBE 3和RBE 4更快地分支直链淀粉和支链淀粉。由重组酶从马铃薯直链淀粉产生的支链α-葡聚糖揭示了寡糖链转移的不同模式; RBE 1主要转移大于10个葡萄糖单元的长链，而RBE 3优选转移6和7个葡萄糖单元的短链。北方印迹分析表明，RBE 4基因在叶片和发育中的种子中都有表达。RBE 4基因与RBE 1和RBE 3基因的区别在于在种子发育的早期阶段表达。因此，RBE 4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.