利用大腸桿菌表現甘藷澱粉粒結合性澱粉合成酵素及其特性分析

An Expression of the Sweet Potato GBSSI Gene in Escherichia coli

本研究的主要目的是利用大腸桿菌（E. coli）表現甘藷之澱粉合成酵素基因（spss67），並藉由氨基酸序列比對、抗體辨認及生化特性分析鑑定此澱粉合成酵素基因究竟是屬於澱粉粒結合性澱粉合成酵素（granule- bound starch synthase; GBSS）或是可溶性澱粉合成酵素（soluble starch synthase; SSS）基因。西方雜合分析結果顯示由E. coli表現的spss67蛋白質產物可被馬鈴薯GBSSI抗體辨認。經氨基酸序列比對顯示此蛋白與其他植物之GBSSI有較高的同源性，而與SSS的相似性則較低。此外，經N端氨基酸序列分析，得知甘藷之GBSSI蛋白具有一段77個氨基酸的導引訊息（transit peptide）。澱粉合成酵素活性分析的結果顯示spss67 cDNA之蛋白產物需以直鏈澱粉（amylose）做為做為引子以進行葡萄糖聚合，但其無法以支鏈澱粉（amylopectin）及肝醣（glycogen）做為引子以進行反應，此結果提供一直接的證據證明spss67 cDNA為甘藷之GBSSI基因。

A starch synthase cDNA clone (spss67) was isolated from tuberous roots of sweet potato. This clone contained an open reading frame corresponding to a protein of 608 amino acid residues, which also included three consensus regions of starch synthases. Amino acid sequence analyses showed that the protein product of spss67 shared a high degree of homology with other plant granule-bound starch synthase I (GBSSI). Comparisons of N-terminal amino acid sequence of spss67 cDNA protein product with that of the native protein isolated from sweet potato indicated that the protein contained a signal peptide of 77 amino acids, and the transit peptide had hydrophobic regions in the initiation and end of peptide that was also present in GBSSI of other plant species. In addition, the protein product of spss67 expressed in E. coli could be recognized by anti-GBSSI antibody of potato. Moreover, this protein exhibited a high activity of starch synthase when amylose was used as a primer, but the activity was not significant when the primer was replaced by amylopectin or glycogen. Studies based on the sequence analyses, western blotting and priming activity indicated that the product of spss67 clone was a GBSSI.