Aminoglycoside 3'-phosphotransferase基因在質體pUC4-KISS中表現之探討

Abstract

質體pUC4-KISS所攜帶的Aminoglycoside 3'-phosphotransferase（APHI）基因在大腸桿菌中的表現量可高達全部蛋白質的百分之四十．然而，發現在不同的方向，不同的載體上，APHI基因的表現量顯著的降低，故推測質體pUC4-KISS中基因上游的某些核酸序列會影響APHI基因的表現．因此在本實驗中利用限制?切割及Exolll-SI方法刪除DNA片段；確實基因上游的核酸序列會影響APHI的表現．此外核酸定序分析發現lacP是位在APHI基因的上游，此發現與前人發表的圖譜不同．因此APHI基因之高表現可能是由於lacP及APHI基因產生Operon fusion之結果．除了lacP外，在更上游的核酸序列中，有兩個區域似乎也能影響APHI的表現，刪除其中41 bp的DNA使得APHI基因表現降低百分之八十．但是如果此41 bp之序列連同上游650 bp之序列一同刪除後，APHI之表現則維持在40％．顯示在41 bp上游序列，可能對APHI基因之表現有抑制作用，本研究主要是證實，基因的表現除了與本身的起動子有關外，基因上游的起動子和核酸序列也扮演著重要的角色．

The expression level of the gene encoding aminoglycoside 3'-phosphotransferase (APHI) in pUC4-KISS reached approximately 40% of total protein in Escherichia coli. The expression level decreased, however, when the orientation of the gene in pUC4-KISS was reverted or when the gene cartridge was cloned into the Pstl site of pUC19. Sequencing analysis revealed that the lac promoter was situated upstream from the APHI gene. This finding indicated that the map of pUC-4-KISS reported previously was incorrect. The high expression of APHI was probably due to an operon fusion between lacZ and APHI gene. The P5 promoter of pBR322 which was located upstream from the lac promoter also affected the expression of APHI. Deletions in this promoter region lowered the gene expression by 25%. However, the DNA upstream from the P5 promter seemed to inhibit the expression. Restriction deletion studies revealed that deletions in this region actually increased the expression of APHI gene in pUC4-KISS. This study demonstrated that the upstream sequence of the APHI was essential for the high expression of the gene.