小麦谷氨酰胺合成酶的原核表达特点

Expression characteristics of glutamine synthetase of wheat in Escherichia coli

摘要:

谷氨酰胺合成酶 (GS) 是植物氮同化的关键酶，为了研究小麦GS同工酶的结构及其表达特点，我们构建了小麦GS1、GSr、GSe、GS2和GS2前体GS2p的原核表达载体，并对表达条件进行了优化。结果表明，尽管小麦GS同工酶氨基酸序列同源性达70%–80%，蛋白质表达却各具特点。30 ℃诱导3 h后，GSr、GSe及GS2表达量达最大，诱导7 h后GS1表达量达最大，GS2p不表达，表达量依次为GS1 (22%) >GSr (15%) >GS2 (12%)>GSe (5%)；且GSe可溶性表达，GS1主要为可溶性表达，而GSr和GS2为包涵体。30 ℃诱导3 h，GS同工酶相对转录量为GSr (7.59) >GS2 (1.84) >GS2p (1.66)>GSe (1.46) >GS1 (1.00)，酶蛋白质翻译水平与转录水平不一致。mRNA结构分析显示，GS同工酶翻译起始区稳定二级结构的自由能不同：GS1 (14.4)GSr (13%) >GS2 (10%) >GSe (7%)，酶活性为GS1>GSe>GS2，GSr无活性。可见，GS同工酶的基因序列决定了蛋白质在原核细胞中的表达量、状态及其活性。

Abstract:

Glutamine synthetase is a key enzyme in plant nitrogen assimilation. To study the structure of wheat glutamine synthetase isoenzymes, GS1, GSr, GSe, GS2 and GS2p of wheat were cloned into pET-21a, and the expression condition was optimized. Although wheat glutamine synthetase isoenzymes had 70%–80% amino acid sequence homology, the isoforms expressed with different characteristics. Induced at 30 °C, the most expression level of GSr, GSe and GS2 was after 3 h, and of GS1 was at the 7 h whereas no GS2p was expressed, and the GS isoenzymes showed different expression level, with the order GS1 (22%)>GSr (15%)>GS2 (12%)>GSe (5%). GSe expressed as soluble protein, and GS1 expressed mainly as soluble protein whereas GSr and GS2 expressed as insoluble proteins. Induced at 30 °C for 3 h, mRNA transcript levels of GS isoforms were different, with the order of GSr (7.59)>GS2 (1.84)>GS2p (1.66)>GSe (1.46)>GS1 (1.00). The levels of mRNA transcription were not consistent with the level of the protein translation. The analysis of mRNA secondary structure showed the free energy of translation initiation region of glutamine synthetase isoforms was different, with the order of GS1 (14.4)GSr (13%)>GS2 (10%)>GSe (7%), and different activities with GS1>GSe>GS2, and the activity of GSr was not detected. The gene sequence of glutamine synthetase isoenzymes determines the amount, status and activity of proteins expressed in prokaryotic cells.