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 of 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 (17.2)<GS2 (22.6) <GSe (25.4) <GS2p (31.6), the smaller freed energy, the more unstable mRNA secondary structure of translation initiation region and the higher level of protein expression. Soluble expression condition of glutamine synthetase isozymes was also different, with GS1, GSr, GSe and GS2 induced at 30 °C for 5 h, 16 °C for 15 h, 37 °C for 5 h, and 25 °C for 7 h respectively. The soluble protein showed different expression level with GS1 (20%)>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.
ABSTRACT
Objective To compare the oil yield of celery seeds and the contents of 3-n-butylphthalide and the total phthalocyanine lactones of celery seed oil extracted by different methods. Methods Three routine extraction methods involving organic solvent extraction, Soxhlet extraction, steam distillation extraction, as well as subcritical extraction method and supercritical fluid extraction method were used to extract the celery seed oil. The contents of 3-n-butylphthalide and total phthalocyanine lactones were respectively detected by high performance liquid chromatography(HPLC) and ultraviolet visible spectrophotometry. Results The ranges of oil yield and the contents of 3-n-butylphthalide and total phthalocyanine lactones of celery seed oil extracted by different methods were 0.30%-20.02%, 1.40%-10.13%, 4.74%-17.65%, respectively, indicating obvious differences. Conclusion With R134a and butane as the solvents, the subcritical extraction method is better than other extraction methods for the extraction of 3-n-butylphthalide. With dimethyl ether as the solvent, the subcritical extraction method is the best for the extraction of total phthalocyanine lactones.