苎麻胞液型谷氨酰胺合成酶基因的克隆和超量表达载体构建

Cloning and Construction of Over-expression Vector of GS1 Gene from Ramie (Boehmeria nivea Gaud)

谷氨酰胺合成酶是植物氮代谢途径中的关键酶,分为胞液型(GS1)和质体型(GS2)两种。本文首次从苎麻中克隆了一个胞液型谷氨酰胺合成酶基因BnGS1-1,并利用生物信息学对其序列和结构特征进行了分析。该基因序列全长1205 bp,含有一个1071 bp的ORF区,编码356个氨基酸残基的多肽,pI和Mw分别为5.64和39.19 KDa,具有beta-Grasp和catalytic两个保守功能域;蛋白序列在56、92、249和297位点分别为Asp、Cys、His和Glu。系统进化分析表明BnGS1-1与大豆(Glycine max)、四季豆(Phaseolus vulgaris)、苜蓿(Medicago sativa)、棉花(Gossypium raimondii)在同一个分支上。同时,将BnGS1-1与pBI121载体利用同源重组技术,成功构建了植物超量表达载体。因此,本研究为了解和改善苎麻饲用特性提供了物质和理论基础。

The glutamine synthetase （GS） incluing cytosolic （GS1） and plastid （GS2） is an essen- tial enzyme in nitrogen metabolism pathway of higher plants. We reported the first cloning of cytosolic glutamine synthetase gene BnGS1 - 1 from ramie （Boehmeria nivea Gaud） . The cDNA of BnGS1 - 1 with length of 1205 bp encoded GS1 polypeptide of 356 amino acids. Sequence and structural analyses showed that BnGS1 - 1 protein contained beta - Grasp and catalytic functional domains that were conser- vative with other plants GS. The pI and Mw was 5.64 and 39. 19 KDa, the residues in site 56, 92,249 and 297 was Asp, Cys, His and Glu respectively. The amino acid sequences derived from BnGS1 - 1 had extensive homology with GSls from other higher plants. The phylogenetic tree displayed that the BnGS1 - 1 and GS1 from Glycine max, Phaseolus vulgaris, Medicago sativa and Gossypium raimondii in- corporated into the same sub - clade. In addition, the plant over - expression vector was successfully constructed according to homologous recombination technology. Therefore, our work provided material and theoretical basis for understanding and improving ramie forage quality.