发菜1,4-α-葡聚糖分支酶(GBE)的基因克隆及其对干旱胁迫的响应

Gene Cloning of 1,4-α-glucan branching enzyme(GBE) from Nostoc flagelliforme and Its Differential Expression in Response to Drought Stress

由glgB基因编码的1,4-α-葡聚糖分支酶(GBE)是催化调控α(1-6)糖苷键分支合成的关键酶。本研究从发菜中克隆获得1,4-α-葡聚糖分支酶glgB基因全长2 292 bp(GeenBank登录号:KX679395),编码763个氨基酸。原核表达得到88.6 kD的glgB基因外源蛋白,MALDI-TOF-TOF/MS质谱分析表明外源蛋白为GBE。发菜glgB与点形念珠藻的glgB序列相似性为94%,氨基酸序列相似性为97%;GBE在第747位的甘氨酸疏水性最强,第168位的组氨酸亲水性最强;Thr有15个磷酸化位点,Tyr有12个磷酸化位点、Ser有27个磷酸化位点;GBE二级结构及三级结构由α螺旋、β折叠、随机卷曲和β转角构成。发菜glgB在干旱胁迫下转录水平的表达量逐渐增加,GBE含量增加,糖原合成酶活性和糖原含量均先增加后降低。研究结果为认识glgB基因信息和发菜响应干旱胁迫的糖原代谢和调控机制提供了依据。

glgB coded 1,4-α-glucan branching enzyme(GBE)is a key enzyme that catalyzes the synthesis of theα(1-6)branch in prokaryote.In this study,the glgB of GBE was cloned from N.flagelliforme,the full-length of glgB is 2 292 bp(GenBank accession number:KX679395),encoding 763 amino acids.glgB was expressed in E.coil then a 88.6 kD heterologous protein was observed and tested by MALDI-TOF-TOF/MS.Bioinformatics analysis showed that nucleotide sequence and deduced amino acid sequence of glgB from N.flagelliforme were similar to that of N.punctiforme PCC 73102 with similarities were 94%and 97%,respectively.Gly is the most hydrophobic in site 747,but His is the most hydrophilic in site 168.Thr,Ser and Tyr have 15,27 and 12 phosphorylation sites,respectively.Secondary structure and tertiary predicted structures of GBE were consisted ofα-helix,β-sheet,β-turn and random coil.The expression level of glgB at the level of transcription gradually increased in N.flagelliforme under drought stress.Relative exression level of 1,4-α-glucan branching enzyme,the glycogen synthase activity and the glycogen content all firstly increased and then decreased.The above results provide basis for further research on glgB molecular information and metabolic mechanism of glycogen of N.flagelliforme in re-sponse to drought stress.