发菜GPR基因(proA)克隆及其干旱胁迫下的差异表达

Cloning of GPR Gene(proA) from Nostoc flagelliforme and Its Differential Expression of Pattern under Drought Stress

proA编码的γ-谷氨酰磷酸还原酶(GPR)是脯氨酸合成途径的关键酶。该研究通过设计特异性引物克隆获得全长1 308 bp的发菜proA基因(GenBank登录号为KX553954)。与点形念珠藻的proA基因及其推译的氨基酸序列相似性分别为91%和93%;氨基酸序列中第123位的Leu疏水性最强,第80位的Lys亲水性最强;Thr、Ser和Tyr分别有19、12、4个磷酸化位点;proA编码的蛋白为膜外蛋白,其二级结构由α螺旋、β折叠、无规卷曲和β转角构成。将proA基因在大肠杆菌表达,获得一个47.22 kD的外源蛋白,MALDI-TOF-TOF/MS分析证明该外源蛋白为γ-谷氨酰磷酸还原酶。随着干旱胁迫程度的增加,发菜proA在转录水平的差异表达及脯氨酸含量均呈现增加的趋势。研究结果对深入研究发菜proA的分子功能及阐明其在干旱胁迫下的调控机制具有重要意义。

By the proA coded gamma-glutamyl phosphate reductase(GPR)is an important enzyme in the proline synthesis pathway.In this study,the 1 308 bp proA(GenBank accession number isKX553954)was cloned by designed specific primers.Nucleotide sequence and deduced amino acid sequence of proA from N.flagelliforme were similar to that of Nostoc punctiforme PCC 73102 with similarities above 91%and 93%,respectively.Leu is most hydrophobic in site 123,and Lys is the most hydrophilic in site 80.Thr,Ser and Tyr have 19,12 and 4 phosphorylation sites,respectively.The GPR encoded by proA is an extramembranous protein and secondary predicted structures of GPR were mainly composed ofα-helix,β-sheet,random coil andβ-turn.proA was expressed in E.coil,then a 47.22 kD heterologous protein was observed and tested byMALDI-TOF-TOF/MS.In addition,both the expression of proA at the transcriptional level and the content of proline increased under drought stress.The results are of great importance for the further study of the molecular function of proA in N.flagelliforme and its regulation mechanism under drought stress.