3份高羊茅材料吡咯琳-5-羧酸合成酶基因的结构及酶活性比较

Characteristics of pyrroline-5-carboxylate synthetase genes and the enzyme activity in three Festuca arundinacea from Guizhou Province

吡咯琳-5-羧酸合成酶(Pyrroline-5-Carboxylate Synthetase,P5CS)是经谷氨酸途径合成脯氨酸的关键酶,脯氨酸的积累有助于提高植物的抗逆性。本研究以黔草一号高羊茅(Festuca arundinacea cv.Qiancao No.1,QC1)、黔草二号细叶高羊茅(F.arundinacea cv.Qiancao No.2n,QC2n)和黔草二号宽叶高羊茅(F.arundinacea cv.Qiancao No.2b,QC2b)为材料,经RT-PCR克隆得到P5CS基因,基因长2 151bp,含有完整的开放读码框(OpenReading Frame,ORF),编码716个氨基酸,与朝鲜碱茅(Puccinellia chinampoensis)P5CS基因的相似性为94%。对3份高羊茅材料P5CS基因编码的氨基酸序列进行比较,有6个氨基酸差异,这些氨基酸均处于P5CS蛋白的谷氨酸激酶(Glu-5-Kinase,G5K)功能区,其中第37、85及142位的氨基酸所带电荷发生了改变。比较干旱处理后3份高羊茅材料叶片的P5CS酶活性及其脯氨酸含量,QC1叶片的P5CS酶活性及其脯氨酸含量最高,其次为QC2n,QC2b最低。说明P5CS中谷氨酸激酶(G5K)功能区重要氨基酸种类的改变,可能影响高羊茅P5CS的酶活性及其脯氨酸的生成量,也是高羊茅抗逆性差异的主要原因。

Pyrroline-5-carboxylate synthetase（P5CS） is the key enzyme in the proline synthesis from glutamate.As the main osmoregulation substance in plants,proline accumulation is positively correlated with the adaptability of plants to drought stress.Samples were chosen from three tall fescue（Festuca arundinacea） plants,Qiancao No.1（QC1）,Qiancao No.2 with narrow leaf（QC2n） and broad leaf（QC2b）.The upstream and downstream of P5CS genes from three samples were cloned by reverse transcription polymerase chain reaction（RT-PCR） method.The complete P5CS genes of QC1,QC2n and QC2b were spliced with open reading frame of 2 151 bp in length and encoded P5CS protein consisted of 717 amino acid residues.Compare to the known genes deposited in GenBank,the highest homology of P5CS genes from QC1,QC2n and QC2b with Puccinellia chinampoensis is 94%.Furthermore,six different amino acids were found from the Glu-5-kinase domain of the encoded P5CS protein among QC1,QC2n and QC2b samples,in which amino acids at site 37,85 and 142 were changed from hydrophobic residues into positively charged hydrophilic ones.The enzyme activities of P5CS and the proline content of leaves in sample QC1 were the highest and those of QC2b were the lowest.The results suggested that the amino acids changes in the Glu-5-kinase domain of P5CS might be pivotal for the enzyme activity,proline biosynthesis and the drought tolerance of F.arundinacea.