安徽农业大学学报2018，Vol.45Issue(1)：1-5,5.DOI:10.13610/j.CnkI.1672-352x.20180302.003
茶树谷氨酸脱羧酶基因(GAD)的原核表达及酶活快速检测
Prokaryotic expression of the glutamic acid decarboxylase gene (GAD) in Camellia sinensis and rapid determination of its enzyme activity
摘要
Abstract
L-glutamic acid can be decarboxylated to produce gamma aminobutyric acid (GABA).In this process,glutamic acid decarboxylase(GAD) catalyzed the reaction.Screening of GAD from a tea plant transcriptome library was performed and the obtained sequence was compared to NCBI to identify the cDNA sequence (GenBank accession No.KT728367.1).Sequence analysis showed that the ORF (open reading frame) of GAD was 1 482 bp,encoding 493 amino acids.And its encoding GAD was a cytoplasmic protein with the molecule weight of 55.49 kDa and the pI of 5.44.Cloning of the GAD gene in tea plant was carried out and the prokaryotic expression system of pMAL-c5X-GAD was built.After IPTG induction,SDS-PAGE result showed that the molecule weight is about 55 kDa.Determination of the enzyme activity in vitro was done using thin-layer chromatography (TLC).The products of this enzymatic reaction were separated by two different TLC solvents,BAW (Butanol∶ acetic acid∶ H2O,4∶1∶2) and 75％ phenol (Phenol∶ H2O,3∶1),individually.The product spot of GABA was detected by these two TLC solvents.The expression level of GAD in different tea tissues was analyzed using qRT-PCR.The results showed that the highest expression of GAD was in the root and the lowest in the bud.The expression of GAD showed a tissue specificity phenomenon,whose expression varied in different tissues.关键词
茶树/谷氨酸脱羧酶/原核表达/TLC/实时荧光定量PCRKey words
Camellia sinensis/glutamic acid decarboxylase/prokaryotic expression/TLC/qRT-PCR分类
农业科技引用本文复制引用
韩洁云,宛晓春,邓威威..茶树谷氨酸脱羧酶基因(GAD)的原核表达及酶活快速检测[J].安徽农业大学学报,2018,45(1):1-5,5.基金项目
安徽省自然科学基金(1608085QC60)和国家自然科学基金(31300576)共同资助. （1608085QC60）
