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

L-谷氨酸经脱羧酶反应可生成γ-氨基丁酸(GABA),此过程受谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)催化。从茶树转录组库中筛选GAD基因,通过NCBI比对获得其cDNA序列(GenBank登录号为KT728367.1)。序列分析表明,GAD基因的ORF全长为1 482 bp,共编码493个氨基酸,该蛋白为细胞质蛋白,分子量为55.49 kDa,理论等电点为5.44。从舒茶早茶树根部中克隆该基因,并构建pMAL-c5X-GAD表达载体,经IPTG诱导,SDS-PAGE检测,得到分子量约为55 kDa的目的蛋白。体外酶活反应后用薄层色谱法(TLC)分析,经BAW(Butanol:Acetic acid:H_2O,4:1:2)和75%苯酚水溶液(Phenol:H_2O,3:1)2种展开剂分离酶促产物,均可检测到GABA的生成。通过实时荧光定量PCR对GAD基因在不同茶树组织的表达水平进行检测,发现GAD在根中表达量最高,而在芽中表达量最低,在其他组织中表达各不相同,其表达具有组织特异性。

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 GADwas 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 p I 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： H_2O, 4：1：2） and 75% phenol（Phenol： H_2O, 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.