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谷氨酸脱羧酶(glutamate decarboxylase,GAD)是生物体内普遍存在的一类胞内酶,主要分布于细胞质中,是催化谷氨酸脱羧反应的关键限速酶,产物γ-氨基丁酸(γ-aminobutyric acid,GABA)具有调节植物生长发育、抵抗非生物胁迫等功能。本研究以小白菜‘五月慢’(Brassica campestris ssp.chinensis)为材料,采用同源序列法克隆了小白菜谷氨酸脱羧酶基因(glutamic acid decarboxylase gene,GAD)(Gen Bank登录号为KP852557);采用浸种和外源添加处理,检测了高氮水平下外源GABA对小白菜生长、GAD基因和酶蛋白表达及酶活性的影响。结果表明,小白菜GAD含有一个长为1 485 bp的开放阅读框,编码494个氨基酸,蛋白保守结构域包含一个磷酸吡哆醛依赖的谷草转氨酶超家族;小白菜GAD基因编码的氨基酸序列与大白菜(B.rapa)GAD2基因氨基酸序列的一致性达到100%,与甘蓝型油菜(B.napus)、萝卜(Raphanus sativus)和芥菜(B.juncea)的GAD2基因氨基酸序列亲缘关系较近,序列一致性分别为99%、99%和98%。外源GABA添加和浸种均提高了高氮水平下小白菜的生长以及叶片GAD基因转录水平、GAD蛋白表达量和GAD酶的活性,但GABA处理间存在着明显的浓度差异,其中3.75~6.25 mmol/L GABA浸种与2.5~3.75 mmol/L添加处理效果较明显(P<0.05)。实验结果初步证实了小白菜的生长、GAD基因、蛋白和活性受GABA施用浓度和施用方式的影响,且与根际环境氮素水平密切相关。该研究为进一步利用Bc GAD基因和GABA调控方法提高叶菜类蔬菜品质提供理论依据。
Glutamate decarboxylase (GAD) is a kind of ubiquitous intracellular enzyme that is mainly distributed in the cytoplasm. It is the key rate-limiting enzyme in the catalytic glutamate decarboxylation reaction, and the product γ-aminobutyric acid (γ -aminobutyric acid, GABA) has the function of regulating plant growth and development and resisting abiotic stress. In this study, Brassica campestris ssp.chinensis was used as a material to clone the glutamic acid decarboxylase gene (GAD) from Brassica campestris (GenBank accession number: KP852557 ). The effects of exogenous GABA on the growth, GAD gene and enzyme protein expression and enzyme activity in Brassica chinensis under high nitrogen level were tested by soaking and exogenous addition. The results showed that the GAD of Chinese cabbage contains an open reading frame of 1 485 bp, encoding 494 amino acids. The conserved protein domain contains a pyridoxal phosphate-dependent transglutaminase superfamily. The amino acid sequence of GAD gene in Chinese cabbage is The amino acid sequence of GAD2 gene in B. rapa was 100% consistent with the amino acid sequence of GAD2 gene in B. napus, Raphanus sativus and B.juncea. The sequence Consistency was 99%, 99% and 98% respectively. Exogenous GABA addition and soaking increased the growth of cabbage and the expression of GAD gene, the expression of GAD protein and the activity of GAD enzyme under high nitrogen level, but there was a significant concentration difference between GABA treatments, of which 3.75 ~ 6.25 mmol / L GABA soaked with 2.5 ~ 3.75 mmol / L added more obvious (P <0.05). The results of the experiment initially confirmed the growth of Chinese cabbage. The GAD gene, protein and activity were affected by the concentration of GABA and the mode of application, and were closely related to the rhizosphere nitrogen level. This study provides a theoretical basis for further utilizing the Bc GAD gene and GABA regulation methods to improve the quality of leafy vegetables.