小白菜GAD基因克隆及高氮条件下外源GABA的诱导表达分析

摘要

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) (GenBank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(Raphanussativus)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BcGAD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) mainly distributes in the cytoplasm and catalyzes the reaction of glutamate decarboxylation as a kind of intracellular enzyme found in many organisms.The metabolite of γ-aminobutyric acid (GABA) has the function of regulating plant growth,and development and improving the resistance to abiotic stress.This study cloned the gene of GAD in pakchoi (Brassica campestris ssp.chinensis)cultivar,cv.Wuyueman by the homologous cloning technology,namely BcGAD (GenBank accession No.KP852557).In addition,the effect of exogenous GABA on growth,expression of BcGAD gene and protein as well as enzyme activity were detected in pakchoi plants treated with application or soaking seeds of GABA under rhizosphere high nitrogen level.The results showed that the BcGAD gene contained a 1 485 bp ORF encoding 494 amino acids,which included a conserved domain of pyridoxal phosphate dependent aspartate aminotransferase superfamily.The homology analysis of amino acid sequence showed that the the similarity of amino acid sequences of BcGAD gene and GAD2 gene in B.rapa reached 100％,which demonstrated that there existed the closest homology between them.Moreover,the amino acid sequence of BcGAD was similar to GAD2 of B.napus,Raphanus sativus and B.juncea with the similarities of 99％,99％ and 98％,respectively.The treatments of soaking seeds at 3.75～7.5 mmol/L and application in nutrient solution at 1.25～5 mmol/L significantly promoted the growth supraterraneous compared with higher nitrogen treatment(P＜0.05).qRT-PCR and enzyme linked immunosorbent assay (ELISA) analysis showed that exogenous GABA increased the BcGAD gene transcriptional level and protein expression in pakchoi leaves treated with applying in nutrient solution and soaking seeds at different GABA concentration under the higher level of rhizosphere NO3.BcGAD gene expression in leaves was significantly increased treated with soaking seeds (2.5～10 mmol/L) and applying GABA in nutrient solution (1.25～5 mmol/L) than that treated with high nitrogen condition.BcGADprotein expression in leaves enhanced accordingly treated with GABA of application in nutrient solution (3.75～10 mmol/L) and soaking seeds (1.25～5 mmol/L).Meanwhile,the GAD activities were significantly increasing by applying 5～6.25 mmol/L GABA in nutrient solution or soaking seeds with 2.5～3.75 mmol/L GABA correspondingly.In general,the treatments of soaking seeds at 3.75～6.25 mmol/L and applying solution at 2.5～3.75 mmol/L showed obvious effects among the different GABA concentration treatments(P＜0.05).Above all,the improved effects were dominant of soaking seeds at 5 mmol/L GABA and application GABA at 2.5 mmol/L.The results primarily confirmed that the growth,BcGAD,BcGAD protein and GAD activity were all affected by exogenous GABA concentration,application method and higher nitrogenlevel of pakchoi rhizosphere,which provides a theoretical basis for further utilization of BcGAD gene and exogenous GABA to improving the quality of leafy vegetables.