生菜硝酸还原酶基因的克隆及高氮水平下外源γ-氨基丁酸对其表达和叶片硝酸盐含量的影响

Cloning of Nitrate Reductase Gene of Lettuce and Effect of Exogenous γ-Aminobutyric Acid on Gene Expression and Nitrate Content in Leaves under High Nitrogen Level

硝酸还原酶(NR)是硝酸盐代谢的关键酶。该研究在成功克隆生菜NR基因的同时,进行了高氮水平下外源γ-氨基丁酸(GABA)对生菜叶片NR基因表达和NO3--N含量的研究。结果表明:(1)生菜NR基因(GenBank登录号为KP122207)序列长1 791bp,编码585个氨基酸残基,蛋白保守结构域含钼辅蛋白超家族、细胞色素b5超家族和FNR超家族;生菜NR基因与菊苣NR基因亲缘关系最近,序列一致性为93%,与烟草、甜菜、黄瓜、大豆等NR序列的一致性均在80%以上。(2)高氮水平下外源2.5mmol/L GABA处理生菜可诱导NR基因上调表达,并显著提高了生菜叶片NR、亚硝酸还原酶(NiR)、谷氨酸脱羧酶(GAD)活性和NH4+-N含量,降低了NO3--N、NO2--N含量;虽然NO3--N含量与NR、GAD、NiR活性、NO2--N、NH4+-N含量均呈显著相关关系,但与NR活性的相关系数最高且达极显著水平。研究认为,高氮水平下外源GABA可通过诱导NR基因上调表达、增强相关酶活性来影响无机氮代谢,从而降低了生菜叶片硝酸盐含量,其中NR在该过程中发挥着至关重要的作用,为生产中GABA的施用及降低叶菜类蔬菜硝酸盐含量提供了理论依据。

Nitrate reductase （NR） is the key enzyme of nitrate metabolism. This paper successfully cloned the gene of NR in lettuce and investigated the effect of exogenous γ-aminobutyric acid （GABA） on the ex- pression of NR gene and NO3^--N content under high nitrogen level in leaves of lettuce cultured with hydroponics culture at the same time. The result showed that： （1）the NR gene （the accession NO. of Genbank is KP122207） sequence of lettuce was 1 791 bp that encoding 585 amino acid residues, and contained the Moco superfamily,Cyt-b5 superfamily and FNR superfamily protein domains. The NR gene sequence of let- tuce had close genetic relationship with Cichorium intybus （93 % consistency） and had relative close genetic relationship with tobacco, sugar beets, cucumber and soybeans （more than 80% consistency）. （2） The exog- enous 2.5 mmol/L GABA induced a marked increase in NR gene expression. Meanwhile, significantly increased the activities of NR, nitrite reductase （NiR）, glutamate decarboxylase （GAD） and the content of NH4^＋-N in lettuce leaves under high nitrogen level of nutrient solution, but reduced the contents of NO3^-- N and NO2^--N. The content of NO3 -N had significant correlation with the activities of NR, NiR, GAD and the contents of NO2^--N, NH4^＋ -N, but the correlation coefficient was the highest between NO3^--N and NR which reaching extremely significant negative correlation. The study suggested that,exogenous GABA could affect inorganic nitrogen metabolism and reduce the nitrate content in lettuce leaves by inducing NR gene up-regulated expression and enhancing enzyme activities under high nitrogen level. Especially the NR played a key role in the process,which provided a theoretical basis for the application of GABA and reduc- ing the nitrate content in leafv vegetables.