The Study Of γ-aminobutyric Acid On Nitrate Metabolism Key Enzymes Regulation Mechanism Of Rape And Lettuce Under High Nitrogen Level

Vegetables are easily accumulating nitrate, especially leafy vegetables. But in vegetable production, the phenomenon of excessive application of fertilizer is becoming more and more serious, especially single nitrogen fertilizer, which results in a excessive level of nitrate content in vegetables and affects the quality of vegetables and people’s health seriously. There are methods for reducing nitrate content in vegetables, for example, rational application of fertilizer, improving the environment for vegetables, selecting appropriate harvest period, adopting of appropriate storage processing and edible treatment. But the methods are easily affected by environmental factors, and the effects are not stable. γ-aminobutyric acid(GABA) is a four-carbon non-protein amino acid, which can regulate the uptake and utilization of nitrate, be a long-distance signal of nitrate uptake, involve in the nitrate metabolism. In this paper, exogenous GABA was used to study the internal mechanism of reducing nitrate content of vegetables.In this study, ’Zhongshu Wu Yue Man’ of rape(Brassica campestris L.) variety and ’Da Su Sheng’ of lettuce(Lactuca sativa L.) variety was used as materials. On the basis of cloning and analyzing key enzyme genes in nitrate metabolism: nitrate reductase(NR) gene and glutamate decarboxylase(GAD) gene, exogenously GABA was adopted in adding and soaking methods to study the effect of GABA on the gene expression, enzyme protein contents and enzyme activities of NR, GAD under high nitrogen level. The results are as follows:1. NR gene(the accession NO. of Genbank is KP852556) of rape was successfully cloned, and nucleotide sequence length was 2863 bp, containing a 2733 bp open reading frame, encoding 910 amino acid residues, conserved protein domains including: Moco superfamily(N-terminal), cytochrome b5 superfamily(center), FNR superfamily(Cterminal). NR gene amino acid sequence of rape had close genetic relationship with Brassica napus(98% consistency) and had relative close genetic relationship with Arabidopsis, soybeans, spinach and other 14 species(more than 70% consistency).2. GAD gene(the accession NO. of Genbank is KP852557) of rape was successfully cloned, and the length of nucleotide sequence was 1566 bp, containing a 1485 bp open reading frame, encoding 494 amino acid residues, conserved protein domain including a pyridoxal phosphate-dependent aspartate aminotransferase superfamily. GAD gene amino acid sequence of rape had close genetic relationship with Brassica napus, Brassica juncea and Arabidopsis(99%, 98%, 92% consistency) and had relative close genetic relationship with tomato, cucumber, soybean and other 8 species(more than 80% consistency).3. Exogenously adding GABA improved the gene expression and enzyme protein contents of NR, GAD in rape leaves under high nitrogen level, and enhanced the activities of NR, GAD at the same time. Meanwhile 2.5 mmol/L GABA treatment had the best effect. GABA soaking also enhanced the gene expression and enzyme protein contents of NR, GAD in rape leaves under high nitrogen level, and the effect of 5 mmol/L GABA treatment was the best.4. NR gene fragment of lettuce was successfully cloned, and the length was 1791 bp, encoding 585 amino acid residues, including 3 NR family conserved domains: Moco superfamily, cytochrome b5 superfamily and FNR superfamily. The NR gene amino acid sequence of lettuce 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).5. Exogenously adding GABA improved the NR gene expression and activity of lettuce leaves under high nitrogen level, and the effect of 2.5 mmol/L GABA treatment was the best.