Carbon And Nitrogen Metabolism In Response To Nitrogen Form And Cloning Of NRT2.2

Nitrogen is one of the three essential element for plant growth, is wideparticipation in biosynthesis, energy transfer, signal transduction and other metabolicprocesses in plants. Nitrogen plays an important role in promoting the growth andmetabolism of tobacco. In order to explore the theory that the effect of differentnitrogen stress on nitrogen absorption mechanism and leaf carbon and nitrogenmetabolism of tobacco. In this experiment,“Zhongyan100” as test materials,mainly to study the different aspects of leaf structure, gene expression, enzymeactivity, the chemical composition, etc of tobacco seedlings under different nitrogentreatments from the laboratory to the field in sand processing plants growing tobacco.smoke on the carbon and nitrogen metabolism strains were compared. The resultsshowed that:1. In order to demonstrate the mechanism of nitrogen stress and differentnitrogen forms on glucose metabolism of tobacco seedlings, Zhongyan100was usedas the material. Sand culture were adopted in the normal supply of nitrogen (CK, NO3-:NH4+=1:1), nitrogen deficiency (T1), single ammonium (T2) and single nitrate (T3)of the four treatments. Detecting the contents of reducing sugar, total sugars, sucrose,starch and the activity of glucose metabolism-related enzymes of functional leaves oftobacco plants. At the same time, we analysis the differences of key glucosemetabolism genes (SUT1, INV, GBSSI, AGPase) in roots, stems, leaves. The resultsshowed that:(1) Content of sugar, total sugar, sucrose and starch in tobacco leaves ofT1were increased significantly (P<0.05) compared with CK during early plantdevelopment; The amylase activity was significantly reduced, however the invertaseactivity was different (P>0.05). The accumulation of sugar in four treatments areT1>T2>T3>CK（.2）The glucose metabolism gene expression showed that SUT1, INV,AGPase, GBSSI were expressed significantly in T1, T2and T3，indicating thatbiosynthesis of grain starch in T1, T2and T3were more active. The SUT1may bevery important in starch biosynthesis under different nitrogen forms.2. The above treatment options were used to test the content of nitrogen andnicotine and the activity of nitrogen metabolism enzyme in tobacco leaf, and toanalyze the differences in three key glucose metabolism genes (Nii2, GDH, NRT2.1,NRT2.2) expression in roots, stems and leaves. The results showed that:(1) Overall,the content of total nitrogen in nitrate treatment (T3) was the highest among the four treatments, ammonium nitrogen (T2) and normal nitrogen (CK) followed by. Andnitrogen deficiency treatment (T1) is the minimum; Compared to normal treatment,ammonium nitrogen treatment (T2) and nitrate treatment (T3) respectively increasedby125.24%and143.87%, which difference was significant (P>0.05). This showedthat single nitrogen forms influence the accumulation of nitrogen metabolites oftobacco seedlings. And the difference in content of nicotine belonged to the latenitrogen metabolites in the four treatments was not obvious (P>0.05).(2) Analysis ofthe expression of key genes for nitrogen metabolism showed that nitrogen deficiencytreatment (T1) could significantly influence the expression of nitrite reductase gene(Nii2), but the difference was not obvious between ammonium treatment (T2) andnitrate treatment (T3). Speculated that nitrogen deficiency may be the key factor ininducing expression of gene Nii2.(3)The detection of two genes of nitrate transporterprotein (NRT2) family showed that stress regardless of what kind of nitrogen, theexpression of NRT2.1and NRT2.2were particularly strong in the roots of tobaccoplants, which were significantly higher than the control treatment (CK). Speculatingthat NRT2.1and NRT2.2play an important role in nitrogen absorption in tobaccoroots.3. Nitrate ion absorption is the first step in the process of plant nitrate nitrogenutilization. High affinity Nitrate transporters (Nitrate from2, NRT2) mainly inexogenous Nitrate nitrogen concentration is lower approximately7-50microns (Km),is one of the key biological macromolecules of plant nitrogen nutrition system.Therefore, there are broad prospect of research and application. This research bymeans of biotechnology, from nitrogen deficiency of tobacco “Zhongyan100” varietyroot cDNA cloning of the gene sequence, through the analysis of the sequence, thegene and reported diploid tobacco NRT2.2nucleotide sequence similarity was96.04%,98.68%amino acid sequence similarity, we suspect that it is a member of the familygenes. Further through the connection, transformation to build the excessiveexpression vector of gene expression vector for the next step in transgenic tobacco.