Cloning And Expression Characteristion Of Nitrate Transporter Genes NRTs From Flowering Chinese Cabbage

In recent years, with the increasing amount of nitrogen fertilizer in vegetable cultivation, the problem of the reduction of nitrogen utilization efficiency of vegetables is gradually outstanding. Nitrate nitrogen is the main source of nitrogen plants absorb, root system to absorb NO₃^- is mainly done by nitrate transporters NRT, its main representative is low affinity nitrate transporters NRT1 and high affinity nitrate transporters NRT2, they are different to the affinity of nitrate nitrogen is influenced by the external environment is also different.Flowering Chinese Cabbage?Brassica campestris L. ssp. chinesisvar.utilis Tsen et Lee? is a specialty vegetables of Southern China area, is a high nitrate-accumulating leaf vegetables. The nitrogen absorption ability and related transporter gene expression research for the promotion of flowering Chinese cabbage nitrogen utilization is of great significance. But at present about the molecular mechanism and absorption of Flowering Chinese Cabbage in different conditions of ammonium and nitrate nitrogen is less.The Flowering Chinese cabbage was as materials, under hydroponic conditions in order study nitrogen absorption ability and related gene expression. Divided into the following aspects:?1?Cloning and function analysis of Bc NRT1;1, Bc NRT1;2and Bc NRT2;1from flowering Chinese cabbage;?2?The absorption ability and NRTs expression of Flowering Chinese cabbage at different growth stages and root;?3?The absorption ability and NRTs expression of Flowering Chinese cabbage at different form and concentration of nitrogen?4?The absorption ability and NRTs expression of Flowering Chinese cabbage at the mixture of nitrate and ammonium condition?5?The nitrogen absorption ability of Flowering Chinese cabbage at decreases of nitrateand increase of ammonium.The results are as follows:1.Nitrate transporter Bc NRT1; 1 and Bc NRT1; 2 and Bc NRT2; 1 has isolated from flowering Chinese cabbage.Bc NRT1; 1 and Bc NRT1; 2 and Bc NRT2; 1 have an open reading framerespectively for 1770 bp, 1638 bp and 1530 bp. The putative protein structure of the Bc NRT1; 1, Bc NRT1; 2 and Bc NRT2; 1 has showed that : the Bc NRT1; 1 encoding 589 aa, relative molecular mass of 64.97 KD, theoretical isoelectric point was 8.44, protein average hydrophobicity 0.353; and Bc NRT1; 2 encoding 545 aaand relative molecular mass was 61.55 KD, theoretical isoelectric point is 8.80. Protein average hydrophobicity 0.321. Bc NRT2; 1 encoding 510 aa, the relative molecular mass of 55.55 k D, theoretical isoelectric point was 9.11, and the average hydrophobicity of protein was 0.373. And the other physical and chemical properties of Bc NRT1; 1, Bc NRT1; 2 and Bc NRT2; 1 protein showed Bc NRT1; 1 and Bc NRT1; 2 belong to the?LATS?low affinity nitrate transport system, Bc NRT2; 1 belong to the?HATS?high affinity nitrate transport system. These three genes encoding both flowering Chinese cabbage nitrate transporter.2. Nitrate uptake capacity of flowering Chinese cabbage in different growth stages was significant differences, the strongest nitrate absorption was happened at early bolting stages. In the full nutrient growth stage Bc NRT1; 1was mainly expressed in roots, Bc NRT1; 1 expression in leaves increased gradually during the flowering period from bolting stage; At the vegetative growth stage Bc NRT1; 2 was mainlyexpressed in root; of contrast, from the beginning of the early bolting, leaves Bc NRT1;2 expression was more than root. flowering Chinese cabbage Bc NRT2;1 gene expression has a trend of increased first and then decreased, and leaves Bc NRT2;1 gene expression is decreased full time, the contrast between the two organs, Bc NRT2;1 expression in root of various growth stages always much higher than in leaves. Bc NRT2;1 expression was the best correlation with the ability of absorbing nitrate in different growth stages of Flowering Chinese cabbage.3. The absorption of flowering Chinese cabbage for nitrate nitrogen and ammonium nitrogen with different characteristics. The nitrate absorption with nutrition increases with the increase of nitrate nitrogen concentration, resulting in the total nitrogen content of flowering Chinese cabbage also with the increase of concentration of nitrate nitrogen increases. Flowering Chinese cabbage of ammonium nitrogen absorption generally also with the increase of the concentration of ammonium nitrogen. And Flowering Chinese cabbage in 1⁸m M ammonium nitrogen concentration under the condition of total nitrogen content had no significant difference. The expression of Bc NRT1;1 and Bc NRT1;2increased with the increase of nitrate concentration. The expression level of Bc NRT2;1 decreased with the increase of nitrogen concentration. The expression level of Bc NRT2;1 decreased with the increase of nitrogen concentration. The low concentration of ammonium nitrogen could induce Bc NRT1;1 gene expression, but with the increase of the concentration of ammonium nitrogen, the degree of this expression increased. Different concentrations of ammonium nitrogen on the expression of Bc NRT1;2 gene in Chinese cabbage roots is similar to Bc NRT1;1. Effect of ammonium nitrogen on the expression of Bc NRT2;1 gene of Brassica chinensis root is the main inhibitory effect.4.In this experiment among ammonium nitrate ratio of 0:100?CK?,25:75?T1?and 50:50?T2?, ammonium nitrate ratio of 25:75?T1? treatment uptake for total nitrogen was significantly higher than the total nitrogen treatment of?CK? and?T2?. In this experiment the expression of the Bc NRT1;1 in Flowering Chinese cabbage root and total nitrogen absorption was positively correlated with a correlation coefficient of 0.919 on absorb 24h; the expression of the Bc NRT2;1 and nitrate uptake absorption was positively correlated with a correlation coefficient of 0.863, while the expression Bc NRT1;2 there is no clear correlation between the amount of change and nitrogen absorption.5. ammonium and nitrate 25:75 and 25:100 two treatment in ensuring the invariant ammonium content reduce nitrate content. The results show that no big changes in the two treatment in flowering Chinese cabbage on the nitrate uptake. 0:100 and 25:100 two experiments confirmed that the addition of ammonium significantly inhibited the absorption of nitrate in the case of nitrate nitrogen content unchanged.To sum up, the increasing amount of ammonium promote the absorption increasing of flowering Chinese cabbage plants total nitrogen, mainly is under the condition of appropriate proportion of the mixture of nitrate and ammonium?25:75? of flowering Chinese cabbage of ammonium absorption to compensate for the reduction of nitrate uptake, resulting in total N uptake was significantly increased. Different treatment Bc NRT1;1 expression and total nitrogen absorption have maximum correlation.