Cloning And Functional Analyzation Of Nitrate Transporter Gene In Fast-growing Plants

Nitrogen is not only nutrition elementnecessary for plant growth,but also the one used mostin fertilizers for agricultural production. At the same time, the amount of N in the soil became limiting factor to the increase in crop production if there were sufficient supply of other elements for the plant growth. Nitrate is the main source of nitrogen in the soil for most plants. Yet it can be easily dissolved in the soil solution, which causes great loss of nitrogen and creates environment pollution. And since nitrate transporter plays a key role in the process of absorbing nitrate in plants, researches on nitrate transporter can help improve nitrogen use efficiency, enhance agricultural production and solve the environmental problems arising from nitrogen loss.The growth rate of 20 aquatic plant species such as Glossostigma elatinoides, Anubias nana, Hydrilla verticillata, Ceratophyllum demersum, were found that Glossostigma elatinoides, Anubias nana, Hydrilla verticillata, Ceratophyllum demersum grow fastest, After 4 weeks the wet weight of 0.5g robust stems were up 362%, 372%, 376% and 402%.In order to study the nitrate transporter, degenerate primers were designed according to the amino acid conserved sequence of some known nitrate transporter. And nitrate transport genes GeNRT1.1, GeNRT2.1 were cloned from Glossostigma elatinoides. Moreover, the function of nitrate transporters GeNRT1.1 and GeNRT2.1 and Cylindrotheca fusiformis nitrate transporter CfNRT were verified through the defected Hansenula polymorpha heterologous gene expression system. The main research progresses were as follows.1. As described in the experimental procedures, a full length GeNRT1.1 clone has been constructed, which contains 1797 bp. Sequencing of the GeNRT1.1 cDNA clone revealed it consists a 1440 bp complete open reading frame for a 584 amino acid polypeptide. There are respectively 74.1% and 62.4% similarity compared with sequences of low-affinity nitrate transporter gene in Arabidopsis and tobacco. pYNR-EX- GeNRT1.1 has been transformed into high-affinity nitrate transporter gene mutant-deficient strain(△ynt)of H. Polymorpha. And growth recovery of Δynt yeast strains can be observed as a result of the GeNRT1.1. It suggestes that GeNRT1.1 can transfer nitrate. Real-time PCR results shows that 3.5 times more GeNRT1.1 has been expressed in stems and leaves than in root. The expressions level of GeNRT1.1, induced by high concentrations of nitrate(2mM) in roots, stems and leaves, are all higher than that induced by a low concentration of 0.5mM, which further indicates that GeNRT1.1 is a low affinity nitrate transporter.2. In this experiment we cloned a new nitrate transporter protein encoding gene from Glossostigma elatinoides. This GeNRT2.1 gene has a total length of 2078 bp, containing the full open reading frame of 1596 bp for a 530 amino acids polypeptide. With biology software we found that GeNRT2.1 is transmembrane protein and belongs to the family of MFS. Determination of GeNRT2.1 kinetic parameters revealed a Km for nitrate of about 140 μM, which was consistent with its expectedhigh-affinity of nitrate. Fungal growth and nitrate uptake were recovered after transfermation of pYNR-EX-GeNRT2.1 into high-affinity nitrate transporter gene mutant-deficient strain(△ynt)of H. Polymorpha, which suggested that GeNRT2.1 is acting in yeast as a high-affinity nitrate transporter. The real-time PCR experimental results revealed that the expression of GeNRT2.1 was upregulated in 0.5μM nitrate, indicating that GeNRT2.1 is high-affinity nitrate transporter.3. Sincenitrate transporters transport nitrate, their location shall have a relationship with their function. The observation via laser scanning confocal microscope showed that pRTL-2-GeNRT1.1 and pRTL-2-GeNRT2.1 are both located on the cell membrane of protoplasts. It suggested that GeNRT1.1 and GeNRT2.1 are membrane protein.4. Separately transformation of pYNR-EX-CfNRT 、 pYNR-EX-GeNRT1.1and pYNR-EXGeNRT2.1 into high-affinity nitrate transporter gene mutant-deficient strain(△ynt)of H. Polymorpha endowed transgene yeasts(△ynt-CfNRT、△ynt-GeNRT1.1 and △ynt-GeNRT2.1)with different uptake sufficiencies of nitrate in the same time, among which the uptake sufficiency of nitrate in △ynt-CfNRT rose most rapidly to the maximum in the same condition, compared with the other two strains.5. The influence of N element on growth of tobacco. To test the function of CfNRT gene in transgenic tobacco, we planted T2 generation of transgenic tobacco and non-transgenic tobacco in MS0 mediums in which nitrate concentration was gradually reduced. 27 d after sowing, there was no significant difference between the gm and non-gm tobaccos in MS0 mediums. Whereas the leaves of transgenic tobaccos were bigger and the color of green leaves was deeper than non-transgenic tobacco in the medium lack of N element. 61 d after sowing,determination of fresh weight and dry weight of transgenic tobacco and non-transgenic tobacco showed that fresh weight and dry weight per plant of two tobaccos maintained at the same level in the excessive N-1/6 N MS0 medium(fresh weight 138.0-201.3 mg;dry weight 16.6-29.9 mg).While tobacco growth was significantly inhibited in 1/16N-1/32 N MS0medium(fresh weight 30.8-65.5 mg;dry weight 5.3-10.0 mg).The fresh weight and dry weight transgenic tobacco were higher than that weight of transgenic tobacco.If we use 1/8 N MS0,transgenic tobacco can save 50% nitrogen.