| Nitrogen is the core member of fruit essential mineral elements and root is the main work center in the agricultural production.Malus hupehensis Rehd. var pinyiensis Jiang is one promising rootstock of apple trees in China.In order to determine the relationship between nitrogen supply and root growth,we studied the effect of NO3--N,NH4+-N,L-glutamate treatments on the growth of root.In order to investigate the mechanism of L-glutamate,we isolated MhGLR3.6 full-length cDNA from new root of Malus hupehensis Rehd. and studied its expression by quantitative real-time PCR.To study the function of MhGLR3.6,we introduced the antisense MhGLR3.6 under the control of 35S promoter of Cauliflower mosaic virus into Malus domestica Borth.cv. Royal Gala plants.The main results were as follows:1. Shoot and root biomass both reached the highest level supplied with 1 mmol/L NO3-,which were 216.7%and 138.5% times of the control's respectively.Length and number of lateral root also reached the highest level supplied with 1 mmol/L NO3-,which were 168.9%and 100.9% higher than that of the control.But above four indexes decreased when NO3- concentration was higher than 1 mmol/L.Above four indexes under NH4+ treatment condition showed the same trend as NO3- treatment,but was lower than that of NO3- treatment when supplied with the same nitrogen concentration.Nitrogen treatments remarkably increased growth of primary root,but differences among different concentration treatments were not marked.Local supplied NO3- and NH4+ could increase lateral root density under fertilizer being bag-controlled release condition.2. The primary root's length of Malus hupehensis Rehd. seedlings increased remarkably with L-glutamate's concentration increasing from 0 to 50μmol/L.The length of primary root decreased when L-glutamate's concentration was higher than 50μmol/L and reached the lowest level supplied with 1 000μmol/L,which was 35.6% times of 50μmol/L treatment.Lower concentration of L-glutamate treatment could stimulate lateral root growth,the lateral root's length of 50μmol/L treatment was 142% times of the control's.The growth of lateral root inhabited with L-glutamate's concentration increasing.From 0μmol/ L to 100μmol/ L L-glutamate treatment could increase the number of lateral root and reached the highest level supplied with 50μmol/L,which was 184% times of the control's.The number of lateral root remarkably decreased with L-glutamate's concentration increasing.3. According to the homologous sequences from other plants,degenerate primers were designed to amplify specific DNA fragment.By RT-PCR and SMART RACE-PCR,we isolated a GLR gene named MhGLR3.6,with the accession number EF432572.Sequencing analysis showed that the full-length of MhGLR3.6 consisted of 3 600 bp with an open reading frame of 2 838 bp that encoded a protein of 946 amino acids.The predicted molecular mass is 107.809 kDa.Sequence alignment of MhGLR with other members of the GLR family showed that MhGLR was closely related to cladeⅢArabidopsis GLRs and was closest to AtGLR3.6.Hydropathy analysis indicated that MhGLR3.6 contains an N-terminal signal peptide (Sp) and has a three-plus-one transmembrane structure (M1-M4).Two large extracellular domains GlnH1 and GlnH2 functioning as ligand-binding domains are predicted between the signal peptide and M1,and between M3 and M4,respectively.4. Quantitative real-time PCR analysis demonstrated that MhGLR3.6 was expressed in roots,stems and leaves.The expression level in leaves was higher than that in roots and stems.L-glutamate and IBA treatments were able to induce the expression of MhGLR3.6 in roots.5. Successfully constructed the expression vector of pBI121-MhGLR3.6 and transformed MhGLR3.6 gene into Malus domestica Borth.cv. Royal Gala plants by Agrobacterium-mediated transformation.
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