Cloning And Functional Characterization Of Nitrate Transporter Genes From Non-Heading Chinese Cabbage

Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) originated in China, widely cultivated in the basin of Yangtze River, as one of the most important leafy vegetables. With the completion of the re-sequencing work, it will be the model plant for molecular breeding of the Brassica vegetables. Nitrogen is the most important nutrient for plant growth and development, having a profound impact on agricultural production. Nitrate is the main form of nitrogen for most of the land plants, as an important nutrient and signal substance for plant growth and development. Non-heading Chinese cabbage, one of the most important horticultural crops in China, is a high nitrate-accumulating leafy vegetable, but few reports were on the nitrate uptake mechanism. In Arabidopsis, nitrate transporter genes NRT1.1, NRT1.2, NRT2.1and NRT2.2were involved in nitrate uptake in roots. It is very important to study mechanism of nitrate uptake for improving the nitrogen use efficiency (NUE) and controling nitrate accumulation in non-heading Chinese cabbage through genetic means.In this study, we cloned the nitrate transporter genes BcNRT1,BcNRT1.1, BcNRT2.1and the promoter sequence of BcNRT1, BcNRT1.1, and analysed their mRNA expression patterns with treatment of different concentration of nitrate. Tissue specific expression of GUS (β-glucuronidase gene) driven by promoter of BcNRT1and BcNRT1.1were performed. The functional characterization of nitrate transporter genes was conducted on oocytes and transgenic plants chl1-5. The main findings are as follows.1. Molecular cloning and expression of BcNRT1from non-heading Chinese cabbageA nitrate transporter gene BcNRTl was isolated from non-heading Chinese cabbage cultivar’Suzhouqing’. The sequence length is2067bp. Its deduced amino sequence was highly homology to Arabidopsis thaliana, Brassica napus and Nicotiana benthamiana. The protein structure domain analysis indicated that BcNRT1possessed12putative transmembrane domains and a hydrophilic loop between TM6and TM7. This sequence has been submitted to the GenBank database and the accession number is JF439305. The BcNRT1expression was amplified by PCR technique and the recombinant plasmid pYNR-EX-BcNRTl was constructed. The recombinant protein was expressed in Hansenula polymorpha (NCYC495) induced by nitrate. SDS-PAGE was used to analyze expression of the target protein. The result showed that there was a specific band at about65kDa, identified with the expected molecular weight of the BcNRTl.2. Functional characteriation of BcNRT1from non-heading Chinese cabbageUsing the GUS marker gene driven by the BcNRT1promoter, we found BcNRT1expression to be concentrated in primary and lateral root tips and in shoots of transgenic Arabidopsis plants. The yellow fluorescent protein (YFP) fused to BcNRT1and transformed into cabbage protoplasts indicated that BcNRT1was localized to the plasma membrane. The expression of BcNRTl in roots was induced by exposure to25mM nitrate, and the BcNRT1cRNA heterologously expressed in Xenopus laevis oocytes showed nitrate conductance when nitrate was included in the medium. Moreover, mutant chl1-5plants harboring35S:.BcNRT1showed sensitivity to chlorate treatment and exhibited restored nitrate uptake.3. Molecular cloning and functional analysis of BcNRT1.1from non-heading Chinese cabbageThe BcNRTl.1gene was isolated from non-heading Chinese cabbage. The BcNRT1.1cDNA contained an open reading frame of1773bp encoding a predicted protein with12putative transmembrane domains and a hydrophilic loop between TM6and TM7. This sequence has been submitted to the GenBank database and the accession number is JQ797419. Using the GUS marker gene driven by the BcNRT1.1promoter, we found BcNRT1expression to be concentrated in primary and lateral root tips and in shoots of transgenic Arabidopsis plants, like BcNRT1gene. To confirm the interaction between BcNRT1.1and BcCIPK3in plant cells, a bimolecular fluorescence complementation (BiFC) assay based on split yellow fluorescence protein was performed. The combination of the N-terminal end of YFP fused to BcNRT1.1or BcCIPK23were transiently coexpressed in non-heading Chinese cabbage protoplasts. A fluorescent signal was seen in the plasma membrane, whereas no significant signals were observed in controls. The result of the protein interaction by the BiFC assay in plant cells show that BcCIPK23interacts with BcNRT1.1in the plasma membrane. To determine whether BcNRT1.1was involved in primary nitrate response, the transgenic plants chll-5over-expressing BcNRT1.1was obtained, and the changes in the expression of the primary nitrate response gene NRT2.1were examined in chll-5plants and transgenic chll-5plants over-expressing BcNRT1.1. During exposure to nitrate for30min, increased expression of NRT2.1was seen in transgenic chl1-5plants over-expressing BcNRT1.1. This indicated that BcNRT1.1was involved in primary nitrate response by regulating the expression of NRT2.1.4. Molecular cloning, expression analysis and subcellular localization of nitrate transport gene BcNRT2.1from non-heading Chinese cabbageA nitrate transporter gene BcNRT2.1was isolated from from non-heading Chinese cabbage. The cDNA got with RACE technique contained an open reading frame of1593bp encoding a predicted protein of530acid residus with12putative transmembrane domains and a hydrophilic loop between TM6and TM7. This sequence has been submitted to the GenBank database and the accession number is JQ797418. The deduced amino sequence was highly homology to Arabidopsis thaliana and Brassica napus. The expression of BcNRT2.1was performed in roots and shoots by relative quantitative RT-PCR and the results showed the expression levels of BcNRT2.1were mainly expressed in roots and the levels of BcNRT2.1in roots were induced when exposed to200μM and25mM nitrate concentration. Compared to low concentration of nitrate, high concentration of nitrate is stronger in the induction of NRT2.1expression and more obvious in the suppression of NRT2.1. A fusion protein of YFP fused to BcNRT2.1expressed in protoplasts indicated that BcNRT2.1was localized on the plasma membrane.5. Silencing of BcNRT1gene in non-heading Chinese cabbage by virus-induced gene silencingIn this study, a viral vector derived from Turnip yellow mosaic virus (TYMV) was used to induce VIGS in non-heading Chinese cabbage on condition that it carries an inverted-repeat fragment of the target gene from plants. Reliable gene silencing was observed when non-heading Chinese cabbage were inoculated simply by abrasion with intact plasmid DNA of pTY harbouring a80bp fragment of PDS gene in leaves. To study the function of BcNRT1in non-heading Chinese cabbage, the VIGS vector inserted with inverted-repeat fragment of BcNRT1gene was used to induce gene silencing, and the result showed the expression of BcNRT1were repressed. Our results indicate that the TYMV-derived vector also has the ability to repress target genes expressed in non-heading Chinese cabbage. The VIGS vector represents an ideal tool for improving high-throughput functional genomics in non-heading Chinese cabbage.In summary, we found that BcNRT1, located on the plasma membrane, induced by the nitrate, involved in lateral root development and expressing in the root tips and shoots, was a low-affinity nitrate transporter. BcNRT1.1was a nitrate-inducible transporter, involved in the lateral roots development and primary nitrate response by regulating the expression of NRT2.1, expressing in the root tips and shoots, interacted with BcCIPK23on the plasma membrane. The BcNRT2.1was a nitrate inducible nitrate transporter located on plasma membrane mainly expressing in roots.