Cloning And Characterization Of The CjMATE Gene From Citrus Junos

Multidrug and toxic compound extrusion(MATE) transporters is a new secondary transporter family, and there are 56 members in Arabidopsis. It plays important role in tolerance of Aluminum toxicity and iron deficiency. The Citrus junos is considered resistant to iron chlorosis, and grow healthily in acid siol. Identifying Citrus MATE gene not only be helpful to understand the molecular mechanism of iron deficiency tolerance in Citrus junos, but also be beneficial to breeding programs on acid soil and iron deficiency tolerance via genetic engineering, thus helping to increase crop yield and quality.After partial sequence of CjMATE gene was produced by homologous PCR, which primer designated as AtFRD3, the full length cDNA of CjMATE gene was obtained by RACE, which containing a 155 lbp ORF encoding a predicted protein of 516 amino acids with 12 transmenbrane helices. Sequence analysis indicated that the deduced amino acid sequence of CjMATE shared high homology to other MATE proteins,60% with SbMATE,59% with AtFRD3,70% with AtMATE. The coding sequence was named as CjMATE (Citrus junos MATE) gene. CjMATE gene was expressed in root, shoot and leaf, and highest level in the root, Fe-deficiency did not affect the expression of CjMATE gene. To characterize CjMATE gene, the overexpression vectors were constructed and introduced into alfalfa and Arabidopsis mutant of Al-tolerant and iron transport.To confirm the function of CjMATE gene, Al-tolerance and resistance to iron deficiency capability of the transgenic Arabidopsis were detected, the content of organic acids in the leaf of overexpressing CjMATE alfalfa were detected, the content of Al were detected. The primary results as followed.1. We introduced CjMATE gene into Arabidopsis mutants(frd3-1, frd3-3, AtALMTl-KO, AtMATE-KD, Double KO/KD), and obtained 85 transgenic plants. frd3-3, an iron-deficiency-sensitive mutant, showed chlorotic in 20μmol/L Fe2+ condition, but frd3-3 transgenic plants of expressing CjMATE was not chlorotic as well as the wild-type in the same condition. The citrate content of the transgenic plants w3 34.2% higher than frd3-3, but iron content was reduced 30.5% compared to frd3-3. This result showed that CjMATE gene play a role in iron transport.2. At 1.5μmol/L Al3+activity, Aluminum-sensitive mutant AtALMTl-KO, AtMATE-KD and Double KO/KD were severely inhibited in root elongation compared to wild-type, but it changed in the transgenic plants of CjMATE to them, in which percentage of relative net root growth(%RNRG) were increased compared to the mutant in the same condition respectively. The RNRG of transgenic plants increased 35% compared to AtALMTl-KO,33% compared to AtMATE-KD,29% compare to Double KO/KD and 13% compared to wild-type. It implied that CjMATE gene play a role in Aluminum tolerance too.3. We introduced CjMATE gene into alfalfa, and 20 transgenic plants were obtained. The relative expression levels of all transgentic plants and wild-type were analyzed, and we got 4 plants overexpressing CjMATE gene. Organic acid of transgenic plants were analyzed. The result showed that:oxalic acid content of transgenic plants were 0.46-0.63mg/g (freash weight), citric acid content were 0.56-0.78mg/g, malic acid content were 0.66-0.97mg/g, the three organic acid content of wild-type were 0.41,0.48 and 0.56mg/g. The three organic acid content of overexpressing CjMATE gene alfalfa plants were 12.0%～53.8%,16.3%-61.6%, 18.8%-72.5% higher than wild-type respectively.4. Aluminum tolerance test was also be done to transgenic alfalfa plants. Under 50μmol/L Al3+stress condition. Aniline blue dying of root tips with or without Al3+ treatment displayed distinctly, the transgenic plants showed less lightness compared with wild-type, no matter with or without Al3+treatment. The result showed that Aluminum toxicity was reduced in transgenic alfalfa plants.5. The content of Al in transgenic alfalfa plants was 3.4%-21.7% higher in shoot but 50.3%-66.3% lower in leaves, and total content of Al in transgenic alfalfa decreased 27.1%～43.8% compared to wild-type. The reduction of the content of Al in leaves is more than the increase in the shoot, the content of Al in transgenic alfalfa plants is lower compared with wild-type.