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| Cloning and Expression Analysis of IRT1 Gene in Pyrus betulaefolia |
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Abstract Abstract Iron-regulated transporter 1 gene (IRT1) has been proved to be involved in plant iron absorption as the main transporter when plants are under iron deficiency stress. In this study, for cloning the IRT1 gene sequence and investigating its expression characteristics of Pyrus betulaefolia, the full length cDNA of iron-regulated transporter gene named PbIRT1 (GenBank accession No. KX355331) was cloned with reverse transcription PCR (RT-PCR) and RACE by roots of Py. betulaefolia seedings. The full length cDNA of PbIRT1 was 1 369 bp, 5'-UTR was 269 bp, 3'-UTR was 490 bp and contained 6 bp PolyA tail, including an 1 095 bp ORF. The protein consisted of 364 amino acids encoded by the PbIRT1, and it had 9 transmembrane domains located in 10~28, 55~74, 89~109, 128~153, 212~232, 242~266 272~290, 303~323, 346~364 of amino acid sequence, and a complete ZRT/IRT-like protein (ZIP) transport family domain architecture which could transport divalent metal ions, whose protein molecular weight was 39.17 kD and isoelectric point was 8.84. Hydrophobicity analysis speculated that PbIRT1 was a hydrophobic protein and there were 10 serine phosphorylation sites and 5 threonine phosphorylation sites in coding region. The secondary structure prediction of Py. betulaefolia IRT1 protein showed that 37.64% α-helix, 29.67% extended strand and 24.45% random coil might be formed. The amino acid identity of PbIRT1 protein among the Py. betulaefolia, Py. bretschneider, Malus xiaojinensis, Prunus mume and Pr. persica were 100%, 97.8%, 87.9% and 87.4%. In addition, subcellular localization prediction analysis indicated that the protein was most likely targeted to the plasma membrane. It was found that the protein sequence of PbIRT1 was the closest with MxIRT1 (M. xiaojinensis) (GenBank accession No. AAO17059.1) and PbrIRT1 (Py. bretschneideri) (GenBank accession No. XP_009357272.1) in genetic relationship by the evolutionary trees analysis of amino acids. RT-PCR and qRT-PCR analysis of PbIRT1 showed that it expressed mainly in the roots of Py. betulaefolia seedings but almost no expression was found in the leaf or the stem, which suggested a typical expression of root specific. The expression levels in the roots of Fe-deficiency treatment were significantly higher than that of Fe-sufficient treatment. Moreover, the expression levels of PbIRT1 in roots decreased firstly (6 h) and then increased over time under the Fe-deficiency stress and reached the highest at 5 d, and then decreased but remained at relatively high level from 5 d to 15 d, being 3.5~5.5 times as much as in the Fe-sufficient treatment, whereas its expression levels were rapidly inhibited when iron resupplied which reduced by 43.1% compared with 1 d and 63.4% compared with 15 d. The expression levels under the Fe-sufficient treatment remained stable throughout. It could be speculated that the PbIRT1 gene could be induced by iron deficiency stress and was involved in the process of iron uptake and transport. This study can contribute to further investigation for the function of IRT1 and the mechanism of Py. betulaefolia iron-deficiency.
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Received: 25 November 2016
Published: 02 May 2017
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