Molecular cloning,tissue expression specificity detection and functional analysis of AMT1;2 from Chinese cabbage Pak-Choi

【Objective】 In this paper, an ammonium transporter gene BcAMT1;2 was cloned from Chinese cabbage Pak-Choi, its bioinformatics and tissue expression characteristics were detected, and its function was analyzed, which provided a the oretical basis for further studying the mechanism of BcAMT1;2 gene in NH₄⁺ absorption and transportation in Chinese cabbagePak-Choi.【Method】 Homologous cloning method was applied to clone BcAMT1;2 gene from Chinese cabbage Pak-Choi variety Shanghaiqing, then its biological information was carried out on its encoded protein. The expression of BcAMT1;2 gene was detected by real-time fluorescence quantitative PCR in different tissues of Chinese cabbage Pak-Choi, and the function of BcAMT1;2 was verified by overexpression BcAMT1;2 in Arabidopsis thaliana.【Result】 The open reading frame(ORF) of BcAMT1;2 gene was1539 bp, encoding 512 amino acids residues, with molecular weights of 54.90 kD, and theoretical isoelectric points(pI) of 8.03. In addition, BcAMT1;2 protein had no signal peptide, but had 9 transmembrane domains. Stability prediction indicated that BcAMT1;2 was stable amphipathic protein, and the subcellular localization prediction showed BcAMT1;2 might locate in the plasmalemma. Conserved domain analysis suggested that BcAMT1;2 included the motif(DFAGSGVVHMVGGIAGLWGALIEGPR), which was the signature motif of AMT1 subfamily. Moreover, amino acid sequence alignment showed that BcAMT1;2 protein had the highest homology with At-AMT1;2(91.21%) in A. thaliana, so BcAMT1;2 was one member of AMT1 subfamily. The expression level of BcAMT1;2 in leaf was significantly higher than in other tissues in Pak-Choi(P<0.05, the same below), followed by the ones of root and petiole, but almost no expression in stem. Under the treatment of 0.25 mmol/L NH₄⁺, the biomass(the fresh weight of root and shoot), the length of primary root, and NH₄⁺-N content of the whole plants were significantly increased in three lines of overexpressing BcAMT1;2, compared with the ones of wild type. At 20 mmol/L methylamine(MeA), the biomass of three lines of overexpressing BcAMT1;2 was significantly lower than that of wild type, with yellow leaves, short roots, and other symptoms of ammonia toxicity.【Conclusion】 BcAMT1;2 gene has obvious tissue specificity and can regulate the absorption and transportation of NH₄⁺ and its analog methylamine, indicating that BcAMT1;2 protein may play an important role in the absorption and transport of NH₄⁺ in Pak-Choi.