关键词: 豆梨, 铵转运蛋白基因, 克隆, 表达特点, 功能互补

Abstract: The objective of this study was to illuminate sequence feature，physiological function and expression characteristic of PcAMT1-1 and PcAMT1-2 from ammonium transport protein gene family in bean pear（Pyrus calleryana Dcne.）. The cDNA sequences of PcAMT1-1 and PcAMT1-2 were cloned from bean pear seedlings by EST splicing and rapid amplification of cDNA ends（RACE）methods. The yeast complementation was adapted to preliminary study PcAMT1-1 and PcAMT1-2 physiological functions.Furthermore，their expression patterns were analyzed by semi-quantitative RT-PCR under different ammonium concentrations. The results showed that PcAMT1-1 cDNA had an open reading frame of 1 518 nucleotides encoding a polypeptide with 505 residues，while PcAMT1-2 cDNA had an opening reading frame of 1 515 nucleotides encoding a polypeptide with 504 residues. The deduced proteins of PcAMT1-1 and PcAMT1-2 had higher homologies（81.96% and 78.31%）with Citrus sinensis × Citrus trifoliata CpAMT（ABI52423）and Camellia sinensis CsAMT1;2（AB114913），respectively. Both PcAMT1-1 and PcAMT1-2 were able to complement the growth defect of yeast（Saccharomyces cerevisiae）ammonium transport mutant strain 31019b. Furthermore，the complementary effect under acid condition（pH 4.8 or 5.8）was slightly better than neutral condition（pH 6.8）. The presence of toxic NH₄⁺ analog methylamine（MeA+）markedly inhibited the growth of yeast with PcAMT1-1，whereas yeast cells transformed with PcAMT1-2 can grow normally. L-methionine sulfoximine（MSX），the glutamine synthetase inhibitors，could effectively inhibit PcAMT1-2 complementary. However，it had no significant effect on PcAMT1-1.Under the normal NH₄⁺ concentration condition，transcription of PcAMT1-1 was mainly in the leaves andPcAMT1-2 expressed mainly in the root. When NH₄⁺ was depleted，PcAMT1-1 and PcAMT1-2 expression levels in roots increased firstly and then decreased. Moreover，their expression levels were recovered upon NH₄⁺ resupply. However，their expression levels in shoots were not observably changed when the NH₄⁺ concentration was changed. Taken together，both PcAMT1-1 and PcAMT1-2 play the roles in absorption or
translocate NH₄⁺ in bean pear，which maybe have different regulatory mechanisms.

Key words: Pyrus calleryana, ammonium transport protein gene, cloning, expression feature, functional complementation