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为探明豆梨(Pyrus calleryana Dcne.)铵转运蛋白基因家族成员的序列特征、生理功能和表达特点,以豆梨幼苗为材料,运用电子克隆与3′-RACE技术获得2个铵转运蛋白基因PcAMT1-1和PcAMT1-2,采用酵母互补和半定量RT-PCR方法研究它们的生理功能与表达特点。结果表明:PcAMT1-1和PcAMT1-2开放阅读框为1 518 bp和1 515 bp,编码的蛋白分别含505和504个氨基酸残基。PcAMT1-1和PcAMT1-2分别与甜橙×枳后代CpAMT(ABI52423)和茶CsAMT1;2(AB114913)的同源性最高(81.96%和78.31%)。PcAMT1-1和PcAMT1-2转入酵母均能使铵转运体缺失突变菌株31019b恢复NH4+吸收能力,在酸性条件下(pH 4.8或5.8)PcAMT1-1和PcAMT1-2对31019b的互补效果均优于中性条件(pH 6.8)。NH4+有毒类似物MeA+可以显著抑制转PcAMT1-1酵母31019b的生长,该物质对转PcAMT1-2酵母31019b无抑制效果;谷氨酰胺合成酶抑制剂MSX可有效抑制PcAMT1-2对酵母31019b的互补效果,对PcAMT1-1的互补效果无显著影响。正常供铵,PcAMT1-1主要在叶中表达,PcAMT1-2主要在根中表达;无氮处理后,PcAMT1-1和PcAMT1-2在根中的表达先上调后下降;重新供铵后,它们的表达量恢复;地上部的表达对上述处理无显著响应。综上所述,PcAMT1-1和PcAMT1-2在豆梨中具有吸收或转运NH4+的功能,并可能具备不同的调控机制。
In order to investigate the sequence characteristics, physiological functions and expression characteristics of the members of the ammonium transporter gene family of Pyrus calleryana Dcne., Two ammonium transporter genes were obtained by electron microscopy and 3’-RACE using the seedlings of Pear PcAMT1-1 and PcAMT1-2, their physiological functions and expression characteristics were studied by yeast complementation and semi-quantitative RT-PCR. The results showed that the open reading frames of PcAMT1-1 and PcAMT1-2 were 1 518 bp and 1 515 bp, encoding 505 and 504 amino acid residues respectively. PcAMT1-1 and PcAMT1-2 had the highest homologies (81.96% and 78.31%) with CpAMT (ABI52423) and tea CsAMT1; 2 (AB114913), respectively. PcAMT1-1 and PcAMT1-2 transformed into yeast, the NH4 + -transfected mutant 31019b could restore NH4 + absorption ability. Under acidic conditions (pH 4.8 or 5.8), the complementarity of PcAMT1-1 and PcAMT1-2 to 31019b was better than that of 31019b Neutral conditions (pH 6.8). NH4 + toxic analogue MeA + can significantly inhibit the growth of the yeast 31019b transformed with PcAMT1-1, which has no inhibitory effect on the transfer of PcAMT1-2 yeast 31019b; the glutamine synthetase inhibitor MSX can effectively inhibit the complementarity of PcAMT1-2 to yeast 31019b , No significant effect on the complementarity of PcAMT1-1. Normal ammonium supply, PcAMT1-1 was mainly expressed in leaves, PcAMT1-2 was mainly expressed in roots. After nitrogen-free treatment, the expression of PcAMT1-1 and PcAMT1-2 in roots first increased and then decreased; The expression of above-ground parts did not show any significant response to the above treatment. In summary, PcAMT1-1 and PcAMT1-2 have the function of absorbing or transporting NH4 + in pears and pear, and may have different regulatory mechanisms.