豆梨磷转运蛋白质基因(PcPht1)的克隆、表达及启动子分析

Cloning,expression and promoter analysis of a phosphate transporter protein gene PcPht1 from Pyrus calleryana Dcne.

为明确梨砧木豆梨(Pyrus calleryana Dcne.)磷转运蛋白质基因的序列特点和表达模式,采用同源克隆、RACE技术和染色体步移法克隆获得1个Pht基因PcPht1的cDNA、DNA及启动子序列,并利用RT-PCR检测在不同供磷水平下该基因在豆梨幼苗中的表达情况。结果表明PcPht1 cDNA序列编码区长1 617 bp,编码1个由538个氨基酸组成的蛋白质,其相对分子质量为5.908×104。该基因编码区DNA序列没有内含子,其启动子除了具有TATA/CAAT-box外还包含防御与胁迫响应元件、光响应元件和茉莉酸甲酯响应顺式作用元件。PcPht1所编码蛋白质由12个疏水的跨膜区域组成,包括H2PO4-/nH+共运体、糖转运体和MFS通用底物转运体3个Pht1蛋白质特征结构域。PcPht1与大豆GmPht1;7和橡胶HbPht1间有较高的一致性(分别为88.0%和87.0%);与拟南芥Pht1蛋白质家族处于系统进化树的同一分支,并与AtPht1;4和AtPht1;7的亲缘关系最近。正常供磷条件下,PcPht1基因主要在根中表达,低磷时该基因的表达上调,提高供磷浓度其表达受到抑制,说明PcPht1的表达受环境磷浓度调控。

This experiment was conducted to investigate the sequence characteristics and expression pattern of phosphate transporter protein （Pht） gene in bean pear （Pyrus calleryana Dcne. ）. The cDNA, DNA and promoter sequences of a PcPht1 gene were isolated from bean pear using homologous cloning, rapid amplification of cDNA ends （RACE） and genome walking method. At the same time, its expression in bean pear seedlings at different phosphate levels was analyzed by semi-quantitative reverse transcription polymerase chain reaction. PcPht1 cDNA coding region length is 1 617 bp, which contains an open reading frame encoding a 5. 908104 protein consisting of 538 amino acids. Its DNA sequence has no intron. In addition to the TATA/ CAAT box, its promoter contains some specific regulatory elements such as cis-acting element involved in defense and stress responsiveness, light responsive element and cis-acting regulatory element involved in the MeJA-responsiveness. Twelve hydrophobic transmembrane regions constitute PcPht1 protein which has three feature domains, H2 PO4- / nH＋ trans-porter, sugar transporter and the major facilitator superfamily （MFS） general substrate transporter. PcPht1 shared high similarities （88. 0% and 87. 0%） with Glycine max GmPht1;7 and Hevea brasiliensis HbPht1, respectively. PcPht1 and Arabidopsis Pht1 proteins belong to the same branch in Pht phylogenetic tree. PcPht1 has the closest relationship with AtPht1;4 and AtPht1;7. Semi-quantitative RT-PCR results revealed that PcPht1 gene was expressed mainly in root under normal inorganic phosphate （Pi） conditions. Its expression was increased at low Pi levels, and was inhibited at high Pi level,suggesting PcPht1 expression was regulated by environmental phosphorus concentration.