水稻液泡ATPaseB亚基基因的克隆及其在低磷胁迫下的表达特征分析(英)

利用抑制性扣除杂交 (SSH)技术构建水稻 (OryzasativaL .)根系磷饥饿诱导cDNA文库 ,获得编码液泡ATPase (V_ATPase)B亚基的克隆 ,通过反转录PCR方法获得该基因的完整序列。该基因编码 4 87个氨基酸 ,含有一个保守的ATP结合位点 ,其蛋白分子量为 5 4 .0 6kD ,等电点为 4 .99。Southern印迹表明 ,V_ATPaseB亚基基因在水稻基因组中以单拷贝形式存在。氨基酸同源性分析发现 ,V_ATPaseB亚基是一个较为保守的蛋白亚基 ,其序列变化伴随生物的进化过程同步进行。Northern印迹表明 ,V_ATPaseB亚基在水稻根系中受到磷饥饿诱导表达 ,磷饥饿 6～ 12h出现表达高峰 ,而在叶片中表达高峰有所滞后 (2 4～ 4 8h)。在缺磷环境条件下 ,ATPaseB亚基可能通过提高其表达量 ,进而提高质子转运活性 ,形成跨膜的电化学梯度 ,为体内储备磷跨液泡膜运输提供能量 ,从而提高植物体内磷的利用效率及其耐低磷的能力。

Identification of the Rice Vacuolar ATPase B Subunit Gene and Its Expression Pattern Analysis Under Phosphorus Deficiency

A vacuolar ATPase (V_ATPase) B subunit gene has been cloned and characterized from a phosphorus starvation induced rice root subtractive cDNA library by suppression subtractive hybridization (SSH) method and RT_PCR amplification. This gene encodes a polypeptide of 487 amino acid residues, containing a conservative ATP binding site and with a molecular weight of 54.06 kD and an isoelectric point of 4.99. Southern analysis of the genomic DNA indicates that V_ATPase B subunit is encoded by a single gene in rice genome. The amino acid homologies of V_ATPase B subunits among different organisms range from 76% to 97% and reveals that the evolution of V_ATPase B subunit is accompanied with the biological evolution. Expression pattern analysis indicated that the maximal expression of V_ATPase B subunit gene occurred at an early stage (6-12 h) after phosphorus starvation in roots, and lately stage (24-48 h) in leaves. Under phosphorus deficiency, the up_regulated expression of V_ATPase gene was presumed to strengthen the proton transport and provide the required energy to maintain an electrochemical gradient across the tonoplast to facilitate phosphorus transport.