刚毛柽柳液泡膜H^+转运无机焦磷酸酶ThVP3的克隆与表达分析

Cloning and Expression Analysis of a Vacuolar Membrane H^+ Transport Inorganic Pyrophosphatase Gene ThVP3 from Tamarix hispida

通过对刚毛柽柳转录组的分析,获得并克隆了1个H^+-PPase基因cDNA全长,命名为ThVP3。序列分析结果表明:ThVP3全长3 500 bp,包含2 406 bp完整的开放读码框,618的5'非编码区和476 bp的3'非编码区。该cDNA编码1个801个氨基酸的多肽,等电点为5.67,推测分子质量为84.86 k D。通过与其他物种的氨基酸多序列比对及系统进化树分析,发现ThVP3氨基酸序列与AtVP2(拟南芥)一致性达88%,属于Ⅱ型VP成员。ThVP3基因有15个跨膜区,并具有高度保守的3个结构域(CS1,CS2,CS3)。相对荧光定量RT-PCR结果表明,ThVP3在高盐及干旱胁迫下均呈现为表达上调,但在各胁迫时间点和不同组织中的表达模式有所差异,0.4 mol/L Na Cl胁迫处理下,地下部分3 h达到最大值后有不同程度的降低,地上部分24 h达到最大值,与地下部分比,地上部分呈现较高的表达量;20%(w/v)PEG 6000胁迫处理下,地下部分在3 h和12 h达到峰值,地上部分表达量逐渐升高,24 h达到最大值后逐渐降低,96 h达到第2个峰值。ThVP3的表达受盐旱胁迫的诱导,推测其可能在柽柳的胁迫应答中起着重要的作用。

A full length cDNA of a vaculor H^+-ppase gene(named ThVP3) was isolated from the transcriptome cDNA library of Tamarix hispida. Sequence analysis indicated that is 3 500 bp in length, containing an open reading frame of 2 406 bp, a 5'-untranslated region(5'-UTR) of 618 bp and a 3'-untranslated region(3'-UTR) of 476 bp.The ThVP3 cDNA encodes a protein of 801 amino acids with a p I of 5.67 and a deduced molecular mass of 84.86 KD. Amino acid sequence alignment with other species and phylogenetic analysis indicates that the T. hispda of ThVP3 displays the highest similarity(88%) to the Arabidopsis thaliana of AtVP2, and belongs to the typeⅡ. ThVP3 has fifteen putative transmembrane(TM) domain structures, and contains three conservative domains(CS1, CS2, CS3).Quantitative real-time PCR assay revealed that the m RNA level of ThVP3 was significantly up-regulated by Na Cl and PEG, and its expression is different at different time and in different organ. The m RNA level of ThVP3 in the root under the salt stress was reached the maximum at 3 h and then gradually decreased, while the shoot under the salt stress was reached the maximum at 24 h. The root has a higher expression compaired with the shoot. The m RNA level of ThVP3 in the root under the drought stress was reached the maximum at 3 h and 12 h, while the shoot under the drought stress was gradually increased until reached the maximum at 24 h, then gradually decreased until reached the second peak(96 h). The expression of ThVP3 was induced by salt and drought stress,suggesting that ThVP3 might play important roles in response to abiotic stresses in Tamarix hispida.