野生毛葡萄水通道蛋白基因VhPIP1的克隆及其在干旱胁迫下的表达分析

Cloning of Aquaporion Gene Vh PIP1 in Vitis heyneana and Its Expression Under Drought Stress

采用RT-PCR和RACE技术从中国野生毛葡萄(Vitis heyneana)‘花溪–9’根中克隆得到1个质膜内在蛋白(plasma membrane intrinsic proteins,PIPs)类的水通道蛋白基因,命名为Vh PIP1(登录号为KM026528)。该基因c DNA全长为1 087 bp,包含1个858 bp的完整开放阅读框,编码286个氨基酸。氨基酸序列分析表明,Vh PIP1含有水通道蛋白家族高度保守的两个NPA(Asn-Pro-Ala)单元和6个跨膜区,其氨基酸残基与MIP家族蛋白保守区序列完全一致。实时荧光定量RT-PCR分析表明,Vh PIP1在野生毛葡萄根、茎、叶中均有表达,在根中的表达量最高,叶片中最少。干旱胁迫下,抗旱性强的毛葡萄‘花溪–9’Vh PIP1的表达量随着胁迫时间的延长先增加后降低,而不抗旱的欧亚种‘红地球’Vv PIP1的表达量呈下降的趋势。与不抗旱的‘红地球’相比,干旱胁迫下极抗旱的‘花溪–9’较高的Vh PIP1转录水平对应较高的叶片相对含水量,同时对应较低的根系细胞膜相对透性。Vh PIP1的表达丰度与毛葡萄抗旱性密切相关。

A plasma membrane intrinsic proteins(PIPs)gene,designated Vh PIP1,was cloned from root on wild grapevine(Vitis heyneana‘Huaxi-9')native to China by RT-PCR and RACE(Rapid amplication of c DNA ends). The full c DNA sequence of Vh PIP1 was 1 087 bp,containing an open reading frame(858 bp)and encoding a putative Vh PIP1 protein with 286 amino acids. Bioinformatics analysis demonstrated that Vh PIP1 exhibited an internal symmetry showing two highly conserved NPA(Asn-Pro-Ala)motifs and a typical structure with six membrane-spanning domains and possessing the MIP family signal consensus sequence. Quantitative real-time PCR results showed that the Vh PIP1 expressed in roots,stems and leaves,and the expression level was the highest in roots,and the lowest in leaves. Under drought stress,the expression level of Vh PIP1 of Huaxi-9 changed with time extension,which was first rised and then dropped,but the expression level of Vv PIP1 of Red Globe was decreased. Compared with drought sensitive genotype Red Globe,the relatively higher expression level of Vh PIP1 in drought resistance genotype corresponds to the higher leaves relative water content and the lower ion leakage of plasma membrane. These results suggested that the expression quantity of Vh PIP1 closely related to drought resistance.