多年生黑麦草质膜型水通道蛋白基因LpAQP的克隆及功能分析

【目的】研究黑麦草在逆境胁迫下水分代谢的基因调控，克隆黑麦草质膜型水通道蛋白基因的全长cDNA序列，并对其表达模式进行研究。【方法】利用RACE技术从黑麦草（品种“顶峰”）中获得LpAQP全长cDNA序列；运用生物信息学软件分析其编码蛋白；构建LpAQP与GFP融合的瞬时表达载体，采用基因枪法转入洋葱表皮细胞，观察其细胞定位；通过real time-PCR分析LpAQP的表达模式，并用Southern杂交分析其拷贝数。【结果】克隆获得LpAQP的cDNA序列，GenBank登录号为JX569791，其开放阅读框（ORF）为867 bp，编码288个氨基酸，蛋白质分子量为30.7 kD。该基因含有2个NPA单元，6个跨膜区，LpAQP含有与MIP家族完全一致的蛋白质保守区，其氨基酸序列与其它禾本科PIP类质膜型水通道蛋白同源性较高。物种间聚类分析表明LpAQP与大麦、小麦质膜型水通道蛋白同源性较高。LpAQP可能定位于细胞质膜上。通过Southern杂交分析，发现LpAQP在黑麦草基因组中以单拷贝存在。real time-PCR分析表明LpAQP在黑麦草茎部表达高于叶和根，持续干旱胁迫会导致LpAQP表达量先上调后下降。【结论】克隆获得黑麦草质膜型水通道蛋白基因LpAQP，其以单拷贝形式存在，在黑麦草根、茎、叶中均有表达，尤其是茎中表达最高。其表达受干旱胁迫影响。

Cloning and Functional Analysis of the Plasma Membrane Aquaporins Gene in Lolium perenne L.

【Objective】To provide an insight into mechanism of drought resistance and cultivar development in Lolium perenne L., the sequence characteristics of the aquaporin gene LpAQP in L. perenne L. was analyzed and the expression profiling was studied.【Method】The full-length cDNA sequence of LpAQP was isolated by RACE. The obtained cDNA sequence and the deduced amino acid sequence was analyzed. In order to follow the intracellular localization of the protein, the GFP sequence was fused downstream to the LpAQP coding region and the fusion gene LpAQP::GFP was transferred into onion epidermal cells by biolistic method. The real time-PCR was adopted to study the expression profile of gene LpAQP. The southern blotting was used to analyze the copies number of the LpAQP. 【Result】The potential open reading frame (ORF) of LpAQP (GenBank Accession No. JX569791) was 867 bp, encoding a protein of 288 amino acids with a predicted molecular mass of 30.7 kD. Bioinformatics analysis demonstrated that LpAQP exhibited a typical structure with an internal symmetry showing two highly conserved Asn-Pro-Ala (NPA) motifs and six membrane-spanning domains, and possessing the MIP family signal consensus sequence. The LpAQP amino acids showed high identity with other plant species PIP subfamily by NCBI homology comparison analysis. Phylogenetic analysis indicated that LpAQP was clustered with the HvAQP from Hordeum vulgare and TaAQP from Triticum aestivum. The result of transient expression showed that LpAQP gene was probably located in the membrane. Southern blotting analysis indicated that LpAQP was a single-copy gene in L. perenne L. Real-time PCR analysis showed higher expression of LpAQP gene was observed in shoot than other tissues. LpAQP expression level was up-regulated firstly, then down-regulated under drought stress.【Conclusion】 An aquaporin gene LpAQP was cloned by RACE from L. perenne L. This gene is regulated by drought stress. This study will provide important information for the future research on the gene-expression regulation during drought stress.