番茄水通道蛋白基因SlAQP的克隆与序列分析

doi:10.3864/j.issn.0578-1752.2012.02.012

摘要/Abstract

摘要： 【目的】通过分析克隆得到的番茄水通道蛋白SlAQP基因的cDNA全长序列特征、编码蛋白的细胞定位及其在番茄材料Mirco Tom中经干旱胁迫后的表达模式，为进一步研究番茄在逆境下的抗逆机制及加快番茄抗逆育种积累资料。【方法】采用RACE 技术克隆番茄水通道蛋白基因的cDNA全长，结合生物信息学软件分析该基因的编码蛋白特性；利用基因枪转化法将SlAQP基因与GFP融合的瞬时表达载体（SlAQP::GFP）转入洋葱表皮细胞，对该基因进行亚细胞定位；利用Real time-PCR 分析该基因在番茄品种Mirco Tom中经干旱胁迫下的表达机制。【结果】SlAQP基因（GenBank登录号：HQ433337）cDNA全长为1 107 bp，编码区含852 bp，共编码283个氨基酸。生物信息学软件分析表明，SlAQP基因含有6 个跨膜区，2 个NPA 单元，其氨基酸残基与MIP 家族蛋白保守区序列完全一致，且该基因氨基酸序列与其它物种PIP 类质膜型水通道蛋白氨基酸序列具有很高的同源性。11个物种间的聚类分析表明，SlAQP基因编码的蛋白与马铃薯质膜型水通道蛋白遗传距离最近。细胞定位结果确认SlAQP基因编码蛋白在细胞质膜上发挥生物学作用。Southern杂交结果显示，SlAQP基因在番茄基因组DNA中呈单拷贝。Real time-PCR 分析结果证实，干旱胁迫下该基因表达量总体呈下降趋势。【结论】对番茄水通道蛋白SlAQP基因在干旱胁迫后的表达模式分析，预示该基因表达受逆境条件的影响，为今后进一步探讨其在干旱胁迫中发挥的作用提供了重要信息。

关键词: 番茄, 水通道蛋白, 序列分析, 细胞定位, Realtime-PCR

Abstract: 【Objective】 To provide basal data for mechanism of tomato’s drought resistance and cultivar development, the sequence characteristics of the aquaporin gene SlAQP in tomato were analyzed, the coded protein was intracellar located and the expression profiling of the Mirco Tom tomato was studied after treatment under drought stress. 【Method】 The rapid-amplification of cDNA ends (RACE) was used to amplify the full-length SlAQP gene, and the bioinformatics software was used to analyze the structures and function of the coded protein. In order to follow the intracellular localisation of the protein, the GFP sequence was fused downstream to the SlAQP coding region and the fusion gene SlAQP::GFP transferred into onion epidermal cells by biolistic method. The Mirco Tom tomato was used as material, the real time-PCR was adopted to study the expression profile of gene SlAQP.【Result】 The full-length cDNA of SlAQP gene (GenBank Accessin No. HQ433337) consists of 1 107 bp and contains a 852 bp open reading frame（ORF） encoding 283 amino acid proteins. Bioinformatics analysis demonstrated that SlAQP gene exhibited a typical structure with six membrane-spanning domains and an internal symmetry showing two highly conserved Asn-Pro-Ala (NPA) motifs, and possessing the MIP family signal consensus sequence. The SlAQP amino acids showed high identity with other 10 plant species PIP subfamily by NCBI homology comparison analysis. Phylogenetic analysis among 11 species indicated that SlAQP was clustered with the StAQP from Solanum tuberosum. The result of transient expression showed that SlAQP gene was located in the membrane. Southern blot analysis indicated that SlAQP was a single-copy gene. Real time-PCR analysis showed that the expression of SlAQP gene was down-regulated by drought stress.【Conclusion】The expression profiling of this gene indicates that the SlAQP gene may regulared by drought stress. This paper provides important information for the future study on the gene-expression regulation during drought stress.

Key words: tomato, aquaporins, sequence analysis, intracellular localization, Real time-PCR

李仁, 吴新新, 李蔚, 杨荣超, 赵永钦, 温常龙, 赵冰, 郭仰东. 番茄水通道蛋白基因SlAQP的克隆与序列分析[J]. 中国农业科学, 2012, 45(2): 302-310.

LI Ren, WU Xin-Xin, LI Wei, YANG Rong-Chao, ZHAO Yong-Qin, WEN Chang-Long, ZHAO Bing, GUO Yang-Dong. Cloning and Sequence Analysis of the Aquaporins Gene SlAQP in Tomato[J]. Scientia Agricultura Sinica, 2012, 45(2): 302-310.

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