马铃薯StZnT11的电子克隆、表达及生物信息学分析

In silico cloning, expression and bioinformatics analysis of StZnT11 in Solanum tuberosum

摘要:

马铃薯锌转运蛋白 (Solanum tuberosum zinc transporter 11，StZnT11) 对于维持细胞中锌稳态至关重要。通过研究StZnT11在非生物胁迫和生物胁迫下的表达情况，为验证马铃薯StZnT11在青枯菌生物胁迫过程中的作用奠定了基础。从前期工作获得的表达文库中得到EST序列，利用NCBI中的Blast工具，对原始序列进行同源性分析，选择与原始序列的相似度、覆盖度、e期望值最高的一条同源对象序列。通过电子克隆，得到StZnT11基因。采用生物信息学方法对StZnT11基因的基因序列及编码的氨基酸组成、理化性质、分子进化、磷酸化位点、高级结构等多角度进行分析。结果表明，该基因cDNA全长1 300 bp，编码348个氨基酸，编码蛋白含23个磷酸化位点，有1个信号肽，有9个跨膜区域，是定位在质膜上的疏水性蛋白。通过氨基酸序列比对，StZnT11蛋白与烟草、番茄和辣椒等植物中的锌转运蛋白同源性较高。实时荧光定量聚合酶链反应检测结果表明，StZnT11在不同浓度的外源植物激素脱落酸 (ABA) 的作用下上调。组织定位检测提示StZnT11主要表达于特定组织 (茎维管系统的韧皮部和叶片维管束)。这些结果为进一步进行该基因的实验克隆及功能验证研究提供了理论依据。

Abstract:

Solanum tuberosum Zinc transporter 11 (StZnT11) is very important for maintaining zinc homeostasis in cells. The study on the expression of StZnT11 under abiotic stress and biotic stress laid a foundation for verifying the role of potato StZnT11 in the process of biotic stress of Ralstonia solanacearum species complex. According to the designated EST sequence, the homology of the original sequence was analyzed by using the Blast tool in NCBI, and a homologous object sequence with the highest similarity, coverage and e expectation value was selected. StZnT11 gene is obtained by Silico Cloning. The sequence and coding amino acid composition, physicochemical properties, molecular evolution, phosphorylation site and advanced structure of Solanum tuberosum StZnT11 gene were analyzed by bioinformatics method. The results showed that the cDNA gene is 1 300 bp in length, encoding a protein containing 348 amino acid residues, including 23 phosphorylation sites, one signal peptide and nine transmembrane regions, and is a hydrophobic protein located on the plasma membrane. Through amino acid sequence alignment, StZnT11 protein has a high homology with zinc transporter from tobacco, tomato, pepper and other plants. The results of real-time fluorescence quantitative polymerase chain reaction showed that, StZnT11 is up-regulated by different concentrations of exogenous plant hormone abscisic acid (ABA). Tissue localization showed that StZnT11 was mainly expressed in specific tissues (phloem and leaf vascular bundles of stem vascular system). These results provide a theoretical basis for further experimental cloning and functional verification of the gene.