新疆黄精凝集素基因的克隆、序列分析和表达

xielirong

摘 要: 用百合科黄精属植物凝集素基因的保守序列为引物,从新疆黄精的叶中克隆出黄精凝集素的全长cDNA。序列分析表明, 克隆获得的新疆黄精凝集素(Polygonatum roseum agglutinin, PRA)基因完整的ORF 片段大小为550 bp, 编码1条长159 个氨基酸肽链, 没有内含子, 其中N 端的28 个氨基酸是信号肽。对新疆黄精凝集素cDNA 序列同源性的分析比较发现, 黄精属植物凝集素基因之间有很高的同源性(92%)。氨基酸序列比对和SWISS-MODEL 同源模建分析表明,PRA 由12 个β- 折叠片组成的β- 桶结构, 具有与单子叶植物甘露糖结合凝集素相似的空间结构。重组质粒pGEX-4T-1-PRA 和pMAL-p2x-PRA, 分别转化E. coli BL21 进行原核表达, 新疆黄精凝集素能够以可溶性融合蛋白形式表达, 分子量约为14 kD。构建真核表达载体pcDNA3-PRA, 免疫小鼠后获得了抗血清。免疫印迹结果显示为单一的条带, 证明该抗血清具有针对PRA 抗原的专一性。新疆黄精凝集素基因的克隆、原核和真核的表达以及抗血清的制备, 为进一步研究凝集素蛋白的性质和功能, 并为植物抗病虫基因工程研究提供有用的实验材料。

关键词: 新疆黄精, 凝集素, 序列比较, 原核表达

Cloning, Sequencing Analysis and Expression of a Putative Mannose-binding Lectin Gene from Polygonatum roseum in Xinjiang

Surong Sun, Zhi Zhang, Suli Li, Jun Hu, and Fuchun Zhang

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China

Abstract: The genomic DNA were extracted from the leaves of Polygonatum roseum (Liliaceae) in Xinjiang and the primers were designed according to conservative sequences of Polygonatum lectins gene. The complete ORF of Polygonatum roseum agglutinin (PRA) gene was amplified as a fragment of 550 bp, which was identical with predicted size. Like most of the plant lectin genes, there was no intron in the PRA gene. The ORF of the gene encoded 159 amino acid residues, in which included a signal sequence of 28 amino acid residues at its N-terminus. The cDNA sequence had 92% identities compared with the published sequence. The amino acid sequence and SWISS-MODEL analysis indicated that the three-dimensional structure of PRA strongly resembled with that of monocot mannose-binding lectins, which comprised with three antiparallel four-stranded β-sheets arranged as a 12-stranded β-barrel. The recombinant pGEX4T-1-PRA and pMAL-p2x-PRA prokaryotic expression vectors were constructed to produce GST-PRA and MBP-PRA fusion proteins in E. coli, respectively. SDS-PAGE of the fusion protein demonstrated that the PRA lectin protein migrated at a size of 14 kD. The immunization was performed by intra-muscular injection of pcDNA3-PRA, and the antiserum wasdetected by ELISA. Western blotting analysis showed the antiserum specifically bound the lectin protein. The establishment of such an expression system might provide materials for further investigation of the properties and functions of PRA proteins. It also laid the basis for plant genetic engineering on its defensive functions to pests and diseases.

Keywords: Polygonatum roseum, agglutinin, sequence compare, prokaryotic expression

植物凝集素是一类非免疫来源、对糖及其缀合物专一识别并可逆结合的蛋白质[1], 它的特异性识别不是针对植物内源多糖, 而是针对动物、微生物等外源多糖, 因此可作为植物的防御物质[2]。植物凝集素根据进化及结构相关性可分为豆科植物凝集素、单子叶植物甘露糖结合凝集素等七个家族, 它们在植物各组织器官, 尤其在种子等营养和繁殖器官中广泛分布。

单子叶甘露糖结合凝集素(monocot mannose-binding lectins)广泛分布于石蒜科、兰科、百合科等植物, 在进化上属于彼此相关的家族, 其序列和结构上具有很高的同源性。通常由2~4 个11~14 kD相同的亚基组成二聚体或四聚体,每个亚基包含有3个典型的甘露糖结合位点(QDNY), 表现出D-甘露糖结合的专一性。单子叶甘露糖结合凝集素由于其生理生化特性, 已被用于分析和分离含甘露糖及其糖缀合物的研究工具[3]; 在医药方面, 由于它能与一些感染人和动物的逆转录病毒(如HIV 等)的外壳蛋白糖链相互作用, 表现出潜在抗病毒功能[4], 如雪花莲凝集素(Galanthus nivalis agglutinin, GNA)[5, 6];由于它也能与糖缀合物结合, 严重干扰小肠消化吸收功能, 尤其是对刺吸式昆虫和线虫等有毒性, 目前在农业植物转基因中得到广泛应用。例如, 雪花莲和苋菜凝集素的转基因作物表现了良好的抗蚜虫特性[6, 7]。

新疆黄精(Polygonatum roseum Kunth)又名玫瑰黄精, 属百合科黄精属, 为多年生单子叶草本植物, 分布在西藏、云南、四川、甘肃、青海、宁夏、陕西和新疆。现代药理学研究认为,黄精有抗衰老、降血压、防止动脉硬化及抗菌消炎、增强免疫力等功效[4, 8]。黄精不仅是传统中药材, 而且也是出口创汇药品种之一。目前已克隆得到多条黄精凝集素cDNA 片段, 但迄今为止,关于新疆黄精的凝集素还缺乏系统深入的研究。

为了筛选到新疆黄精的凝集素基因并进行凝集素的功能活性检测, 有必要对新疆黄精的凝集素基因进行克隆并进行序列分析。同时将克隆获得新疆黄精的凝集素基因ORF 片段, 进行原核和真核表达,制备相应的鼠抗血清, 为深入研究凝集素的结构、性质和生物学活性及功能奠定基础。