生物工程學報 Chin J Biotech 2008, March 25; 24(3): 387-394
journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061
[email protected] . 2008 Institute of Microbiology, CAS & CSM, All rights reserved
新疆黃精凝集素基因的克隆,、序列分析和表達
孫素榮, 張智, 李素麗, 胡俊, 張富春
新疆大學生命科學與技術(shù)學院, 新疆生物資源基因工程重點實驗室, 烏魯木齊 830046
摘 要: 用百合科黃精屬植物凝集素基因的保守序列為引物,從新疆黃精的葉中克隆出黃精凝集素的全長cDNA,。序列分析表明, 克隆獲得的新疆黃精凝集素(Polygonatum roseum agglutinin, PRA)基因完整的ORF 片段大小為550 bp, 編碼1條長159 個氨基酸肽鏈, 沒有內(nèi)含子, 其中N 端的28 個氨基酸是信號肽。對新疆黃精凝集素cDNA 序列同源性的分析比較發(fā)現(xiàn), 黃精屬植物凝集素基因之間有很高的同源性(92%),。氨基酸序列比對和SWISS-MODEL 同源模建分析表明,PRA 由12 個β- 折疊片組成的β- 桶結(jié)構(gòu), 具有與單子葉植物甘露糖結(jié)合凝集素相似的空間結(jié)構(gòu),。重組質(zhì)粒pGEX-4T-1-PRA 和pMAL-p2x-PRA, 分別轉(zhuǎn)化E. coli BL21 進行原核表達, 新疆黃精凝集素能夠以可溶性融合蛋白形式表達, 分子量約為14 kD。構(gòu)建真核表達載體pcDNA3-PRA, 免疫小鼠后獲得了抗血清,。免疫印跡結(jié)果顯示為單一的條帶, 證明該抗血清具有針對PRA 抗原的專一性,。新疆黃精凝集素基因的克隆、原核和真核的表達以及抗血清的制備, 為進一步研究凝集素蛋白的性質(zhì)和功能, 并為植物抗病蟲基因工程研究提供有用的實驗材料,。
關(guān)鍵詞: 新疆黃精, 凝集素, 序列比較, 原核表達
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
植物凝集素是一類非免疫來源,、對糖及其綴合物專一識別并可逆結(jié)合的蛋白質(zhì)[1], 它的特異性識別不是針對植物內(nèi)源多糖, 而是針對動物、微生物等外源多糖, 因此可作為植物的防御物質(zhì)[2],。植物凝集素根據(jù)進化及結(jié)構(gòu)相關(guān)性可分為豆科植物凝集素,、單子葉植物甘露糖結(jié)合凝集素等七個家族, 它們在植物各組織器官, 尤其在種子等營養(yǎng)和繁殖器官中廣泛分布。
單子葉甘露糖結(jié)合凝集素(monocot mannose-binding lectins)廣泛分布于石蒜科,、蘭科,、百合科等植物, 在進化上屬于彼此相關(guān)的家族, 其序列和結(jié)構(gòu)上具有很高的同源性。通常由2~4 個11~14 kD相同的亞基組成二聚體或四聚體,每個亞基包含有3個典型的甘露糖結(jié)合位點(QDNY), 表現(xiàn)出D-甘露糖結(jié)合的專一性,。單子葉甘露糖結(jié)合凝集素由于其生理生化特性, 已被用于分析和分離含甘露糖及其糖綴合物的研究工具[3]; 在醫(yī)藥方面, 由于它能與一些感染人和動物的逆轉(zhuǎn)錄病毒(如HIV 等)的外殼蛋白糖鏈相互作用, 表現(xiàn)出潛在抗病毒功能[4], 如雪花蓮凝集素(Galanthus nivalis agglutinin, GNA)[5, 6];由于它也能與糖綴合物結(jié)合, 嚴重干擾小腸消化吸收功能, 尤其是對刺吸式昆蟲和線蟲等有毒性, 目前在農(nóng)業(yè)植物轉(zhuǎn)基因中得到廣泛應(yīng)用,。例如, 雪花蓮和莧菜凝集素的轉(zhuǎn)基因作物表現(xiàn)了良好的抗蚜蟲特性[6, 7]。
新疆黃精(Polygonatum roseum Kunth)又名玫瑰黃精, 屬百合科黃精屬, 為多年生單子葉草本植物, 分布在西藏,、云南,、四川,、甘肅、青海,、寧夏,、陜西和新疆。現(xiàn)代藥理學研究認為,黃精有抗衰老,、降血壓,、防止動脈硬化及抗菌消炎、增強免疫力等功效[4, 8],。黃精不僅是傳統(tǒng)中藥材, 而且也是出口創(chuàng)匯藥品種之一,。目前已克隆得到多條黃精凝集素cDNA 片段, 但迄今為止,關(guān)于新疆黃精的凝集素還缺乏系統(tǒng)深入的研究。
為了篩選到新疆黃精的凝集素基因并進行凝集素的功能活性檢測, 有必要對新疆黃精的凝集素基因進行克隆并進行序列分析,。同時將克隆獲得新疆黃精的凝集素基因ORF 片段, 進行原核和真核表達,制備相應(yīng)的鼠抗血清, 為深入研究凝集素的結(jié)構(gòu),、性質(zhì)和生物學活性及功能奠定基礎(chǔ)。
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