黑麦籽粒Waxy蛋白亚基的高效分离与鉴定技术体系的构建  

Seperation and Identification Systems for Waxy Protein Originated from Rye(Secale cereale L.) Seeds

作　　者：孟敏[1] 董剑[1,2] 高翔[1,2] 赵万春[1,2] 李晓燕[1,2] 陈其皎[1,2] 陈良国[1,2] 石引刚[1,2] 

机构地区：[1]西北农林科技大学农学院,陕西杨凌712100 [2]陕西省小麦工程技术研究中心/陕西省小麦新品种培育工程研究中心,陕西杨凌712100

出　　处：《中国农业科学》2013年第21期4496-4505,共10页Scientia Agricultura Sinica

基　　金：国家现代农业产业技术体系专项(CARS-3-2-47);国家自然科学基金项目(30900896);中央高校基本科研业务费专项资金项目(QN2009007);西北农林科技大学唐仲英育种基金项目(A212020912)

摘　　要：【目的】构建用于制备级分离及高效鉴定黑麦籽粒Waxy蛋白亚基的技术体系,为该亚基理化特性的分析、功能的发掘及Waxy种质的快速筛选等提供理论参考。【方法】设计特异引物从灌浆期间的奥地利黑麦籽粒cDNA中克隆获得Waxy,并进行体外表达;利用DuoFlow对籽粒蛋白提取物进行纯化,对峰值收集物、原核表达产物及其Ni柱纯化的重组Waxy蛋白进行SDS-PAGE分离,挖取蛋白条带进行MALDI-TOF质谱鉴定,结合经由基因序列所推导的氨基酸序列,确定回收产物的Waxy蛋白属性;在优化电泳条件的基础上构建针对Waxy蛋白的毛细管电泳体系;同时,利用部分黑麦品种对DuoFlow及CE体系的有效性进行验证。【结果】核苷酸序列分析表明,从奥地利黑麦cDNA中克隆获得一条覆盖Waxy全长的编码序列(GenBank登录号:KF559182),其理论编码产物约为60 kD;利用DuoFlow Q1阳离子交换柱,以低浓度Tris-Hcl(20 mmol·L-1Tris-Hcl,pH 9.5)进行蛋白挂柱,高浓度NaCl(20 mmol·L-1Tris+1 mol·L-1NaCl,pH 9.5)进行蛋白洗脱,214 nm波长检测,实现了对籽粒Waxy蛋白的DuoFlow高效分离;纯化产物的SDS-PAGE条带大小、质谱鉴定肽段与黑麦、小麦Waxy蛋白序列相似性的统计结果表明,DuoFlow峰值回收产物具有黑麦Waxy亚基一致的特征序列,故属于Waxy蛋白;以DuoFlow纯化产物为标准样,以0.05 mmol·L-1硼砂+15%乙腈+1%SDS溶液(pH 9.5)为缓冲液,采用分离电压20 kV,温度25℃,检测波长214 nm,压力进样,0.5 psi×5 s,在11—12 min处获得峰值约12 mAU单一主峰,图形清晰,基线水平,从而构建了Waxy蛋白的CE鉴定体系,且籽粒Waxy蛋白提取物能直接用于对该亚基的检测;同时,利用黑麦品种对上述体系的验证结果表明,DuoFlow及CE体系适于对Waxy亚基的高效分离与通量检测。【结论】从奥地利栽培黑麦cDNA文库中克隆获得Waxy完整编码序列,构建了能特异分离黑麦籽粒Waxy蛋白的DuoFlow制备级纯化体系,Abstract： [ Objective ] The purpose of the present investigation was to construct the large-scale isolation and high-throughput identification systems for the Waxy proteins in rye seeds so as to provide more information about how to understand its bio-chemical features, functions and genetic diversity in rye cultivars. [ Method ] Waxy gene was isolated by using a set of gene specific primers from the full-length cDNA library derived from the immature seeds of Austria rye, followed by prokaryotic expression in Escherichiacoli system. The extracted product of the DuoFlow system from Austria rye seeds, together with the purified recombinant Waxy protein by Ni-NTA resin, were confirmed by SDS-PAGE and matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF MS）, and then followed by the alignment of protein sequences which deduced from the Waxy gene originated from Austria rye and common wheat. The above Waxy protein purified by DuoFlow workstation was then employed as the sample to construct the optimal capillary electrophoresis （CE） system for the waxy of rye seeds. Both the DuoFlow isolation system and CE identification system were verified based on the analysis results of rye cultivars. [Result] Sequence analysis revealed that the gene isolated from Austria cDNA （KF559182） encoded a 60 kD mature Waxy protein. The optimal isolation system for DuoFlow Workstation was the following： fixation Waxy proteins using 20 mM Tris-Hcl buffer （pH 9.5）, and then elution the target components using 20 mmol-L1 Tris ＋ 1 mol-L1 NaCI buffer （pH 9.5） from the UNO Q1 anion exchange column. All the above results, including SDS-PAGE, MS identified peptides and seqeunces semilarity of deduced amino acids based on the Waxy sequence, confirmed that the purified products by DuoFlow system in the present study was consistant with the characters of rye Waxy and therefore belongs to the Waxy protein itself. The CE was carried out using the purified protein by DuoFlow system under the co

关 键 词：奥地利栽培黑麦 WAXY蛋白 毛细管电泳 DuoFlow蛋白纯化系统 基质辅助激光解析电离飞行时间质谱 

分 类 号：S512.5[农业科学—作物学]