小麦品种陕253新型avenin-like的克隆与原核表达分析  

Cloning and Prokaryotic Expression of an avenin-like from Wheat Variety Shaan 253

作　　者：陈瑞佶[1] 高翔[1,2,3] 陈其皎[1,2,3] 董剑[1,2,3] 赵万春[1,2,3] 李艳亮[1] 王明霞[1] 李敏[1] 庞红喜[1,2,3] 李哲清[1,2,3] 刘俊[1,2,3] 

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,陕西杨凌712100 [3]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》2010年第9期1954-1962,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金青年科学基金项目(30900896);陕西省"13115"科技创新工程重大项目(2007ZDKG-01);现代农业产业技术体系建设专项(NYCYTX-001);西北农林科技大学唐仲英育种基金(A212020912)

摘　　要：【目的】克隆小麦品种陕253中的新型avenin-like(类燕麦储藏蛋白),并构建该基因的原核表达载体,在大肠杆菌中诱导表达融合蛋白。【方法】利用PCR方法从小麦品种陕253中克隆新型avenin-like,将克隆的avenin-like亚克隆至表达载体,经酶切及测序鉴定后,转化宿主菌Escherichia coli Rosetta-gami B(DE3),经IPTG诱导表达融合蛋白,并利用Western blot进行检测。【结果】从小麦品种陕253克隆获得3个新avenin-like,GenBank登录号分别为GQ903577、GQ903578和GQ903579。序列分析表明,其中GQ903579为假基因,利用所构建的大肠杆菌表达载体,经IPTG诱导,实现了对登录号为GQ903578的基因在原核系统的特异性表达。【结论】新型avenin-like的克隆及原核表达的成功,为小麦品质改良提供了研究基础。Objective Molecular cloning and sequence characterization analysis of avenin-like from wheat cultivar Shaan 253, which were then constructed their prokaryotic expression vector and expressed in Escherichia coli Rosetta-gami B （DE3）. Method The novel avenin-like were amplified from wheat variety Shaan 253 by PCR and then cloned into the pMD19-T vector, one of which was subcloned into the expression vector. The recombinant plasmid was identified by sequencing and digestion of restriction enzymes. The fusion protein was finally expressed by IPTG-induction in host bacteria-E. coli Rosetta-gami B （DE3） and detected by Western blot. Result Three novel avenin-like were obtained, and submitted to GenBank, and their accession No. are GQ903577, GQ903578, and GQ903579, respectively. Sequence analysis revealed that GQ903579 was a pseudogene. The gene GQ903578 was successfully expressed in the prokaryotic expression system. Conclusion It could provide a foundation for quality improvement of wheat.

关 键 词：avenin-like 基因克隆 载体构建 融合蛋白 原核表达 

分 类 号：S512.1[农业科学—作物学]