中国农业科学2012，Vol.45Issue(10)：2076-2084,9.DOI:10.3864/j.issn.0578-1752.2012.10.021
小麦胚乳14-3-3基因的克隆及其重组蛋白的原核表达
Cloning of a 14-3-3 Gene from Developing Wheat Endosperm and Expression of its Recombinant Protein in Escherichia coli
摘要
Abstract
[Objective] This research was conducted to clone 14-3-3 genes from developing wheat endosperm and express their recombinant proteins in Escherichia coli aiming to investigate their functions in wheat development [ Method 1 Specific primers with restriction enzymes cut site were designed according to conserved sequence of homologous genes registered in NCBI. The target genes were cloned by RT-PCR from filling wheat grain. Then the cloned genes were inserted into expressing vectors after confirmation by sequencing and multiple alignments. The recombinant protein was expressed in E. coli and purified for further research. [Result] A 14-3-3 gene was amplified from developing endosperm of 13-15 d after anthesis of bread wheat cultivar Jimai22 and inserted into Top plasmid vector then transformed into E. coli strain DH5α by heat shock. The cloned gene was sequenced after the recombinant plasmid was extracted. The results of sequencing analysis showed that the gene belongs to nonε group and contained an open reading frame of 777 bp in length encoding a protein with 259 aa with predicted molecular weight about 29 kD. According to multiple alignments using DNAMAN program, the gene was highly homologous to other 14-3-3 genes from main crops such as wheat rice, maize, barley, soybean and model plant Arabidopsis with the maximum homology of 98%, as well as their encoding proteins. Furthermore, the heterologous protein with molecular weight about 30 kD expressed in E. coli was coincident with predicted size based on deduced amino acid sequence. All results suggested that the cloned gene is a 14-3-3 gene and it was correctly inserted into the vector and expressed heterologously. The cloned gene was inserted into expressing vector pET29c possessing a S-tag specific bounding to S-protein agarose. The recombinant vector was transformed into E. coli strain BL21-CodonPlus (DE3)-RP supplied with additional rare codes to improve heterologous expression. The recombinant protein was expressed at very high level, however, existed mainly as an insoluble inclusion body (about 80%) after extraction by BugBuster Protein Extraction Reagent. The soluble fusion protein was purified directly by bounding to S-protein agarose followed by washing out of non-specific bounding proteins and other impurities, while the inclusion body should be dissolved in 8 mol L-1 urea and refolded firstly. [ Conclusion ]A 14-3-3 gene was cloned from developing wheat endosperm through RT-PCR and heterologously expressed at high level in E. coli. Purified 14-3-3 recombinant protein with bioactivity was harvested for further study by purification with S-protein agarose.关键词
小麦/14-3-3蛋白/基因克隆/重组蛋白/原核表达Key words
wheat (Triticum aestivum L.)/ 14-3-3 protein/ gene clone/ recombinant protein/ heterologous expression引用本文复制引用
戴双,李豪圣,程敦公,刘爱峰,曹新有,刘建军,宋健民..小麦胚乳14-3-3基因的克隆及其重组蛋白的原核表达[J].中国农业科学,2012,45(10):2076-2084,9.基金项目
国家自然科学基金(30700492、31171551)、现代农业产业体系建设专项(CARS-03-1-8)、转基因生物新品种培育科技重大专项(2011ZX08002-003)、国家科技支撑计划(2011BAD07B02-3、2011BAD35B03)、山东省农业良种产业化工程项目 （30700492、31171551）
