水稻逆境响应蛋白OsSGL的生物信息学分析及原核表达条件优化  

Bioinformatics Analysis and Optimization of Protein Expression in Prokaryotic System of Rice Stress Responsive Protein OsSGL

作　　者：周炎[1] 樊帆[1] 雷东阳[1] 卢学丹 Zhou Yan;Fan Fan;Lei Dongyang;Lu Xuedan(College of Agronomy,Hunan Agricultural University,Changsha,410128)

机构地区：[1]湖南农业大学农学院,长沙410128

出　　处：《分子植物育种》2021年第5期1531-1540,共10页Molecular Plant Breeding

基　　金：湖南农业大学南方粮油作用协同创新中心水稻种质资源团队项目科研经费(30552520190100005);湖南省自然科学基金(2019JJ50228);湖南省教育厅科学研究项目(18A110)共同资助。

摘　　要：水稻OsSGL(Oryza sativa stress tolerance and grain length)基因表达响应多种非生物逆境,参与调控水稻产量及耐旱性。该基因CDS全长768 bp,编码一个包含DUF1645(Domain of unknown function protein family 1645)结构域的功能蛋白。生物信息学分析预测显示该蛋白分子量为26.73 kD,理论等电点为9.35,为亲水性蛋白且没有跨膜区域,蛋白二级结构中α螺旋占15.69%,β转角占3.14%,延伸链占14.51%,无规则卷曲占66.67%。OsSGL基因CDS序列中稀有密码子的比例高达29.69%,且有多个串联稀有密码子。为进一步在生化水平研究OsSGL蛋白,本研究拟在原核表达系统中大量表达、纯化并鉴定His标签融合表达的Os-SGL蛋白。在不改变氨基酸序列的前提下,通过全基因合成技术,根据大肠杆菌密码子偏好性对OsSGL CDS序列进行优化、合成并连接到pET-32a表达载体中;然后将重组质粒pET-32a-OsSGL转化大肠杆菌,经体外表达条件优化大量合成融合His标签表达的OsSGL蛋白。结果显示,OsSGL基因在大肠杆菌中可实现诱导表达,融合蛋白分子质量约为45.7 kD。最优诱导表达温度、时间、IPTG浓度和摇床转速分别为16℃、16 h、0.5 mmol/L和120 r/min。利用His抗体进行Western blotting进一步检测到纯化的His-OsSGL融合蛋白。OsSGL蛋白体外表达体系的建立有利于在生化水平上深入解析OsSGL参与的信号调控通路。The expression of rice OsSGL(Oryza sativa stress tolerance and grain length)gene responds to various stresses and is involved in the regulation of rice yield and drought tolerance.Its CDS is 768 bp,encoding a functional protein containing DUF1645 domain.Bioinformatics analysis showed that the molecular weight of OsSGL protein was 26.73 kD,the theoretical isoelectric point was 9.35,the protein was hydrophilic and had no transmembrane region.In the secondary structure of the protein,αhelix,βturn,extended strand and random coil accounted for 15.69%,3.14%,14.51%,and 66.67%,respectively.The proportion of rare codons in the CDS is as high as 29.69%with multiple tandem rare codons.In order to further study OsSGL protein at the biochemical level,protein fused with His tag was expressed and purified in the prokaryotic expression system.Without changing the amino acid sequence,the OsSGL CDS sequence was optimized,synthesized and connected to pET32a expression vector by the whole gene synthesis technology according to the codon preference of E.coli.The recombinant plasmid pet-32a-OsSGL was transformed into E.coli,and a large number of OsSGL proteins expressed by His-tag were synthesized through optimized in vitro expression conditions.It was shown that OsSGL could be induced and expressed in E.%coli,and the molecular weight of the fusion protein was about 45.7 kD.The optimal induction temperature,time,IPTG concentration and shaking table speed were 16℃,16 h,0.5 mmol/L and 120 r/min,respectively.The purified His-OsSGL fusion protein was further detected by Western blotting with His antibody.This study with establishment of OsSGL protein expression system in vitro is conducive to further analysis of signal regulation pathway in which OsSGL involves at biochemical level.

关 键 词：OsSGL 稀有密码子 原核表达系统 条件优化 生物信息学分析 

分 类 号：S511[农业科学—作物学]