摘要
超氧化物歧化酶(Superoxide dismutase,SOD)是逆境条件下清除细胞内活性氧的关键酶,而铁型超氧化物歧化酶(FeSOD)作为该酶系中的关键酶之一,与植物的抗病性关系密切。根据板栗(Castanea mollissimaBl)转录组数据中分析得到FeSOD基因EST序列设计PCR扩增引物,以板栗幼叶的cDNA为模板,采用RT-PCR技术克隆获得CmFeSOD基因cDNA序列,通过生物信息学方法分析该基因的cDNA序列,并推定其氨基酸序列,同时将该基因片段连接到原核表达载体pET28a中,转化大肠杆菌BL21(DE3)并进行不同条件的诱导表达。结果表明,板栗CmFeSOD基因开放阅读框(ORF)大小为705bp,共编码234个氨基酸,推测其蛋白分子质量为26.0165ku,理论等电点(pI)为6.86,具有FeSOD家族的特征基序和保守结构域,GenBank登录号为KY312852。遗传进化分析表明,板栗CmFeSOD与核桃的亲缘关系最近。SDS-PAGE电泳分析表明,通过原核表达获得CmFeSOD蛋白的分子质量约为29ku,CmFeSOD蛋白在30℃,添加0.4mmol/LIPTG,诱导6h表达量最高,主要以包涵体的形式存在。
Uperoxide dismutase(SOD)is a key enzyme in removal of intracellular reactive oxygen species under stress conditions.Iron-containing superoxide dismutase(FeSOD)is one of the key enzymes in this kind of enzyme system,which is closely related to plant disease resistance.Based on EST sequences derived from the FeSOD gene in the transcriptome database of the Castanea mollissima Bl.,the primers used for PCR amplification of the gene were designed.cDNA clones of CmFeSOD gene in C.mollissima Bl.were conducted by RT-PCR using cDNA of young leaves and then we obtained complete sequence.According to the information of cDNA sequence of CmFeSOD gene,putative amino acid sequence of the gene was performed via to bioinformatic analysis.Segment of the gene was inserted into prokaryotic expression vector pET28a and transformed into Escherichia coli BL21(DE3).Induced expressions of the gene were determined under different conditions.The results showed that CmFeSOD gene contained a 705 bp-sized open reading frame(ORF),which 234 amino acids were encoded residues with a predicted molecular weight of 26.016 5 ku and a theoretical isoelectric point of 6.86,characterizing a motif and conserved domain of the FeSOD family.The GenBank accession number of the gene is KY312852.Genetic evolution analysis showed that the CmFeSOD in C.mollissima Bl.has the closest relationship with that of Juglans regia L..SDS-PAGE analysis indicated that the molecular weight of the CmFeSOD protein produced by prokaryotic expression was approximately 29 ku and the optimal expression condition of the CmFeSOD protein was IPTG of a concentration of 0.4 mmol/L at 30℃for 6 h with existence of inclusion body as main form.In this study,the CmFeSOD gene in C.mollissima BL was cloned and expressed in a high level in E.coli transformation system,providing a basis for further study on biological function of the gene.
作者
韩珊
刘裕峰
朱天辉
刘应高
谯天敏
李姝江
汪煜伶
徐缨络
莫义英
HAN Shan;LIU Yufeng;ZHU Tianhui;LIU Yinggao;QIAO Tianmin;LI Shujiang;WANG Yuling;XU Yingluo;MO Yiying(College of Forestry,Sichuan Agricultural University,Chengdu611130,China)
出处
《西北农业学报》
CAS
CSCD
北大核心
2019年第6期935-944,共10页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家自然科学基金(31400547)
四川省科技计划(19ZDYF2270)~~
关键词
板栗
超氧化物歧化酶
基因克隆
原核表达
Castanea mollissima Bl
Superoxide dismutase(SOD)
Gene cloning
Prokaryotic expression
