摘要
类黄酮7-O-甲基转移酶(F7-OMT)具有甲基转移酶功能,能催化底物类黄酮甲基化产生植物抗毒素,提高植物的抗菌性。为了给进一步研究该酶蛋白的功能奠定基础,以青稞"94-19-1"为材料,采用同源克隆方法得到青稞F7-OMT基因编码片段。生物信息学分析表明,青稞F7-OMT开放阅读框(ORF)全长为1173bp,编码390个氨基酸,蛋白分子质量为42 207.8Da,等电点pI为5.36,无信号肽序列,具有甲基转移酶2(Methyltransf_2,pfam00891)和二聚化(Dimerization,pfam08100)保守域。通过亚克隆将F7-OMT连接到表达载体pET-32a上,构建pET-32a-F7-OMT融合表达载体,在E.coli BL21(DE3)pLysS中诱导表达,并采用亲和层析纯化法得到纯化蛋白。
Flavonoid 7-O-methyltransferase （F7-OMT） can produce phytoalexin with the ability of methylating flavonoids for its methyltransferase function so that it can improve the antibacterial property in plant. In order to lay further research foundation for the protein,the objective gene sequence of FT-OMTwas cloned from the hulless barley material ＂94-19-1＂ by using the homology-based cloning technique in this research, bioinformatics analysis showed the full length of open reading frame （ORF） with 1173 bp inFT-OMTthat encoded 390 amino acid residues. The calculated molecular mass of the putative protein without signal peptide was 42207. 8 Da and the isoelectric point was predicted as 5.36. And the gene had Methyltransf_2 and Dimerization domains that were highly conserved, pET- 32a-FZ-OMTfusion expression vector, which could be induced to express in E. coli BL21（DE3）pLysS was constructed. The purified protein was obtained through affinity chromatography purification method.
出处
《麦类作物学报》
CAS
CSCD
北大核心
2016年第1期18-26,共9页
Journal of Triticeae Crops
基金
国家现代农业产业技术体系(大麦青稞体系)建设专项(CARS-05)
关键词
青稞
类黄酮7-O-甲基转移酶
克隆
生物信息学分析
原核表达
Hulless barley
Flavonoid 7-O-methyltransferase
Cloning
Bioinformatics analysis
Prokaryotic expression
