摘要
本研究对菘蓝CYP83A1基因进行了克隆、表达特性及原核表达研究。结果表明,IiCYP83A1基因组DNA全长为1 622 bp,包含2个外显子和1个内含子;cDNA全长为1 512 bp,编码503个氨基酸。IiCYP83A1编码的蛋白包含2个跨膜结构域,无信号肽,主要定位于内质网膜,属于亲水性蛋白,二级结构主要由α-螺旋和无规则卷曲组成,与萝卜、欧洲油菜、西兰花和芜菁等植物的CYP83A1蛋白具有较高的同源性。qRT-PCR结果表明,IiCYP83A1在不同组织中的表达具有显著性差异,表现为茎〉叶〉果〉花和根;在幼苗期、生长期和花期的表达量显著高于萌芽期;茉莉酸甲酯（MeJA）和伤害处理能够显著促进该基因的表达,而低温（4℃）和水杨酸（SA）处理则对其表达具有一定的抑制作用。将克隆到的IiCYP83A1基因连接到表达载体pET-28a上,构建原核表达载体pET-28a-IiCYP83A1并对其进行原核表达。SDS-PAGE结果显示,在E.coli BL21中成功诱导表达了CYP83A1蛋白,与预期蛋白分子量大小一致。本研究结果可为进一步研究IiCYP83A1的功能提供实验依据。
This research analyzed cloning, expression characteristics and prokaryotic expression of CYP83 A1 gene from Isatis indigotica. The results showed that the genomicDNA of IiCYP83 A1 was 1 622 bp in length, and contained 2 exons and 1 intron. Its cDNA was 1 512 bp in length which coded a protein of 503 amino acids.IiCYP83 A1 was a hydrophilic protein located in the endoplasmic reticulum with two transmembrane domains,with no signal peptide. Its secondary structure mainly included alpha helix and irregular curly. Homologous comparison illustrated that IiCYP83 A1 had close relationships with Raphanus sativus L., Brassica napus L.,Brassica oleracea var. and Brassica rapa L.. The result of q RT-PCR showed that expression of IiCYP83 A1 was significantly different in different tissues, ranking as stem leaf fruit root and flower. Moreover, it was more highly expressed in the seedling, vegetative growth and flowering stage, compared with the germination period.Gene expression of IiCYP83 A1 could be induced significantly by methyl jasmonate（MeJA） and wounding treatments, but repressed by low temperature（4℃） and salicylic acid（SA）. The cloned IiCYP83 A1 gene was ligated to pET-28 a then the prokaryotic expression vector of pET-28 a-IiCYP83 A1 was constructed for expressing.SDS-PAGE result showed the target protein could be well expressed by the induction of E. coli BL21 and was consistent with the expected size. The obtained result in this study might provide some effective references for the further functional study on IiCYP83 A1.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2018年第1期302-312,共11页
Genomics and Applied Biology
基金
国家自然科学基金项目(31200221)
陕西省自然科学基金项目(2016JQ3007)
陕西师范大学研究生培养创新基金(2015CXS025)共同资助
关键词
菘蓝
CYP83A1
基因克隆
表达特性
原核表达
Isatis indigotica Fort., CYP83A 1, Gene clone, Expression characteristics, Prokaryotic expression
