日本蛇根草DFR3基因的克隆分析及其编码蛋白的分离纯化

Cloning and Analysis of DFR3 Gene from Ophiorrhiza japonicus(OjDFR3)and Isolation and Purification of Its Coding Protein

二氢黄酮醇4-还原酶(DFR)作为花色苷代谢途径下游的关键酶,对植物花色苷的合成具有重要调控作用。该研究以日本蛇根草(Ophiorrhiza japonica)为材料,采用RT-PCR方法克隆获得一个DFR基因(OjDFR3),利用生物信息学方法对OjDFR3蛋白的性质进行了分析,通过实验完成该基因原核表达载体的构建及其重组蛋白的制备与纯化,为深入揭示日本蛇根草DFR基因的功能以及花色苷的合成与调控研究奠定基础。结果表明:(1)成功克隆获得一个DFR基因(OjDFR3);序列分析显示,OjDFR3基因cDNA全长为1071 bp,可编码356个氨基酸,蛋白分子量为39.52 kD。(2)生物信息学分析表明,OjDFR3基因编码形成的蛋白由20种氨基酸组成,其中亮氨酸含量最多,不存在信号肽,是一种亲水性蛋白,定位在细胞质的可能性最大,三级结构由α螺旋、延伸链、无规则卷曲组成。(3)原核表达分析显示,重组质粒pET32a-OjDFR3可在大肠杆菌BL21(DE3)中表达,其最佳诱导表达条件为37℃、4 h、IPTG浓度为0.8 mmol/L,同时在100和200 mmol/L咪唑洗脱下的蛋白纯度最好。(4)按照上述最佳条件,制备并获得了大量浓度和纯度较好的蛋白。

As a key enzyme downstream of anthocyanin metabolism pathway,dihydroflavonol 4-reductase plays an important role in regulating anthocyanin synthesis.In this study,a DFR gene was successfully cloned by RT-PCR method with Ophiorrhiza japonica as materials.Subsequently,the properties of OjDFR3 protein were analyzed through bioinformatics methods.Meanwhile,the prokaryotic expression vector of OjDFR3 was constructed and its recombinant protein was prepared and purified which would lay a foundation for the further study on OjDFR3 function as well as the synthesis and regulation of anthocyanins in O.japonica.The results showed that:(1)a DFR gene was successfully cloned(OjDFR3),and sequence analysis displayed that the full-length cDNA of OjDFR3 was 1071 bp,encoding 356 amino acids,and the putative protein molecular weight was 39.52 kD;(2)Bioinformatics analysis showed that the protein encoded by OjDFR3 gene was composed of 20 kinds of amino acids,of which leucine was the most.OjDFR3 was a hydrophilic protein without signal peptide,and was likely located in cytoplasm.Simultaneously,structure prediction showed that OjDFR3 was composed ofα-helix,extended chain and irregular coil;(3)Prokaryotic expression analysis exhibited that the recombinant plasmid pET32a-OjDFR3 could be expressed in E.coli BL21(DE3),and the optimal expression conditions were 37℃,4 h,IPTG concentration of 0.8 mmol/L.Meanwhile,the purity of recombinant protein was best at 100 mmol/L and 200 mmol/L imidazole concentrations.(4)According to the optimal conditions,a large number of proteins with good concentration and purity were obtained.In conclusions,the results of this study will lay a foundation for further study on the function of this gene as well as the synthesis and regulation of anthocyanin in O.japonica.