地黄糖基转移酶基因克隆表达及表达分析

Cloning and Expression Analysis of RgUGT Gene in Rehmannia glutinosa

目的:从地黄中克隆尿苷二磷酸糖基转移酶(uridine diphosphate glycosyltransferase,UGT)基因,构建其原核表达体系,为后续分析验证其功能奠定基础。方法:在转录组测序的基础上,通过RT-PCR克隆全长cDNA序列。运用生物信息学的方法对该序列进行分析。通过荧光定量PCR检测该基因在不同品种地黄不同部位中的表达情况。构建重组表达载体pET-32a-RgUGT,并转化大肠杆菌BL21,获得重组蛋白。结果:克隆得到RgUGT基因长度为1 374 bp,编码457个氨基酸,RgUGT基因具有尿嘧啶二磷酸-糖基转移酶PSPG盒子,荧光定量PCR结果显示RgUGT基因在根和叶中的表达水平较高。该基因成功在大肠杆菌BL21细胞中表达。结论:该研究从地黄中克隆获得了糖基转移酶基因,可为进一步研究其在地黄次生代谢生物合成中的功能奠定基础。

Objective:To clone the uridine diphosphate glycosyltransferase(UGT)gene in Rehmannia glutinosa,and to construct the prokaryotic expression system in order to provide the foundation for its further function verification.Methods:RT-PCR was used to clone the full-length cDNA of RgUGT based on the transcriptome dataset of Rehmannia glutinosa.The physical and chemical properties and secondary structure of the RgUGT protein were analyzed.And the expression profiles under different tissues were analyzed by QPCR.The recombinant plasmid pET-32 a-RgUGT was expressed in a prokaryotic expression system after they were transformed into Escherichia coli BL21.Results:The full length of ORF was 1 374 bp,encoding 457 amino acids.The PSPG motif domain of glycosyltransferases was presented in RgUGT.PCR analysis indicated that RgUGT had the higher transcript abundance in the roots and leaves.The recombinant RgUGT protein was successfully expressed in Escherichia coli BL21 cells.Conclusion:The RgUGT gene is isolated from Rehmannia glutinosa.The results of this study provide a scientific basis for functional characterization of RgUGT gene involved in secondary metabolic biosynthesis of Rehmannia glutinosa.