Full-length Cloning and Protein Expression Analysis of Glycosyltransferases Gene SaUGT1/SaUGT2 in Sorbus aucuparia
10.13422/j.cnki.syfjx.20190514
- VernacularTitle: 欧洲花楸糖基转移酶基因的全长克隆与表达分析
- Author:
Jia-xing LI
1
;
Ge MO
2
;
Liang-yun ZHOU
1
;
Ya-hui LIU
3
;
Jing-yi JIANG
3
;
Yu-ping TAN
1
;
Jin-fu TANG
3
;
Lan-ping GUO
1
Author Information
1. School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China
2. Tibetan Traditional Medical College, Lhasa 850000, China
3. State Key Laboratory of Dao-di Herbs Breeding Base, National Resources Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700
- Publication Type:Research Article
- Keywords:
Sorbus aucuparia;
glycosyltransferases;
full-length cDNA cloning;
expression analysis
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2019;25(5):167-172
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To obtain the glycosyltransferase gene involved in modification reaction of phytoalexin from Sorbus pohuashanensis suspension cell,and conduct sequence analysis and prokaryotic expression analysis. Method: Based on the transcriptome data,specific primers were designed to obtain 2 cDNA sequences of SaUGTs genes,construct prokaryotic expression vector HIS-MBP-pET28a-SaUGTs and induce the expression of recombinant SaUGTs protein. Result: SaUGT1 and SaUGT2 sequences were cloned and obtained from glycosyltransferases,then bioinformatic analysis of the sequence and prokaryotic expression analysis were conducted. SaUGT1 gene contained 1 458 bp open reading frame (ORF),encoding a polypeptide of 485 amino acids,with a relative molecular weight of 54.27 kDa and theoretical isoelectric point (pI) of 5.50.SaUGT2 gene contained 1 431 bp ORF,encoding a polypeptide of 476 amino acids,with a relative molecular weight of 53.49 kDa and theoretical pI of 5.63. Bioinformatics analysis indicated that SaUGT1 and SaUGT2 protein had no signal peptide,and the conserved domains of glycosyltransferase family were detected. Phylogenetic results showed that SaUGT1 and SaUGT2 proteins had the closest relationship with the UGT85 family of A. thaliana. Differential expression analysis revealed that the relative expression levels of SaUGT1 and SaUGT2 were increased significantly after being induced by yeast extract (YE), with the highest expression level found at 24 h and 12 h. The recombinant SaUGT1 and SaUGT2 proteins were successfully expressed in Escherichia coli DE3 cells and finally,the recombinant SaUGT1 and SaUGT2 proteins were purified through Ni2+ affinity chromatography. Conclusion: The glycosyltransferase gene was cloned from the S. aucuparia for the first time,and the prokaryotic expression vector was successfully constructed,laying foundation for further study of the function of this gene.
