Full-length Cloning and Protein Expression Analysis of Glycosyltransferases Gene SaUGT1/SaUGT2 in Sorbus aucuparia

Jia-xing LI; Ge MO; Liang-yun ZHOU; Ya-hui LIU; Jing-yi JIANG; Yu-ping TAN; Jin-fu TANG; Lan-ping GUO

doi:10.13422/j.cnki.syfjx.20190514

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Full-length Cloning and Protein Expression Analysis of Glycosyltransferases Gene SaUGT1/SaUGT2 in Sorbus aucuparia

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- Full-length Cloning and Protein Expression Analysis of Glycosyltransferases Gene SaUGT1/SaUGT2 in Sorbus aucuparia
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 5, Pages: 167-172(2019)
- 作者机构：
  
  1.广东药科大学　中药学院，广州　 510006；
  2.中国中医科学院　中药资源中心，道地药材国家重点实验室培育基地，北京　 100700；
  3.西藏藏医学院，拉萨　 850000
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20190514
  CLC： R284.1;R289;R22;R2-03
- Received：19 July 2018，
  
  Published Online：21 November 2018，
  
  Published：05 March 2019
- 稿件说明：
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Jia-xing LI, Ge MO, Liang-yun ZHOU, et al. Full-length Cloning and Protein Expression Analysis of Glycosyltransferases Gene SaUGT1/SaUGT2 in Sorbus aucuparia[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(5): 167-172.
DOI：

Jia-xing LI, Ge MO, Liang-yun ZHOU, et al. Full-length Cloning and Protein Expression Analysis of Glycosyltransferases Gene SaUGT1/SaUGT2 in Sorbus aucuparia[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(5): 167-172. DOI： 10.13422/j.cnki.syfjx.20190514.

摘要

目的：

研究欧洲花楸植保素糖基化修饰的相关基因，从欧洲花楸悬浮细胞中克隆糖基转移酶(glycosyltransferase

GT)基因，进行序列分析和原核表达。

方法：

基于欧洲花楸转录组数据设计特异性引物，克隆得到2条

SaUGTs

基因的cDNA序列，构建HIS-MBP-pET28a-SaUGTs原核表达载体，诱导表达SaUGTs重组蛋白。

结果：

克隆得到2条糖基转移酶基因

SaUGT

1和

SaUGT

2序列，完整开放阅读框为1 458bp和1 431bp，分别编码485和476个氨基酸，相对分子质量为54.27kDa和53.49kDa，理论等电点为5.50和5.63。生物信息学分析显示，无信号肽，含有糖基转移酶家族保守结构域(PSPG)。系统进化结果显示SaUGT1和SaUGT2蛋白与拟南芥UGT85家族亲缘关系较近。实时荧光定量PCR检测显示，欧洲花楸悬浮细胞经酵母提取物(YE)诱导后，

SaUGT

1和

SaUGT

2的相对表达量明显上调，分别在24h和12h达到最大值。通过IPTG诱导，在大肠埃希菌中成功表达SaUGT1和SaUGT2重组蛋白，并得到了纯化的重组蛋白。

结论：

该研究首次在欧洲花楸中克隆得到糖基转移酶基因，并成功构建了原核表达载体，为进一步研究该类基因的功能奠定了基础。

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:

SaUGT

1 and

SaUGT

2 sequences were cloned and obtained from glycosyltransferases

then bioinformatic analysis of the sequence and prokaryotic expression analysis were conducted.

SaUGT

1gene contained 1 458bp open reading frame (ORF)

encoding a polypeptide of 485 amino acids

with a relativemolecular weight of 54.27kDa and theoretical isoelectric point (pI) of 5.50.

SaUGT

2gene contained 1 431bp ORF

encoding a polypeptide of 476 amino acids

with a relativemolecular weight of 53.49kDa 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

thaliana

. Differential expression analysis revealed that the relative expression levels of

SaUGT

1 and

SaUGT

2 were increased significantly after being induced by yeast extract (YE)

with the highest expression level found at 24h and 12h. 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 Ni

affinity chromatography.

Conclusion:

The glycosyltransferase gene was cloned from the

aucuparia

for the first time

and the prokaryotic expression vector was successfully constructed

laying foundation for further study of the function of this gene.

关键词

Keywords

references

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