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Gene cloning and target gene identification of transcription factor SmERF081 from
Salvia miltiorrhiza - Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 38, Issue 3, Pages: 7-14(2024)
- 作者机构：
1.上海中医药大学中药研究所，国家中医药管理局中药新资源与品质评价重点研究室，教育部中药标准化重点研究室，上海市复方中药重点实验室（上海 201203）
2.上海中医药大学中药学院（上海 201203）
- 作者简介：
- 基金信息：
DOI：10.16306/j.1008-861x.2024.03.002
CLC：Published：25 May 2024，
Received：28 April 2023，
Revised：15 April 2024，
扫 描 看 全 文
肖亦菽,陈敏,谈荣慧等.丹参SmERF081转录因子基因克隆与靶基因鉴定[J].上海中医药大学学报,2024,38(03):7-14.
XIAO Yishu,CHEN Min,TAN Ronghui,et al.Gene cloning and target gene identification of transcription factor SmERF081 from Salvia miltiorrhiza[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2024,38(03):7-14.
肖亦菽,陈敏,谈荣慧等.丹参SmERF081转录因子基因克隆与靶基因鉴定[J].上海中医药大学学报,2024,38(03):7-14. DOI： 10.16306/j.1008-861x.2024.03.002.
XIAO Yishu,CHEN Min,TAN Ronghui,et al.Gene cloning and target gene identification of transcription factor SmERF081 from Salvia miltiorrhiza[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2024,38(03):7-14. DOI： 10.16306/j.1008-861x.2024.03.002.
摘要
目的
2
克隆丹参（
Salvia miltiorrhiza
Bge.） 转录因子
SmERF081
，分析
SmERF081
的靶基因。
方法
2
以丹参转录组数据Unigene（c48961-g1）序列为参考，利用PCR扩增获得
SmERF081
基因序列；通过生物信息学预测
SmERF081
编码蛋白的理化性质、蛋白质结构等分子特征；使用MEGA 7软件构建系统进化树；对
SmERF081
进行异源表达和蛋白纯化；通过酵母单杂、双荧光素酶报告系统和化学发光法凝胶迁移实验（EMSA）鉴定
SmERF081
与
SmCPS5
启动子是否结合。
结果
2
克隆得到
SmERF081
基因，cDNA全长453 bp（Genbank登陆号：OQ466089），编码151个氨基酸，相对分子质量16.46 kDa，等电点9.75。该蛋白不含信号肽，无跨膜区，其高级结构主要由无规则卷曲构成。进化树分析表明
SmERF081
与拟南芥At1G28360.1、丹参SmERF8同源关系最近。原核表达结果显示，经IPTG诱导后
SmERF081
在大肠杆菌中成功异源表达，并通过纯化获得目的蛋白。酵母单杂、双荧光素酶报告基因检测和EMSA确认
SmERF081
可与丹参
SmCPS5
启动子结合。
结论
2
鉴定到丹参中一个新转录因子
SmERF081
，生物信息学分析及分子互作初步证实其靶基因为丹参
SmCPS5
二萜环化酶。
Abstract
Objective： To clone the transcription factor
SmERF081
from
Salvia miltiorrhiza
Bunge and analyze its target gene.
Methods
2
Using the UniGene （c48961-g1） sequence of
S.
miltiorrhiza
transcriptome data as reference， the
SmERF081
gene sequence was obtained by PCR amplification. Bioinformatics was used to predict the physicochemical properties， protein structure， and other molecular characteristics of the protein encoded by
SmERF081
. MEGA 7 was used to build the phylogenetic tree. Heterologous expression and protein purification on
SmERF081
were performed. Whether
SmERF081
binds to the promoter of
SmCPS5
（
copalyl diphosphate synthase 5， CPS5
）was identified by yeast one-hybrid system， dual-luciferase reporter system and EMSA.
Results
2
The
SmERF081
gene was cloned， with a total length of cDNA 453 bp （GenBank accession number： OQ466089）， encoding 151 amino acids， the relative molecular mass of 16.46 kDa， and the isoelectric point of 9.75. The protein contains no signal peptide and no transmembrane region， and its higher-order structure is mainly composed of irregular curls. Phylogenetic tree analysis showed that
SmERF081
had the closest homology with
Arabidopsis
At1G28360.1 and SmERF8. The results of Prokaryotic expression showed that
SmERF081
induced by IPTG was successfully heterologously expressed in
Escherichia coli
and the target protein was obtained by purification.Yeast one-hybrid system， dual-luciferase reporter gene assay and EMSA verified that
SmERF081
could bind to the promoter of
SmCPS5
.
Conclusion
2
A new transcription factor SmERF081 in
S. miltiorrhiza
was identified. Bioinformatics analysis and molecular interactions preliminarily confirmed its target gene was
SmCPS5
diterpene cyclase.
关键词
丹参SmERF081生物信息学靶基因
Keywords
Salvia miltiorrhizaSmERF081bioinformaticstarget gene analysis
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