Cloning and Transcriptional Activity Analysis of Endogenous U6 Promoters in Artemisia annua
10.13422/j.cnki.syfjx.20251417
- VernacularTitle:黄花蒿U6启动子克隆及转录活性分析
- Author:
Yuting PU
1
;
Bohan CHENG
1
;
Mengyue WANG
1
;
Jun ZOU
1
;
Ranran GAO
1
;
Lan WU
1
;
Qinggang YIN
1
;
Li XIANG
1
;
Yuhua SHI
1
Author Information
1. State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs，Artemisinin Research Center，Institute of Chinese Materia Medica，China Academy of Chinese Medical Sciences， Beijing 100700，China
- Publication Type:Journal Article
- Keywords:
Artemisia annua;
U6 promoter;
transcriptional activity
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2025;31(24):161-167
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveThe U6 promoter is an essential element for driving sgRNA expression in the clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9（CRISPR/Cas9）gene editing system in dicotyledonous plants. Endogenous U6 promoters typically exhibit higher transcriptional activity， which can significantly improve gene editing efficiency. This study aims to identify endogenous U6 promoters in Artemisia annua to optimize its CRISPR/Cas9 gene editing system， which holds significant importance for its molecular breeding. MethodsOn the basis of the highly conserved U6 snRNA sequences in Arabidopsis thaliana， endogenous U6 promoters were screened in the A. annua genome. Expression vectors were constructed with candidate AaU6 promoter driving the firefly luciferase （LUC） reporter gene， and then transiently transformed into Nicotiana benthamiana. Transcriptional activities of the promoters were measured and compared by in vivo imaging and the Dual Luciferase Reporter assay. ResultsEight endogenous U6 promoters were successfully cloned from A. annua. Sequences alignment revealed that all these promoters contained the two conserved cis-acting elements， upstream sequence element （USE） and TATA-box， which affected their transcriptional activity. Dual-luciferase activity assays indicated that the transcriptional activities of AaU6-3， AaU6-1， and AaU6-5 were significantly higher than that of the Arabidopsis AtU6-26 promoter， with AaU6-3 exhibiting the highest activity. ConclusionThis study identified three endogenous AaU6 promoters with high transcriptional activity in A. annua， providing key functional elements for establishing an efficient gene editing system in A. annua. These findings will contribute to advancing precision molecular breeding and high-quality germplasm innovation in A. annua.
