金银花U6启动子的克隆及转录活性分析

doi:10.3976/j.issn.1002-4026.2022.02.002

山东科学 ›› 2022, Vol. 35 ›› Issue (2): 11-17.doi: 10.3976/j.issn.1002-4026.2022.02.002

金银花U6启动子的克隆及转录活性分析

唐志强¹(),李小丽¹,许小涵¹,蒲高斌^1,^2,^*()

1.山东中医药大学,山东济南 250355
2.山东省中药质量控制与全产业链建设协同创新中心,山东济南 250355

收稿日期:2021-07-10 出版日期:2022-04-20 发布日期:2022-04-07
通信作者: 蒲高斌 E-mail:tzq15194181751@163.com;gbpu@163.com
作者简介:唐志强(1996—),男,硕士研究生,研究方向为中药资源。E-mail: tzq15194181751@163.com
基金资助:
国家自然科学基金(81872963);中央本级重大增减支项目(2060302);山东省高等学校优秀青年创新团队支持计划(2019 KJE004);山东省重大科技创新工程(2019JZZY011020)

Analysis of the cloning and transcriptional activity of U6 promoter from Lonicera japonica

TANG Zhi-qiang¹(),LI Xiao-li¹,XU Xiao-han¹,PU Gao-bin^1,^2,^*()

1. Shandong University of Traditional Chinese Medicine, Jinan 250355,China
2. Shandong Provincial Collaborative Innovation Center for Quality Control and Construction of the Whole Industrial Chain of Traditional Chinese Medicine, Jinan 250355, China

Received:2021-07-10 Online:2022-04-20 Published:2022-04-07
Contact: Gao-bin PU E-mail:tzq15194181751@163.com;gbpu@163.com

摘要/Abstract

摘要：

U6启动子是构建CRISPR/Cas9体系中驱动sgRNA转录的重要元件。利用聚合酶链式反应方法,从华金6号金银花中克隆到3种U6启动子,并分别构建5个不同长度的启动子驱动GUS的植物融合表达载体。以CaMV35S作为阳性对照实验,农杆菌作为阴性对照实验,采用农杆菌瞬时转化法对烟草原生质体进行转染,GUS染色后置于显微镜下观察原生质体的染色数量和效率。根据染色结果,成功筛选出Chr01 U6-F1这一高效转录的启动子,为后期开展金银花的基因编辑奠定了基础。

关键词: 金银花, U6启动子, 克隆, 基因编辑

Abstract:

U6 promoter is an important element that drives sgRNA transcription in the construction of the CRISPR/Cas9 system. In this study, three types of U6 promoters were cloned from Huajin 6 Lonicera japonica using polymerase chain reaction(PCR), and five promoters of different lengths were constructed to drive the GUS expression vector. CaMV35S was used in a positive control experiment, and Agrobacterium tumefaciens was used in a negative control experiment. Tobacco protoplasts were transfected through the transient transformation method using A. tumefaciens. After GUS staining, the quantity and efficiency of protoplasts were observed under a microscope. The staining results revealed that the highly efficient transcription promoter Chr01 U6-F1 was screened successfully, which laid a foundation for the gene editing of Lonicera japonica in the later stage.

Key words: Lonicera japonica, U6 promoter, clone, gene editing

中图分类号:

唐志强,李小丽,许小涵,蒲高斌. 金银花U6启动子的克隆及转录活性分析[J]. 山东科学, 2022, 35(2): 11-17.

TANG Zhi-qiang,LI Xiao-li,XU Xiao-han,PU Gao-bin. Analysis of the cloning and transcriptional activity of U6 promoter from Lonicera japonica[J]. Shandong Science, 2022, 35(2): 11-17.

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引物名称	正向引物序列	反向引物序列
Chr01 U6-F1	AAGCTTGAAACTAATGTGTATAT	GGATCCGCTTAATTTCCCACCC
Chr02 U61-F1	AAGCTTTCAGGTGGGATTAAAAT	GGATCCTGTCTTATTGTTGTGCG
Chr02 U61-F2	AAGCTTGAGTGTTCGTATCAGTT	GGATCCTGTCTTATTGTTGTGCG
Chr02 U62-F1	AAGCTTTATGTTTGAACTTGATT	GGATCCTACATTGTTGTTGTCCG
Chr02 U62-F2	AAGCTTCTCACATGGGAACTCTA	GGATCCTACATTGTTGTTGTCCG

金银花U6启动子的克隆及转录活性分析

Analysis of the cloning and transcriptional activity of U6 promoter from Lonicera japonica

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