月季‘月月粉’与野蔷薇U6启动子的克隆及活性分析

doi:10.3864/j.issn.0578-1752.2024.13.012

中国农业科学 ›› 2024, Vol. 57 ›› Issue (13): 2651-2661.doi: 10.3864/j.issn.0578-1752.2024.13.012

月季‘月月粉’与野蔷薇U6启动子的克隆及活性分析

覃语祺¹^,³(), 苏健馨¹^,³(), 曹雪敏¹^,³, 朱婉¹^,³, 程文翰²(), 张蔚¹^,²^,³()

¹ 华中农业大学园艺林学学院/果蔬作物种质创新与利用全国重点实验室，武汉 430070
² 荆楚理工学院/特色花卉生物育种湖北省工程研究中心，湖北荆门 448000
³ 华中农业大学花卉研究所，武汉 430070

收稿日期:2023-11-22 接受日期:2024-01-18 出版日期:2024-07-09 发布日期:2024-07-09
通信作者:
程文翰，E-mail：kaven_53@163.com。
张蔚，E-mail：zhangw@mail.hzau.edu.cn
联系方式: 覃语祺，E-mail：476818962@qq.com。苏健馨：1551701554@qq.com。覃语祺与苏健馨为同等贡献作者。
基金资助:
国家自然科学基金(32072617); 湖北省教育厅科学规划项目(Q20224301); 湖北省中央引导地方科技发展专项(2022BGE262)

Cloning and Activity Analysis of U6 Promoter in Rosa chinensis Old Blush and Rosa multiflora

QIN YuQi¹^,³(), SU JianXin¹^,³(), CAO XueMin¹^,³, ZHU Wan¹^,³, CHENG WenHan²(), ZHANG Wei¹^,²^,³()

¹ College of Horticulture & Forestry Sciences of Huazhong Agricultural University/National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Wuhan 430070
² Jingchu University of Technology, Hubei Engineering Research Center for Specialty Flowers Biological Breeding, Jingmen 448000, Hubei
³ Institute of Flowers Research, Huazhong Agricultural University, Wuhan 430070

Received:2023-11-22 Accepted:2024-01-18 Published:2024-07-09 Online:2024-07-09

摘要/Abstract

摘要：

【背景】 U6启动子是CRISPR/Cas9基因编辑系统中驱动sgRNA转录的重要元件，而不同物种的U6启动子转录活性可能存在差异，且物种内源U6启动子相比物种外源U6启动子可能具有更高效的启动效率。迄今对蔷薇属（Rosa）植物的U6启动子少有报道，且尚未获得转录活性强于拟南芥U6的启动子。【目的】筛选转录活性更高的蔷薇属植物U6启动子，为后续月季和野蔷薇等蔷薇属植物CRISPR/Cas9基因编辑系统的优化和分子育种奠定基础。【方法】利用拟南芥保守的102 bp U6 snRNA序列，从中国古老月季‘月月粉’（Rosa chinensis Old Blush，OB）基因组中克隆相似性最高的6个OBU6启动子，从野蔷薇（R. multiflora）基因组中克隆相似性最高的7个RmU6启动子，并构建OBU6启动子和RmU6启动子分别驱动LUC和GUS报告基因的融合表达载体，利用农杆菌介导的瞬时转化法转染本氏烟草（Nicotiana benthamiana）叶片和月季‘萨曼莎’（R. Samantha）组培苗，通过双荧光素酶活性检测和GUS组织化学染色对月季和野蔷薇的启动子转录活性分别进行比较。【结果】 ‘月月粉’6个OBU6启动子和野蔷薇7个RmU6启动子均具有影响U6启动子转录活性的两个必要元件USE和TATA box。双荧光素酶活性检测和GUS组织化学染色结果显示，月季‘月月粉’OBU6启动子和野蔷薇RmU6启动子均具有转录活性，但OBU6启动子的转录活性均弱于拟南芥AtU6-1启动子。考虑到过长的U6启动子可能会削弱其转录活性，于是选取其中转录活性相对较高的OBU6-4进行5′端截短（1 507、1 076、574和187 bp），但截短后的启动子未能提高转录活性；而在野蔷薇中，成功筛选到一个长度为630 bp的RmU6-2启动子，其转录活性显著高于拟南芥AtU6-1启动子。【结论】从月季‘月月粉’中克隆得到6条U6启动子，从野蔷薇中克隆得到7条U6启动子，并筛选出一条转录活性显著高于拟南芥AtU6-1启动子的野蔷薇U6启动子RmU6-2，为构建蔷薇属植物CRISPR/Cas9基因组编辑系统提供了极具应用潜力的启动子。

关键词: U6启动子, 月季, 蔷薇属, 报告基因, 瞬时表达

Abstract:

【Background】 The U6 promoter is an important element for driving sgRNA transcription in the CRISPR/Cas9 gene editing system, and the transcriptional activity of U6 promoter in various species may be different, while endogenous U6 promoters of species may have higher efficiency than that of exogenous U6 promoters. So far, there have been only sporadic reports on the U6 promoter in genus Rosa, and no promoter with stronger transcriptional activity than Arabidopsis U6 has been obtained. 【Objective】 This study aimed to screen out the U6 promoter of Rosa plants with high transcriptional activity to optimize the CRISPR/Cas9 gene editing system, so as to lay the foundation for molecular breeding in Rosa plants, such as R. chinensis and R. multiflora in future. 【Method】 Six OBU6 promoters with the highest similarity were cloned from the genome of R. chinensis Old Blush (OB), and seven RmU6 promoters with the highest similarity were cloned from the genome of R. multiflora using conserved 102 bp U6 snRNA sequence of Arabidopsis thaliana. The fusion expression vectors of LUC and GUS reporter genes driven by OBU6 promoter and RmU6 promoter were constructed, respectively. Tobacco (Nicotiana benthamiana) leaves and tissue cultured plantlets of R. Samantha were transfected through Agrobacterium-mediated transient transformation. The transcriptional activities of promoters were determined according to the luciferase activity and GUS histochemical staining. 【Result】 Six OBU6 promoters and seven RmU6 promoters all had two essential elements that affected the transcriptional activity of U6 promoter, the USE and TATA boxes. The results of the luciferase activity and GUS histochemical staining showed that all of these U6 promoters had transcriptional activity, however, the transcriptional activity of OBU6 promoters were weaker than that of AtU6-1 promoter. Considering that an excessively long U6 promoter might weaken its transcriptional activity, OBU6-4 with relatively high transcriptional activity was selected for 5' end truncation (1 507 bp, 1 076 bp, 574 bp and 187 bp), but the truncated promoter failed to increase the transcriptional activity. And yet, RmU6-2 promoter (630 bp) was successfully identified from R. multiflora, with significantly higher transcriptional activity compared with AtU6-1 promoter. 【Conclusion】 Six OBU6 promoters were cloned from the genome of R. chinensis Old Blush and seven RmU6 promoters were cloned from the genome of R. multiflora. Finally, an RmU6-2 promoter with significantly higher transcriptional activity than AtU6-1 was obtained, which could be served as a highly promising promoter for the construction of CRISPR/Cas9 genome editing system in Rosa plants.

Key words: U6 promoter, R. chinensis, Rosa, reporter gene, transient expression

覃语祺, 苏健馨, 曹雪敏, 朱婉, 程文翰, 张蔚. 月季‘月月粉’与野蔷薇U6启动子的克隆及活性分析[J]. 中国农业科学, 2024, 57(13): 2651-2661.

QIN YuQi, SU JianXin, CAO XueMin, ZHU Wan, CHENG WenHan, ZHANG Wei. Cloning and Activity Analysis of U6 Promoter in Rosa chinensis Old Blush and Rosa multiflora[J]. Scientia Agricultura Sinica, 2024, 57(13): 2651-2661.

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引物名称 Primer name	上游引物 Forward sequence (5′-3′)	下游引物 Reverse sequence (5′-3′)
OBU6-1-LUC	ctatagggcgaattgggtaccAACCAGAGCATTATCATCTTTTGAA	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
OBU6-2-LUC	ctatagggcgaattgggtaccTGTTTAGTGATAGTTCTTATGTATT	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
OBU6-3-LUC	ctatagggcgaattgggtaccATAGAACGATCCCTATTTACTTTAT	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
OBU6-4-LUC	ctatagggcgaattgggtaccTCTGTTTTGTGGTTAGAGTCGTG	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
OBU6-5-LUC	ctatagggcgaattgggtaccTATAGGTGAGATGGTATGTGGTAAT	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
OBU6-6-LUC	ctatagggcgaattgggtaccCCAATTGCGGATCTACTAACGTCTA	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
OBU6-7-LUC	ctatagggcgaattgggtaccAGTTGGGCTCCAGTTAGGAACAGTA	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
OBU6-8-LUC	ctatagggcgaattgggtaccTCAGGACACTCACATGATTAGTCAC	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
OBU6-1-GUS	cttgcatgcctgcaggtcgacAACCAGAGCATTATCATCTTTTGAA	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
OBU6-2-GUS	cttgcatgcctgcaggtcgacTGTTTAGTGATAGTTCTTATGTATT	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
OBU6-3-GUS	cttgcatgcctgcaggtcgacATAGAACGATCCCTATTTACTTTAT	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
OBU6-4-GUS	cttgcatgcctgcaggtcgacTCTGTTTTGTGGTTAGAGTCGTG	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
OBU6-5-GUS	cttgcatgcctgcaggtcgacTATAGGTGAGATGGTATGTGGTAAT	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
OBU6-6-GUS	cttgcatgcctgcaggtcgacCCAATTGCGGATCTACTAACGTCTA	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
OBU6-7-GUS	cttgcatgcctgcaggtcgacAGTTGGGCTCCAGTTAGGAACAGTA	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
OBU6-8-GUS	cttgcatgcctgcaggtcgacTCAGGACACTCACATGATTAGTCAC	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
AtU6-1	gtcgacGAAATCTCAAAATTCCGGCAGAAC	cccgggCAATCACTACTTCGTCTCTAACCATAT

引物名称 Primer name	上游引物 Forward sequence (5′-3′)	下游引物 Reverse sequence (5′-3′)
RmU6-1-LUC	ctatagggcgaattgggtaccCAGGAGTGATTCTTCTGGATTTTGT	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
RmU6-2-LUC	ctatagggcgaattgggtaccTCCAAGTATATTTTGCACAGTTTGT	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
RmU6-3-LUC	ctatagggcgaattgggtaccCCTTTAAGGAGCGTTTGAGACGTAT	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
RmU6-4-LUC	ctatagggcgaattgggtaccTTTGAATCTTAGAGTTGGAAAGCAA	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
RmU6-5-LUC	ctatagggcgaattgggtaccACAGAATGATGATGTTAATGGTGGT	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
RmU6-6-LUC	ctatagggcgaattgggtaccCAAGTATGTTTTCTGAACCATTCTC	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
RmU6-7-LUC	ctatagggcgaattgggtaccATGAAGTAAATCAGAGCCACGAACA	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
RmU6-8-LUC	ctatagggcgaattgggtaccTATGGTCTCTAGGCTCAACAAGCTA	aagcttatcgataccgtcgacAATTTTATCGGATGTCCCCGA
RmU6-1-GUS	cttgcatgcctgcaggtcgacCAGGAGTGATTCTTCTGGATTTTGT	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
RmU6-2-GUS	cttgcatgcctgcaggtcgacTCCAAGTATATTTTGCACAGTTTGT	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
RmU6-3-GUS	cttgcatgcctgcaggtcgacCCTTTAAGGAGCGTTTGAGACGTAT	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
RmU6-4-GUS	cttgcatgcctgcaggtcgacTTTGAATCTTAGAGTTGGAAAGCAA	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
RmU6-5-GUS	cttgcatgcctgcaggtcgacACAGAATGATGATGTTAATGGTGGT	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
RmU6-6-GUS	cttgcatgcctgcaggtcgacCAAGTATGTTTTCTGAACCATTCTC	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
RmU6-7-GUS	cttgcatgcctgcaggtcgacATGAAGTAAATCAGAGCCACGAACA	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
RmU6-8-GUS	cttgcatgcctgcaggtcgacTATGGTCTCTAGGCTCAACAAGCTA	ataagggactgaccacccgggAATTTTATCGGATGTCCCCGA
AtU6-1	gtcgacGAAATCTCAAAATTCCGGCAGAAC	cccgggCAATCACTACTTCGTCTCTAACCATAT

试剂 Reagent	100 mL所需的量 The amount required for 100 mL
0.2 mol∙L^-1 NaH₂PO4	19 mL
0.2 mol∙L^-1 Na₂HPO4	31 mL
100 mmol∙L^-1 K₃Fe(CN)₆	0.5 mL
100 mmol∙L^-1 K₄Fe(CN)₆	0.5 mL
0.5 mol∙L^-1 Na₂EDTA	2 mL
0.1% Triton X-100	100 µL
20%甲醇	20 mL
X-Gluc	100 mg

基因名称 Gene name	染色体定位 Chromosomal localization	E-值 E-Value
OBU6-1	Chr03: 27486852…27486954	7.55e-48
OBU6-2	Chr07: 28384012…28384114	7.55e-48
OBU6-3	Chr02: 37794875…37794977	7.55e-48
OBU6-4	Chr03: 27477144…27477246	7.55e-48
OBU6-5	Chr03: 29980675…29980777	7.55e-48
OBU6-6	Chr04: 48001111…48001213	7.55e-48
OBU6-7	Chr07: 28143401…28143503	7.55e-48
OBU6-8	Chr07: 28464723…28464825	7.55e-48

月季‘月月粉’与野蔷薇U6启动子的克隆及活性分析

Cloning and Activity Analysis of U6 Promoter in Rosa chinensis Old Blush and Rosa multiflora

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