Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (13): 2651-2661.doi: 10.3864/j.issn.0578-1752.2024.13.012

• HORTICULTURE • Previous Articles     Next Articles

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. 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 Online:2024-07-09 Published:2024-07-09
  • Contact: CHENG WenHan, ZHANG Wei

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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

Table 1

Relative primers used for OBU6 promoter analysis"

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

Table 2

Relative primers used for RmU6 promoter analysis"

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

Table 3

Preparation of GUS staining solution"

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

Table 4

Mapping of U6 snRNA chromosome in genome of Rosa chinensis Old Blush"

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

Fig. 1

PCR amplification of the OB U6 promoters"

Fig. 2

Sequence analysis of OBU6 promoters from R. chinensis Old Blush"

Fig. 3

Sequence analysis of RmU6 promoters from R. multiflora"

Fig. 4

Comparison of transcriptional activity between OBU6 Promoters and the AtU6 Promoter A: LUC/REN ratios of OB U6 and AtU6-1; B: Analysis of cis-acting elements of OBU6-4; C: LUC/REN ratios of OBU6-4, its truncated fragments and AtU6-1; D: GUS staining results of OB U6 and AtU6-1"

Fig. 5

Comparison of transcriptional activity between RmU6 promoter and the AtU6 promoter A: LUC/REN ratios of RmU6 and AtU6-1; B: LUC fluorescence detection of RmU6 and AtU6-1; C: GUS staining of RmU6 and AtU6-1 in N. benthamiana"

Fig. 6

Transcriptional activity identification of RmU6 promoters in rose"

[1]
TODA E, KOISO N, TAKEBAYASHI A, ICHIKAWA M, KIBA T, OSAKABE K, OSAKABE Y, SAKAKIBARA H, KATO N, OKAMOTO T. An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice. Nature Plants, 2019, 5: 363-368.

doi: 10.1038/s41477-019-0386-z pmid: 30911123
[2]
MAO Y F, BOTELLA J R, LIU Y G, ZHU J K. Gene editing in plants: progress and challenges. National Science Review, 2019, 6(3): 421-437.

doi: 10.1093/nsr/nwz005
[3]
MALI P, YANG L H, ESVELT K M, AACH J, GUELL M, DICARLO J E, NORVILLE J E, CHURCH G M. RNA-guided human genome engineering via Cas9. Science, 2013, 339(6121): 823-826.

doi: 10.1126/science.1232033 pmid: 23287722
[4]
TANG X, ZHONG Z H, ZHENG X L, ZHANG Y. Construction of a single transcriptional unit for expression of Cas9 and single-guide RNAs for genome editing in plants. Bio-protocol Journal, 2017, 7(17): e2546.
[5]
KOR S D, CHOWDHURY N, KEOT A K, YOGENDRA K, CHIKKAPUTTAIAH C, SUDHAKAR REDDY P. RNA Pol III promoters-Key players in precisely targeted plant genome editing. Frontiers in Genetics, 2022, 13: 989199.
[6]
DAS G, HENNING D, REDDY R. Structure, organization, and transcription of Drosophila U6 small nuclear RNA genes. The Journal of Biological Chemistry, 1987, 262(3): 1187-1193.
[7]
NISHITANI C, HIRAI N, KOMORI S, WADA M, OKADA K, OSAKABE K, YAMAMOTO T, OSAKABE Y. Efficient genome editing in apple using a CRISPR/Cas9 system. Scientific Reports, 2016, 6: 31481.

doi: 10.1038/srep31481 pmid: 27530958
[8]
CASARIN T, FREITAS N C, PINTO R T, BREITLER J, RODRIGUES L A Z, MARRACCINI P, ETIENNE H, DINIZ L E C, ANDRADE A C, PAIVA L V. MULTIPLEX CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canephora. Scientific Reports, 2022, 12: 17270.
[9]
ZHOU J H, WANG G M, LIU Z C. Efficient genome editing of wild strawberry genes, vector development and validation. Plant Biotechnology Journal, 2018, 16(11): 1868-1877.

doi: 10.1111/pbi.12922 pmid: 29577545
[10]
雷建峰, 李月, 徐新霞, 阿尔祖古丽∙塔什, 蒲艳, 张巨松, 刘晓东. 棉花不同GbU6启动子截短克隆及功能鉴定. 作物学报, 2016, 42(5): 675-683.
LEI J F, LI Y, XU X X, AERZUGULI∙Tashi, PU Y, ZHANG J S, LIU X D. Cloning and functional analysis of different truncated GbU6 promoters in cotton. Acta Agronomica Sinica, 2016, 42(5): 675-683. (in Chinese)
[11]
蒲艳, 刘超, 李继洋, 阿尔祖古丽·塔什, 胡燕, 刘晓东. 番茄U6启动子的克隆及CRISPR/Cas9基因编辑体系的建立. 中国农业科学, 2018, 51(2): 315-326. doi: 10.3864/j.issn.0578-1752. 2018.02.011.
PU Y, LIU C, LI J Y, AERZUGULI∙Tashi, HU Y, LIU X D. Different SlU6 promoters cloning and establishment of CRISPR/Cas9 mediated gene editing system in tomato. Scientia Agricultura Sinica, 2018, 51(2): 315-326. doi: 10.3864/j.issn.0578-1752.2018.02. 011. (in Chinese)
[12]
LONG L, GUO D D, GAO W, YANG W W, HOU L P, MA X N, MIAO Y C, BOTELLA J R, SONG C P. Optimization of CRISPR/ Cas9 genome editing in cotton by improved sgRNA expression. Plant Methods, 2018, 14: 85.
[13]
BERNARD G, GAGNEUL D, ALVES DOS SANTOS H, ETIENNE A, HILBERT J L, RAMBAUD C. Efficient genome editing using CRISPR/Cas9 technology in chicory. International Journal of Molecular Sciences, 2019, 20(5): 1155.
[14]
MASSEL K, LAM Y, HINTZSCHE J, LESTER N, BOTELLA J R, GODWIN I D. Endogenous U6 promoters improve CRISPR/Cas9 editing efficiencies in Sorghum bicolor and show potential for applications in other cereals. Plant Cell Reports, 2022, 41(2): 489-492.
[15]
王丹, 王谧, 刘军, 周晓慧, 刘松瑜, 杨艳, 庄勇. 茄子U6启动子克隆及CRISPR/Cas9介导的基因编辑体系建立. 园艺学报, 2022, 49(4): 791-800.

doi: 10.16420/j.issn.0513-353x.2020-0265
WANG D, WANG M, LIU J, ZHOU X H, LIU S Y, YANG Y, ZHUANG Y. Cloning of U6 promoters and establishment of CRISPR/ Cas9 mediated gene editing system in eggplant. Acta Horticulturae Sinica, 2022, 49(4): 791-800. (in Chinese)

doi: 10.16420/j.issn.0513-353x.2020-0265
[16]
RIU Y S, KIM G H, CHUNG K W, KONG S G. Enhancement of the CRISPR/Cas9-based genome editing system in lettuce (Lactuca sativa L.) using the endogenous U6 promoter. Plants, 2023, 12(4): 878.
[17]
卞书迅, 韩晓蕾, 袁高鹏, 张利义, 田义, 张彩霞, 丛佩华. 苹果U6启动子的克隆及功能分析. 中国农业科学, 2019, 52(23): 4364-4373. doi: 10.3864/j.issn.0578-1752.2019.23.016.
BIAN S X, HAN X L, YUAN G P, ZHANG L Y, TIAN Y, ZHANG C X, CONG P H. Cloning and functional analysis of U6 promoter in apple. Scientia Agricultura Sinica, 2019, 52(23): 4364-4373. doi: 10.3864/j.issn.0578-1752.2019.23.016. (in Chinese)
[18]
ZHANG S, WU S P, HU C H, YANG Q S, DONG T, SHENG O, DENG G M, HE W D, DOU T X, LI C Y, SUN C K, YI G J, BI F C. Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct. PeerJ, 2022, 10: e12664.
[19]
陈景鲜, 卢超, 王国霞, 李春阁, 李玉华, 苏芳谊, 汪琛颖, 张耀广. 西洋参中U6启动子的克隆及表达分析. 中国中药杂志, 2023, 48(11): 2931-2939.
CHEN J X, LU C, WANG G X, LI C G, LI Y H, SU F Y, WANG C Y, ZHANG Y G. Cloning and expression analysis of U6 promoters in Panax quinquefolius. China Journal of Chinese Materia Medica, 2023, 48(11): 2931-2939. (in Chinese)
[20]
LI X, JIANG D H, YONG K L, ZHANG D B. Varied transcriptional efficiencies of multiple Arabidopsis U6 small nuclear RNA genes. Journal of Integrative Plant Biology, 2007, 49(2): 222-229.
[21]
PAUL C P, GOOD P D, WINER I, ENGELKE D R. Effective expression of small interfering RNA in human cells. Nature Biotechnology, 2002, 20: 505-508.

pmid: 11981566
[22]
KWAK Y D, KOIKE H, SUGAYA K. RNA interference with small hairpin RNAs transcribed from a human U6 promoter-driven DNA vector. Journal of Pharmacological Sciences, 2003, 93(2): 214-217.
[23]
REN C, LIU Y F, GUO Y C, DUAN W, FAN P G, LI S H, LIANG Z C. Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters. Horticulture Research, 2021, 8: 52.

doi: 10.1038/s41438-021-00489-z pmid: 33642575
[24]
WANG P C, ZHANG J, SUN L, MA Y Z, XU J, LIANG S J, DENG J W, TAN J F, ZHANG Q H, TU L L, DANIELL H, JIN S X, ZHANG X L. High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system. Plant Biotechnology Journal, 2018, 16(1): 137-150.

doi: 10.1111/pbi.12755 pmid: 28499063
[25]
HUDZIK C, MAGUIRE S, GUAN S X, HELD J, AXTELL M J. Trans-species microRNA loci in the parasitic plant Cuscuta campestris have a U6-like snRNA promoter. The Plant Cell, 2023, 35(6): 1834-1847.
[26]
JEFFERSON R A, KAVANAGH T A, BEVAN M W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal, 1987, 6(13): 3901-3907.
[27]
CHIU W, NIWA Y, ZENG W, HIRANO T, KOBAYASHI H, SHEEN J. Engineered GFP as a vital reporter in plants. Current Biology, 1996, 6(3): 325-330.

doi: 10.1016/s0960-9822(02)00483-9 pmid: 8805250
[28]
ZHANG G, GURTU V, KAIN S R. An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells. Biochemical and Biophysical Research Communications, 1996, 227(3): 707-711.

pmid: 8885998
[29]
WELSH S, KAY S A. Reporter gene expression for monitoring gene transfer. Current Opinion in Biotechnology, 1997, 8(5): 617-622.

pmid: 9353237
[30]
WANG C P, LI Y, WANG N, YU Q, LI Y H, GAO J P, ZHOU X F, MA N. An efficient CRISPR/Cas9 platform for targeted genome editing in rose (Rosa hybrida). Journal of Integrative Plant Biology, 2023, 65(4): 895-899.
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