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Acta Agronomica Sinica ›› 2025, Vol. 51 ›› Issue (5): 1156-1165.doi: 10.3724/SP.J.1006.2025.44161

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and transcriptional activity analysis of U6 promoter in jute (Corchorus capsularis)

HUANG Meng-Xin1,2(), ZHUANG Ling-Ling1,2, CHENG Pei-Pei1,2, LI Qin1,2, XU Jian-Tang1,2, TAO Ai-Fen1,2, FANG Ping-Ping1,2, QI Jian-Min1,2, ZHANG Li-Wu1,2,*()   

  1. 1Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University / Key Laboratory of Ministry of Agriculture and Rural Affairs for Biological Breeding of Fujian and Taiwan Crops / Fujian Key Laboratory for Crop Breeding by Design, Fuzhou 350002, Fujian, China
    2Experiment Station of Ministry of Agriculture and Rural Affairs for Jute and Kenaf Scientific Observation in Southeast China, Fujian Agriculture and Forestry University / Public Platform of Fujian for Germplasm Resources of Bast Fiber Crops / Fujian International Science and Technology Cooperation Base for Genetics, Breeding and Multiple Utilization Development of Southern Economic Crops, Fuzhou 350002, Fujian, China
  • Received:2024-09-25 Accepted:2025-01-23 Online:2025-05-12 Published:2025-02-07
  • Contact: *E-mail: lwzhang@fafu.edu.com
  • Supported by:
    National Natural Science Foundation of China(32472219);Fujian Provincial Natural Science Foundation(2023J01443);China Agriculture Research System of MOF and MARA(CARS-16);Science and Technology Innovation Project of Fujian Agriculture and Foresty University(KFB23001);Science and Technology Innovation Project of Fujian Agriculture and Foresty University(KFB24080)

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Abstract:

The U6 promoter is a critical element for driving the transcription of single guide RNA (sgRNA) in the CRISPR/Cas9 system, with endogenous U6 promoters often exhibiting higher efficiency than exogenous ones. However, no studies to date have focused on endogenous U6 promoters in jute (Corchorus L.). In this study, two candidate U6 promoters, CcU6.1 and CcU6.3, were cloned from the genome of the jute cultivar “Meifeng 4” using conserved sequences from the Arabidopsis thaliana U6-26 sgRNA promoter (AtU6-26). Fusion expression vectors carrying GUS reporter genes driven by the CcU6.1 and CcU6.3 promoters were constructed, and the transcriptional activities of these promoters were evaluated through Agrobacterium-mediated transformation of tobacco (Nicotiana benthamiana) leaves and jute hairy roots. Promoter activity was determined based on GUS histochemical staining. Homology analysis revealed that both CcU6.1 and CcU6.3 promoters contained two essential elements for U6 promoter activity: the USE and TATA boxes. GUS staining demonstrated that both jute U6 promoters exhibited transcriptional activity, although the CcU6.1 promoter showed weaker activity compared to the CcU6.3 promoter in both Nicotiana benthamiana leaves and jute hairy roots. Quantitative PCR further confirmed these findings. Since excessively long U6 promoters may reduce transcriptional efficiency, a comparative cis-regulatory element analysis of the CcU6.3 promoter and the AtU6-26 promoter were conducted. This analysis suggested that a truncated version of the CcU6.3 promoter, spanning from the transcriptional start site to the -550 bp region, could enhance transcriptional activity. This study is the first to identify and characterize the CcU6.3 promoter, which exhibits relatively high transcriptional activity in jute. The CcU6.3 promoter holds significant potential as a strong and efficient promoter for constructing CRISPR/Cas9 gene-editing systems in Corchorus species.

Key words: jute, U6 promoter, CRISPR/Cas9, Nicotiana benthamiana, hairy roots, transcriptional activity

Table 1

Primer sequences for cloning the U6 promoter in jute"

引物名称Primer name 引物序列Primer sequence (5'-3')
Cc.U6.1-F ggccgcccccttcaccCTAACAGAACAAGCTTCTTATGTTTGC
Cc.U6.1-R cggcgcgcccacccttATCTTGAGTTGCTACTTTCTTATATAAAAAGGC
Cc.U6.3-F ggccgcccccttcaccCTATATGCAGCAAATGTTTTCCTCC
Cc.U6.3-R cggcgcgcccacccttACCCTCTTTGGTACTTTCTGATATATAAAC

Fig. 1

Comparison of the promoter sequences of jute CcU6 and Arabidopsis AtU6-26"

Fig. 2

PCR amplification of the jute CcU6 promoter and identification of the intermediate vector a: PCR amplification products of jute U6 promoter; M: DL2000 DNA marker; 1: CcU6.1; 2: CcU6.3. b: positive identification of CcU6.1 ligated into the intermediate vector. c: positive identification of CcU6.3 ligated into the intermediate vector."

Fig. 3

PCR identification of jute U6 promoter expression vector M: DL2000 DNA marker. a: positive identification of the expression vector in Escherichia coli TOP10; b: positive identification of the expression vector in Agrobacterium tumefaciens GV3101; c: positive identification of the expression vector in Agrobacterium rhizogenes K599; d: schematic diagram of PRNTR carrier; e: schematic diagram of PGWB533 carrier."

Fig. 4

GUS histochemical staining of jute U6 promoter in Nicotiana benthamiana leaves"

Fig. 5

GUS histochemical staining of jute U6 promoter in jute hairy roots"

Fig. 6

Quantitative fluorescence PCR detection and comparison of GUS gene driven by two jute U6 promoters The data represent the transcriptional activities of the three promoters. **, *** indicate significant differences at the 0.01 and 0.001 probability levels, respectively."

Fig. 7

Prediction of cis-acting elements in CcU6.3 TATA-Box: core promoter element of the transcription start site; CAAT-box: cis-acting element common to promoter and enhancer regions; Box 4: part of the conserved DNA module of cis-acting regulatory elements involved in light response; ARE: anaerobic cis-acting regulatory element; AuxRR-core: cis-acting regulatory element involved in auxin response."

Fig. 8

Comparison of transcription-related elements between CcU6.3 and AtU6-26"

[1] Bashar K K, Tareq M Z, Kabir S M T, Hossain M S, Ahmed R, Ahmed B, Islam M S. Comparative transcriptomics discovers the genetic basis of contrasting waterlogging tolerance between two cultivated jute species. Ind Crops Prod, 2023, 199: 116701.
[2] Hossain M S, Ahmed B, Ahmed R, Ullah M W, Kabir S M T, Bashar K K, Emdad E M. The lignin riddle in jute: a comparative genomic investigation identifies targets for improving fiber quality. Gene Rep, 2024, 36: 101972.
[3] 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, et al. High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system. Plant Biotechnol J, 2018, 16: 137-150.
doi: 10.1111/pbi.12755 pmid: 28499063
[4] 唐维, 后猛, 宋炜涵, 闫会, 王欣, 李臣, 高闰飞, 张允刚, 李强. 甘薯U6启动子克隆及其转录活性分析. 江苏农业学报, 2024, 40: 969-974.
Tang W, Hou M, Song W H, Yan H, Wang X, Li C, Gao R F, Zhang Y G, Li Q. Cloning and transcriptional activity analysis of U6 promoter in sweetpotato. Jiangsu J Agric Sci, 2024, 40: 969-974 (in Chinese with English abstract).
[5] Li J F, Norville J E, Aach J, McCormack M, Zhang D D, Bush J, Church G M, Sheen J. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol, 2013, 31: 688-691.
[6] Feng Z Y, Zhang B T, Ding W N, Liu X D, Yang D L, Wei P L, Cao F Q, Zhu S H, Zhang F, Mao Y F, et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res, 2013, 23: 1229-1232.
doi: 10.1038/cr.2013.114 pmid: 23958582
[7] Jiang W Z, Zhou H B, Bi H H, Fromm M, Yang B, Weeks D P. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, Sorghum and rice. Nucleic Acids Res, 2013, 41: e188.
[8] Di Y H, Sun X J, Hu Z, Jiang Q Y, Song G H, Zhang B, Zhao S S, Zhang H. Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean. Biochem Biophys Res Commun, 2019, 519: 819-823.
[9] Wang C G, Rollins J A. Efficient genome editing using endogenous U6 snRNA promoter-driven CRISPR/Cas9 sgRNA in Sclerotinia sclerotiorum. Fungal Genet Biol, 2021, 154: 103598.
[10] 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.
doi: 10.1186/s13007-018-0353-0 pmid: 30305839
[11] Sun X J, Hu Z, Chen R, Jiang Q Y, Song G H, Zhang H, Xi Y J. Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep, 2015, 5: 10342.
doi: 10.1038/srep10342 pmid: 26022141
[12] Jiang S L, Li Q, Meng X X, Huang M X, Yao J Y, Wang C Y, Fang P P, Tao A F, Xu J T, Qi J M, et al. Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute (Corchorus capsularis). Crop J, 2024, 12: 1266-1270.
[13] 王丽珊, 王珅, 王菲, 王海燕, 刘大群. 小麦TaPR1基因启动子的克隆及启动活性分析. 河北农业大学学报, 2022, 45(1): 7-11.
Wang L S, Wang S, Wang F, Wang H Y, Liu D Q. Cloning and activity analysis of wheat TaPR1 promoter. J Hebei Agric Univ, 2022, 45(1): 7-11 (in Chinese with English abstract).
[14] 左春阳, 李亚玮, 李焱龙, 金双侠, 朱龙付, 张献龙, 闵玲. 陆地棉漆酶基因家族成员表达模式分析. 作物学报, 2023, 49: 2344-2361.
doi: 10.3724/SP.J.1006.2023.24246
Zuo C Y, Li Y W, Li Y L, Jin S X, Zhu L F, Zhang X L, Min L. Relative expression patterns of laccase gene family members in upland Gossypium hirsutum L. Acta Agron Sin, 2023, 49: 2344-2361 (in Chinese with English abstract).
[15] Yang Y, Li X R, Li C Y, Zhang H, Tuerxun Z, Hui F J, Li J, Liu Z G, Chen G, Cai D R, et al. Isolation and functional characterization of a constitutive promoter in upland cotton (Gossypium hirsutum L.). Int J Mol Sci, 2024, 25: 1917.
[16] Seki H, Nishizawa T, Tanaka N, Niwa Y S, Yoshida S, Muranaka T. Hairy root-activation tagging: a high-throughput system for activation tagging in transformed hairy roots. Plant Mol Biol, 2005, 59: 793-807.
doi: 10.1007/s11103-005-1008-x pmid: 16270231
[17] 杨昕, 李玉, 刘传兵, 张力岚, 何青垚, 祁建民, 张立武. 黄麻内参基因筛选及次生细胞壁合成相关基因的表达分析. 作物学报, 2022, 48: 1614-1624.
doi: 10.3724/SP.J.1006.2022.14119
Yang X, Li Y, Liu C B, Zhang L L, He Q Y, Qi J M, Zhang L W. Reference genes screening for expression analysis of secondary cell wall synthesis related genes in jute (Corchorus capsularis). Acta Agron Sin, 2022, 48: 1614-1624 (in Chinese with English abstract).
[18] Qi X T, Dong L, Liu C L, Mao L, Liu F, Zhang X, Cheng B J, Xie C X. Systematic identification of endogenous RNA polymerase III promoters for efficient RNA guide-based genome editing technologies in maize. Crop J, 2018, 6: 314-320.
doi: 10.1016/j.cj.2018.02.005
[19] Wei Y D, Qiu Y, Chen Y H, Liu G G, Zhang Y X, Xu L W, Ding Q R. CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter. RNA, 2017, 23: 1-5.
pmid: 27742910
[20] Yan L, Aymerick E, Sasha Y, Bei B J, Veronica T B, Reo Y, Clarabelle C Y, Edward E B, Jenny C M, Henrik V S, et al. A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction. Biotechnology for Biofuels, 2019, 12(1): 1-15.
[21] 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 (Basel), 2023, 12: 878.
[22] Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/cas system. Plant Methods, 2013, 9: 39.
doi: 10.1186/1746-4811-9-39 pmid: 24112467
[23] 郭静远, 赵辉, 屈静, 张丽丽, 郭安平. 狗尾草U6启动子的克隆及功能鉴定. 热带作物学报, 2021, 42: 3156-3164.
doi: 10.3969/j.issn.1000-2561.2021.11.0014
Guo J Y, Zhao H, Qu J, Zhang L L, Guo A P. Cloning and functional identification of setaria viridis U6 promoter. Chin J Trop Crops, 2021, 42: 3156-3164 (in Chinese with English abstract).
[24] 张立武. 专题导读: 加强麻类作物基因组学研究, 推动优异等位基因发掘及种质创新. 作物学报, 2021, 47: 993-996.
doi: 10.3724/SP.J.1006.2021.04993
Zhang L W. Editorial: strengthening the researches of genomics of bast fiber crops to promote elite allele mining and germplasm innovation. Acta Agron Sin, 2021, 47: 993-996 (in Chinese with English abstract).
[25] Li X, Jiang D H, Yong K L, Zhang D B. Varied transcriptional efficiencies of multiple Arabidopsis U6 small nuclear RNA genes. J Integr Plant Biol, 2007, 49: 222-229.
[26] 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: 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. Sci Agric Sin, 2019, 52: 4364-4373 (in Chinese with English abstract).
doi: 10.3864/j.issn.0578-1752.2019.23.016
[27] 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: 675-683.
doi: 10.3724/SP.J.1006.2016.00675
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 Agron Sin, 2016, 42: 675-683 (in Chinese with English abstract).
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