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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"

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