Honeysuckle U6 promoter and its application

Abstract

The present invention discloses a honeysuckle U6 promoter, and its nucleotide sequence is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.4. The present invention cloned 4 LjU6 promoters from the honeysuckle genome, connected the LjU6 promoter to the pBI121 vector carrying the β-glucuronidase gene, transformed tobacco leaves by Agrobacterium transient transformation, and subjected the leaves to GUS histochemical staining, and the staining results showed that 3 of the promoters had transcriptional activity, among which LjU61-F1 had the highest transcriptional activity and the shortest length. The honeysuckle U6 promoter of the present invention is suitable for the honeysuckle CRISPR / Cas9 editing system, and can be used to initiate the expression of sgRNA guide sequences. The present invention lays a foundation for the establishment of honeysuckle CRISPR / Cas9 genome editing technology.

Description

Technical Field

[0001] The invention relates to honeysuckle U6 promoter cloning and application thereof, and belongs to the technical field of gene engineering. Background Art

[0002] In recent years, CRISPR / Cas9 technology has become the most commonly used gene editing technology due to its simple operation, short experimental cycle, high efficiency, and low off-target rate. It has shown great potential in crop genetic breeding, plant genetic modification, and crop variety improvement. The CRISPR / Cas9 gene editing system includes the Cas9 protein that can cut double-stranded DNA as a nuclease, and an artificially fused single guide RNA (sgRNA). The sgRNA determines the specificity of the target sequence and plays an important role in the CRISPR / Cas9 gene editing system. Existing studies have shown that the editing efficiency of CRISPR / Cas9 technology depends on the high expression of Cas9 and sgRNA. Strong constitutive RNA polymerase II promoters such as cauliflower mosaic virus 35S (CaMV35S) are usually used to drive Cas9 expression, while sgRNA is usually driven by the U6 promoter. The U6 promoter is an RNA polymerase III promoter with a clear transcription start site that can recognize a highly conserved start site starting from G. It has been frequently used to drive high-level expression of plant small RNAs and has become the first choice for driving sgRNA expression in CRISPR / Cas9 vectors.

[0003] Existing studies have shown that there are multiple differently expressed U6 genes in the genome of the same species. Not all U6 promoters can drive gene expression and the transcription efficiency of different promoters is not the same. The high conservation of the U6 promoter makes it still have transcriptional activity between different species, but there are certain differences in transcriptional activity between different species with distant homologous relationships. It has been reported that the endogenous U6 promoter can increase the expression of sgRNA, thereby improving the editing efficiency. In soybean, the mutation rate of the pCas9-GmU6-sgRNA vector constructed using the soybean endogenous U6 promoter GmU6-10 is much higher than the mutation rate of the pCas9-AtU6-sgRNA vector constructed using Arabidopsis AtU6-26. Lu et al. used the cotton endogenous GhU6.3 promoter to drive sgRNA and found that its expression level was 6 to 7 times that of the Arabidopsis AtU6-29 promoter, and the editing mutation efficiency was increased by 4 to 6 times. At present, the CRISPR / Cas9 genome editing system has been established in many plants such as Arabidopsis, rice, wheat, soybean, corn, cotton, and tomato using the plant's own U6 promoter to achieve efficient editing of the target gene.

[0004] Honeysuckle is a commonly used bulk medicinal material with the effects of clearing away heat and detoxifying, and evacuating wind-heat. Its main active ingredients are flavonoids, organic acids, volatile oils, triterpenoid saponins, etc. It has broad prospects for development and utilization, and its demand is increasing year by year. However, no research on the U6 promoter of honeysuckle has been reported, which seriously restricts the development of honeysuckle gene editing technology. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a honeysuckle U6 promoter and its application in transgenic technology. The present invention clones and screens a honeysuckle U6 promoter with high transcriptional activity by PCR and Agrobacterium infection of tobacco leaves transient transformation method, which is of great significance for constructing a honeysuckle CRISPR / Cas9 gene editing system and realizing molecular breeding of honeysuckle.

[0006] The present invention is achieved through the following technical solutions:

[0007] The honeysuckle U6 promoter, whose nucleotide sequence is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.4.

[0008] The application of the honeysuckle U6 promoter in transgenic technology and in honeysuckle gene editing, for example, is specifically used in the honeysuckle CRISPR / Cas9 editing system to drive the expression of sgRNA.

[0009] A gene editing vector containing the honeysuckle U6 promoter.

[0010] The gene editing vector is used in transgenic technology and in honeysuckle gene editing, for example, it is specifically used in the honeysuckle CRISPR / Cas9 editing system to drive the expression of sgRNA.

[0011] A pBI121 fusion expression vector is formed by connecting a plant expression vector pBI121 and a GUS gene, wherein the promoter of the plant expression vector pBI121 is the honeysuckle U6 promoter.

[0012] In this study, four honeysuckle U6 promoters were cloned for the first time, and their LjU6-pBI121 fusion expression vectors were constructed respectively. Among the four promoters, three promoters had transcriptional activity, among which LjU61-F1 had the highest transcriptional activity and the shortest length, which was suitable for the honeysuckle CRISPR / Cas9 editing system and could be used to initiate the expression of sgRNA guide sequences.

[0013] The present invention uses honeysuckle genomic DNA as a template to clone and screen out honeysuckle U6 promoters with high transcriptional activity. Four LjU6 promoters with lengths of 336bp, 708bp, 359bp and 602bp were cloned from the honeysuckle genome by PCR method. PlantCARE analysis found that the four promoters all contained typical promoter cis elements such as TATA box and CAAT box, and contained regulatory elements related to light response, stress response, etc. After the cloned products were sequenced correctly, the LjU6 promoter was connected to the pBI121 vector carrying the β-glucuronidase (GUS) gene, and four LjU6-pBI121 fusion expression vectors were successfully constructed. The tobacco leaves were transformed by Agrobacterium transient transformation method, and the leaves were subjected to GUS histochemical staining and GUS quantitative determination. The results all showed that LjU61-F1 had the highest transcriptional activity. This study preliminarily screened out the honeysuckle U6 promoter with high transcriptional activity, laying a foundation for the establishment of honeysuckle CRISPR / Cas9 genome editing technology.

[0014] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Construction of fusion expression vector of GUS driven by different LjU6 promoters from honeysuckle.

[0016] Figure 2 : Enzyme digestion identification of different T-LjU6 expression vectors, where M: 2kb Plus DNA standard molecular weight; 1: T-LjU61-F1; 2: T-LjU61-F2; 3: T-LjU62-F1; 4: T-LjU62-F2; 5: 35S-PBI121 enzyme digestion fragment.

[0017] Figure 3 : PCR detection of LjU6-pBI121 expression vector in Trans5α competent cells, where M: 2kb Plus DNA standard molecular weight; 1: LjU61-F1-pBI121; 2: LjU61-F2-pBI121; 3: LjU62-F1-pBI121; 4: LjU62-F2-pBI121.

[0018] Figure 4 : PCR detection of LjU6-pBI121 expression vector in Agrobacterium LBA4404 competent cells, wherein, M: 2kbPlus DNA standard molecular weight; 1: LjU61-F1-pBI121; 2: LjU61-F2-pBI121; 3: LjU62-F1-pBI121; 4: LjU62-F2-pBI121.

[0019] Figure 5 : Different LjU6 promoters drive the transient expression of GUS in tobacco leaves, where A: CaMV35S-pBI121 positive control; B: LjU61-F1: GUS; C: LjU61-F2: GUS; D: Agrobacterium negative control; E: LjU62-F1: GUS; F: LjU62-F2: GUS.

[0020] Figure 6 : GUS enzyme activity of different LjU6 promoters transiently expressed in tobacco leaves. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0022] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0023] Study on the U6 promoter of honeysuckle

[0024] 1. Materials and Methods

[0025] 1.1 Test materials

[0026] Honeysuckle and tobacco are cultivated at Shandong University of Traditional Chinese Medicine and were identified as Lonicera japonica and Nicotiana tabacum by Professor Pu Gaobin of Shandong University of Traditional Chinese Medicine.

[0027] The pBI121 vector used in the experiment was preserved by the laboratory of the School of Pharmacy, Shandong University of Traditional Chinese Medicine; plant DNA extraction kit and 5minTM TA / Blunt-Zero Cloning Kit (Nanjing Novozyme Biotechnology Co., Ltd.); universal DNA purification and recovery kit, plasmid mini-extraction kit and DNA molecular weight MakerDL2000 (Tiangen Biochemical Technology (Beijing) Co., Ltd.); T4 DNA ligase (NEB Beijing Co., Ltd.); Trans 5αChemically Competent Cell (Quanshijin Biotechnology); restriction endonucleases HindⅢ and BamHI (Thermo Fisher Scientific); LBA4404 Chemically CompetentCell (Shanghai Weidi Biotechnology Co., Ltd.); cellulase R-10, cleavage enzyme R-10, X-Gluc, 10×PBS buffer (Beijing Solebold Technology Co., Ltd.); primers were synthesized by Boshang Biotechnology (Shanghai) Co., Ltd.

[0028] 1.2 Methods

[0029] 1.2.1 Cloning of the Honeysuckle U6 promoter

[0030] Take the leaves of honeysuckle, use the plant genomic DNA extraction kit to extract the DNA of honeysuckle, and use the designed U6 promoter primers (as shown in Table 1, the primer column represents the primers of a certain promoter) for PCR amplification. The PCR reaction program is: 95℃3min; 95℃30s, 55℃30s, 72℃1min, 32 cycles; 72℃5min; 4℃ storage. The reaction system is ddH2O11μL, 2×M5 Hiper plus Taq HiFi 10μL, DNA template 1μL, upstream and downstream primers 0.5μL each, a total of 20μL system.

[0031] 1% agarose gel electrophoresis was used for detection, and the target DNA fragments were obtained by gel excision and recovery, namely LjU61-F1, LjU61-F2, LjU62-F1, and LjU62-F2. The promoter fragments were connected to the TA / Blunt-Zero Cloning Kit, transformed into Trans5α competent cells, and sequenced. The sequencing results were analyzed by DNAMAN for sequence alignment, and the correct plasmids were named T-LjU61-F1, T-LjU61-F2, T-LjU62-F1, and T-LjU62-F2.

[0032] Table 1 Primer sequences used in this study

[0033]

[0034] 1.2.2 Analysis of promoter cis-acting elements

[0035] The sequencing results were submitted to PlantCARE, an online promoter element analysis website, to analyze the cis-acting elements in the honeysuckle U6 promoter.

[0036] 1.2.3 Construction of fusion expression vector

[0037] HindⅢ and BamHⅠ were used to double-digest T-LjU61-F1, T-LjU61-F2, T-LjU62-F1, T-LjU62-F2 plasmids and plant expression vector pBI121, respectively. The target fragment was recovered by gel cutting, and the cloned LjU6 promoter replaced the CaMV35S promoter in the pBI121 vector and connected with the GUS gene to construct the pBI121 fusion expression vector. The fusion expression vector was transformed into Trans5α competent cells. After the primary activation bacterial solution was verified to be correct by PCR, the plasmid was extracted from the secondary activation bacterial solution. The plasmid was transferred into Agrobacterium competent LBA4404, inverted and cultured at 28℃ for 2 days, and a single clone was picked and incubated in LB liquid medium (Kan 50μg.mL -1 , Rif 20μg.mL -1 ) for one activation, and perform PCR verification on the activated bacterial solution. After successful verification, the activated bacterial solution is stored at -80°C for future use.

[0038] 1.2.4 Analysis of transient expression of different promoters in tobacco leaves

[0039] The GUS histochemical staining method was based on the method of Pu Yan et al. (Pu Yan, Liu Chao, Li Jiyang, et al. Cloning of tomato U6 promoter and establishment of CRISPR / Cas9 gene editing system [J]. Chinese Agricultural Science, 2018, 51: 315-326.), with Agrobacterium without target gene added as negative control and CaMV35S-pBI121 as positive control. The four constructed LjU6-pBI121 fusion expression vectors, positive control CaMV35S-pBI121, and negative control pure Agrobacterium were streaked and cultured once activated Agrobacterium culture liquid, and single clones were picked and cultured in LB liquid medium (Kan 50μg.mL -1 , Rif 20μg.mL -1 ), 28℃, 200r.min -1 Activate and culture until the bacterial solution OD 600 =0.6~0.8; Different volumes of activated bacterial solution were inoculated into 5mL LB medium (containing 50μg.mL -1 Kan and 20 μg.mL -1 Rif) to make the OD value of each Agrobacterium starting bacterial solution the same, and incubate again at 28°C, 200 r.min -1Shake the cells until the OD of all bacterial cultures reaches 600 =1.2, centrifuge at 12 000×g for 5 min to collect the cells, discard the supernatant, and resuspend in Buffer (50 mmol.L -1 MgCl2, 200mmol.L -1 MES, 20mmol.L -1 Acetosyringone) was added to 1 mL, placed at room temperature for 3 hours, and then injected into tobacco leaves grown for 2 to 3 weeks. After the injection, the leaves were marked and cultured overnight in the dark. The tobacco leaves cultured overnight in the dark were cut with scissors and immersed in 100 μL GUS dye solution (20 mmol.L -1 X-Gluc, 50mmol.L -1 PBS buffer) and vacuumed for 30 min. Each Agrobacterium transformation was repeated 3 times for technology and 3 times for biology. The tobacco leaves after vacuuming were placed at 37°C and 180 r.min. -1 After 6-7 hours of oscillation, the GUS dye solution was completely absorbed. Finally, 300 μL of 95% ethanol was added for decolorization. During decolorization, the decolorization solution was replaced every 3-4 hours until the green color of the leaves faded. The decolorized leaves were observed and photographed.

[0040] GUS quantitative determination was based on the method of Zhang Lina et al. (Zhang Lina, Liu Yu, Lin Yongjun. Expression pattern of rice CDPK12 gene promoter and analysis of stress response elements [J]. Journal of Agricultural Biotechnology, 2015, 23(10): 1261-1272.). The GUS protein activity was determined by the method reported by Jefferson (Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants [J]. The EMBO Journal, 1987, 6(13): 3901-3907.). The ratio of the amount of 4-methylumbel-liferone (4-MU) produced to the total protein content and time (pmol.mg -1 .min -1 )express.

[0041] The U6 promoter with high transcription efficiency was screened out based on the staining conditions and quantitative measurement results.

[0042] 2. Results and Analysis

[0043] 2.1 Identification of the honeysuckle promoter fragment

[0044] Four LjU6 promoter fragments, LjU61-F1, LjU61-F2, LjU62-F1, and LjU62-F2, were successfully cloned from the genomic DNA of Flos Lonicerae, with lengths of 336 bp, 708 bp, 359 bp, and 602 bp, respectively, and the nucleotide sequences are shown in SEQ ID NOs. 1 to 4. The promoter fragments were connected to the TA / Blunt-Zero Cloning Kit, transformed into Trans5α competent cells, and sequenced, and the sequencing results showed that the sequences were correct.

[0045] 2.2 Promoter sequence analysis

[0046] Through sequence analysis (Table 2 and Table 3), it can be found that the four promoters all contain typical promoter cis-elements such as TATA box and CAAT box. LjU61 also has AT-rich regions, abscisic acid response cis-elements ABRE (ACGTG), defense and stress response cis-elements TC-rich repeats (ATTCTCTAAC), auxin response element AuxRE (TGTCTCAATAAG), etc. In addition, it also contains multiple light response or light response related elements ACE (GACACGTATG), G-box (TACGTG), Box II (TGGTAATAA), GT1-motif (GGTTAA), etc. Compared with LjU61, LjU62 contains fewer cis-elements, mainly light response or light response related elements 4cl-CMA1b (ATTCCGATAAACT), Box 4 (ATTAAT) and GA-motif (ATAGATAA), etc. In addition, it also contains CCAAT-box, an element involved in stress response.

[0047] Table 2 Analysis of cis-acting elements in the LjU61 promoter

[0048]

[0049] Table 3 Analysis of cis-acting elements in the LjU62 promoter

[0050]

[0051]

[0052] 2.3 Construction of fusion expression vector

[0053] Schematic diagram of fusion expression vector of GUS gene driven by different promoters of honeysuckle Figure 1HindⅢ and BamHI double digested T-LjU61-F1, T-LjU61-F2, T-LjU62-F1, T-LjU62-F2 plasmids and plant expression vector pBI121, and 1% agarose gel electrophoresis obtained the target fragment, whose size was consistent with the predicted fragment size ( Figure 2 ); Gel excision was used to recover different LjU6 fragments and the large pBI121 vector fragment after removing the CaMV35S promoter. The target fragments were ligated with T4 ligase and transformed into Trans5α competent cells. The activated bacterial solution was used for PCR identification. 1% agarose gel electrophoresis showed that the promoter band could be amplified and the size was consistent with the expected value ( Figure 3 ), indicating that the fusion expression vector was successfully constructed and the next step of the experiment could be carried out; the plasmid was extracted after the secondary activation of the bacterial solution, and the fusion expression vector plasmid was transformed into Agrobacterium competent LBA4404. The primary activated bacterial solution was identified by PCR, and 1% agarose gel electrophoresis showed that the target band could be amplified and the size was consistent with the expected value ( Figure 4 ), indicating that the conversion was successful.

[0054] 2.4 Analysis of transcriptional activity of different promoters

[0055] The constructed fusion expression vectors LjU61-F1-pBI121, LjU61-F2-pBI121, LjU62-F1-pBI121, LjU62-F2-pBI121, positive control CaMV35S-pBI121, and negative control pure Agrobacterium were transiently transformed into tobacco leaves by Agrobacterium injection. After decolorization by GUS histochemical staining, it was found that no blue was observed in the leaves treated with the negative control and LjU62-F1 promoter, while blue was observed in the tobacco leaves treated with CaMV35S, LjU61-F1, LjU61-F2, and LjU62-F2 promoters, indicating that 3 of the 4 cloned LjU6 promoters can drive the expression of the reporter gene GUS, and the transcriptional activities are different ( Figure 5 ).

[0056] In order to further accurately analyze the expression differences of GUS driven by different promoters, the GUS enzyme activity was quantitatively measured. Figure 6 As shown, the results showed that almost no enzyme activity was detected in the negative control and tobacco leaves treated with the LjU62-F1 promoter. The activity of LjU61-F1 was similar to that of CaMV35S, and the activity was higher, 1.5 times that of LjU61-F2. LjU62-F2 had the lowest activity, which was consistent with the GUS staining results.

[0057] 3. Discussion

[0058] This study cloned the LjU6 promoter of honeysuckle for the first time and analyzed its cis-acting elements. Existing studies have shown that the structure of the promoter, including the arrangement position and distance of the cis-acting elements, affects its recognition and binding with RNA polymerase, thereby affecting the gene expression level; at the same time, the cis-acting element CAAT box has a strong activation effect on gene transcription, and the presence of a CAAT box upstream of the TATA box will significantly increase the expression of the gene. In this study, it was found that LjU61 contained more CAAT boxes than LjU62, which may be one of the reasons why its transcriptional activity is higher than that of LjU62.

[0059] In this study, GUS was used as a reporter gene. The promoter activity was determined by the color depth of the dyed tobacco leaves after transient transformation and the quantitative determination results. The GUS histochemical staining results and quantitative determination results showed that the transcription efficiency of the four cloned U6 promoters in tobacco leaves was different. Compared with LjU61-F2, LjU61-F1 had higher transcription activity, and LjU62-F2 had lower transcription activity than LjU61-F2. LjU62-F1 did not show blue color in GUS staining and almost no enzyme activity was detected. Sequence observation revealed that LjU62-F1 had no other structures except a few TATA and light response sequences. Whether it lost its transcription activity due to the lack of certain elements remains to be further studied.

[0060] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims. Sequence Listing <110> Shandong University of Traditional Chinese Medicine <120> Honeysuckle U6 promoter and its application <141> 2021-11-24 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 336 <212> DNA <213> Lonicera japonica <400> 1 gatcctgtct tcatctcact catcgttata tcaacccctt caatggttct ttttccctcg 60 atgtgggact ctgtctctct ctctccactt tctttctgtt cgcttaaccc acaaaattat 120 tccacacgta tgtatactaa aatttgtgaa tttgatatat aatagttttt cacactatgc 180 atagaaaaca aaataatcat accatatggt ataatcaagt ggtaataata tattattgag 240 acataaagtc atagatttaa ctcattgttt aaacattgtt ttaatgtatt cttattgtca 300 aaatattat ctctttgtta gagtatgaca ttgcta 336 <210> 2 <211> 708 <212> DNA <213> Lonicera japonica <400> 2 gatcctgtct tcatctcact catcgtata tcaacccctt caatgtttct ttttccctcg 60 atgtgggact ctgtctctct ctctccactt tctttctgtt cgcttaaccc acaaaattat 120 tccacacgta tgtatactaa aatttgtgaa tttgatatat aatagttttt cacactatgc 180 atagaaaaca aaataatcat accatatggt ataatcaagt ggtaataata tattattgag 240 acataaagtc atagatttaa ctcattgttt aaacattgtt ttaatgtatt cttattgtca 300 aaatattat ctctttgtta gagtatgaca ttgctttgaa aagtggtatt gtatattttt 360 atattatac attgctttga caataaccat aattattggg agcaagtaaa agcatcctta 420 sttyttccttctssssssssssssssssssssssssssssssssssssssssssnaughtssssssssssssssstaaaaattttaaaaag 480 cacccttaaa tgtaagagca ccttataaa actgcaaaa ttctcatc tctaatgctt 540 gtggcagaat tccaattggc taatagaatt tctagagtgt attctactga gttgattttt 600 aaagtgag tctaaatta gtgtaaaga agatattgac atttaattga aaagtaat 660 tcaatca ttcaattata gatatata tcaactcaa ccgagaca 708 <210> 3 <211> 359 <212> DNA <213> Japonica bench <400> 3 gatccacgtt tcttttgaag tagttctata ttaagtagtc gttttctgta aaatttagcg 60 atgtgggacg aagaggaga gtattgcttt gactgatgtc cagacaaagc gcaatgtgct 120 gggcagagtc tggggtggac cttatgaaat ttgatgact tgttgggctt ttaatttttc 180 atggacacat tttctaaatt aaaagata attackccgt ataattagc atataaatt 240 aaagtattgt acacaaaaaa tattaatgcc ccttccttat tattaata acataataat 300 cctaatataa catattaagt ctctagaaga cacttagcac tgatcacaat caaaattta 359 <210> 4 <211> 602 <212> DNA <213> Lonicera japonica <400> 4 agcttttagc acattgaatt tggctgttat ctcttgggtt gttgttgcta aaaccctttt 60 agcaccatta tagtattgga taatcggtgc tatattgtt ttagcagcac ataattagtt 120 atttgtttt ggacaattag tactaaactg ttttattaa cacatgaatc agagttgatc 180 ggaatgattt tggtgctggt gctcgaccgt tgtaaatttc gtgagagaga aggttgtttg 240 gattttgaaa atttgattg tgatcagtgc taagtgtctt ctagagactt aatatgttat 300 attaggatta tttgttatt attaaataa ggaaggggca ttaatatttt ttgtgtacaa 360 tactttaatt tattatgcta attatacgg agtagttatc ttttataatt tagaaaatgt 420 gtccatgaaa aattaaaagc ccaacaagtc atcaaaattt cataaggtcc accccagact 480 ctgcccagca cattgcgctt tgtctggaca tcagtcaaag caatactctt cctcttcgtc 540 ccacatcgct aaattttaca gaaaacgact acttaatata gaactacttc aaaaagaaacg 600 tg 602

Claims

1. The honeysuckle U6 promoter, whose nucleotide sequence is shown in SEQ ID NO.

1.

2. The honeysuckle U6 promoter, whose nucleotide sequence is shown in SEQ ID NO.

2.

3. The honeysuckle U6 promoter, whose nucleotide sequence is shown in SEQ ID NO.

4.

4. Use of the honeysuckle U6 promoter according to any one of claims 1 to 3 in honeysuckle gene editing.

5. The use according to claim 4, characterized in that: Specially used in the Honeysuckle CRISPR / Cas9 editing system to drive the expression of sgRNA.

6. A gene editing vector, characterized in that: The method comprises the honeysuckle U6 promoter according to any one of claims 1 to 3.

7. Use of the gene editing vector according to claim 6 in honeysuckle gene editing.

8. The use according to claim 7, characterized in that: It is specifically used in the Honeysuckle CRISPR / Cas9 editing system to drive the expression of sgRNA.

9. A pBI121 fusion expression vector, characterized in that: The invention is formed by connecting the plant expression vector pBI121 and the GUS gene, wherein the promoter of the plant expression vector pBI121 is the honeysuckle U6 promoter according to any one of claims 1 to 3.

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