Mango U6 promoter and application thereof
By screening and constructing the mango U6 promoters MiU6-6P-3 and MiU6-7P-2, the problem of promoter deficiency in mango CRISPR/Cas9 genome editing was solved, enabling efficient transcription of sgRNA and promoting mango functional genomics research and genetic improvement.
Patent Information
- Application Number
- CN202511583647.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2026-01-23
AI Technical Summary
Currently, there is no mango U6 promoter, which limits the application of CRISPR/Cas9 genome editing technology in mangoes and makes it difficult to efficiently drive sgRNA transcription.
The mango U6 promoters MiU6-6P-3 and MiU6-7P-2 were screened and constructed into a CRISPR/Cas9 genome editing vector. They were then transformed into mango plants by Agrobacterium and drove the transcription of sgRNA.
This study has enabled efficient CRISPR/Cas9 genome editing in mangoes and provided efficient promoter resources. In particular, MiU6-6P-3 serves as a candidate promoter for efficiently driving sgRNA transcription, which has promoted mango functional genomics research and genetic improvement.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of genetic agriculture, and in particular to a mango U6 promoter and application thereof. BACKGROUND
[0002] In recent years, genome editing technology has developed rapidly. CRISPR / Cas genome editing technology is a highly efficient genome editing tool. The core components of the CRISPR / Cas9 genome editing system include a Cas9 protein with double-strand break ability and an artificially fused small guide RNA. Among them, sgRNA is responsible for targeted binding to the target gene site, and Cas9 protein cuts the target DNA under the guidance of sgRNA. Therefore, selecting a suitable promoter to efficiently drive the transcription of sgRNA is crucial for the study of the CRISPR / Cas9 genome editing system. The U6 promoter is an RNA polymerase III (Pol III) dependent promoter with a clear transcription initiation site, can recognize a highly conserved sequence starting with 'G', and is widely used to drive the efficient expression of plant sgRNA. In the CRISPR / Cas9 genome editing system, the heterologous expression of sgRNA is usually driven by Arabidopsis U6 (AtU6) and rice U6 (OsU6) promoters.
[0003] The U6 promoter applied across species is usually limited to closely related plants. The endogenous U6 promoters of wheat, soybean, chicory, cotton and grape plants can significantly improve the expression efficiency of sgRNA and the genome editing effect. Therefore, in order to improve the efficiency of CRISPR / Cas9 in plant genome editing, the plant endogenous U6 promoter should be selected first.
[0004] Mango is an evergreen fruit tree of the Anacardiaceae family, is an important tropical and subtropical fruit, and has the reputation of "King of Tropical Fruits". Currently, there is no related content of mango U6 promoter disclosed. In order to create new mango germplasm using CRISPR / Cas9 genome editing technology, an efficient MiU6 promoter is needed to lay the foundation for mango CRISPR / Cas9 genome editing. SUMMARY
[0005] In order to solve the above technical problems existing in the prior art, the present application provides a mango U6 promoter and application thereof, which is realized by the following technical scheme: A mango U6 promoter, the mango U6 promoter is one of MiU6-6P-3, MiU6-7P-2, and the specific sequence is as follows: MiU6-6P-3: AATAAAATAATGATTTTATTCCTGCTTTTAACAAAAATTTTAACTTAAATTAATTAATGAATAAAATTTTGAATTTTTTAAACTCTGTAAATATGAGTTTGAAAACATATTAAAACTTGGGCCTTTGGCCAAAAATTAATAATAAGAATGGTCACATTATTACCTGGCTTTGGTGTAGATTTGTCGGGCTTCGAGCCCAACATCAGGACTACTCAAATTCATTTAAAAGTGAACAGATCACGTGACGTGTAGAATGAGTCATCTAGTTAGAAACCAACTATGCCTCCCACATCGTCCAGTTGAGAAACAGATGAATATATTATAAGCAAAGACAAGGTATCAGGTTACTG; MiU6-7P-2: ATTACGGGAGATGTGATAACATTATTTTCCATTTTCGGTTACCGGAAAGCAAGAAAATTGGAGGAAATGACGTTCGCCGTGGGATTGCCGTGGGATGTGGGAAAAATTCGAATTTTCTTTGAATTGCACAGTGAAAATTTGCTGTGTTTATATATGGGGGCAGTTTCGTCATTTCACAAAACATGGGCATTTTTGCTTTAACCTGAATTTTTTGGGCAATTTCGCTTAGACCCCTTTAATTTTGTCTAGTTTTTAAAATTTCCCAGTTAGAAACGAACTATCCCACATTGTTTAGTTGCGAAACAGTGCGATGGTTTATAAACAAAGAAGAAGCGGCAGGAGAGTG.
[0006] Further, the mango U6 promoter is MiU6-6P-3.
[0007] The mango U6 promoter described above is applied in CRISPR / Cas9 genome editing.
[0008] Further, the application in CRISPR / Cas9 genome editing is specifically driving the transcription of sgRNA.
[0009] Further, the CRISPR / Cas9 genome editing is mango CRISPR / Cas9 genome editing.
[0010] A mango CRISPR / Cas9 genome editing vector, a MiU6 promoter sequence is introduced into a pBI121 vector to obtain a MiU6 promoter expression vector.
[0011] The mango CRISPR / Cas9 genome editing vector is applied to mango gene editing.
[0012] A mango CRISPR / Cas9 genome editing method, the promoter sequence is introduced into the mango CRISPR / Cas9 genome editing vector, and then the vector is transformed into Agrobacterium, and finally introduced into a mango plant through immersion.
[0013] Further, the mango CRISPR / Cas9 genome editing vector is the vector of claim 6.
[0014] Compared with the prior art, the technical effects of the present application are embodied in: Based on the genome data of 'Sijimi mango', seven U6 promoter sequences are screened from the mango genome using the conserved sequence of Arabidopsis thaliana U6 promoter, the full length of the conserved region of which is 102 bp, and sequence alignment shows that the conserved sequences of the seven MiU6 promoters of mango are highly similar to the U6 promoter sequences of other species, but the flanking sequences are quite different. A fusion expression vector driven by the MiU6 promoter to drive the GUS reporter gene is constructed, and wild-type Arabidopsis thaliana is transformed by inflorescence infection. The GUS staining and enzyme activity identification results show that among the seven MiU6 promoters, MiU6-2P-1, MiU6-6P-1, MiU6-6P-3 and MiU6-7P-2 successfully drive the GUS reporter gene to express in Arabidopsis thaliana, among which MiU6-6P-3 has the highest transcriptional activity, followed by MiU6-7P-2 and MiU6-2P-1. Further transient expression in transgenic tobacco and mango leaves is detected, and it is found that the GUS activity of MiU6-6P-3 and MiU6-7P-2 is higher. Combined with the GUS activity detection of the three different transgenic plants, MiU6-6P-3 is preferred as the U6 promoter, followed by MiU6-7P-2. This study provides a candidate U6 promoter for editing mango using CRISPR / Cas9 genome editing technology. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 Comparison of mango MiU6 and U6 promoter sequences of other species.
[0016] Figure 2 Analysis of the cis-acting elements of the mango MiU6 promoter.
[0017] Figure 3GUS staining results of MiU6 promoter in different developmental stages of Arabidopsis.
[0018] Figure 4 GUS staining analysis of MiU6 promoter in different organs of Arabidopsis in flowering stage.
[0019] Figure 5 MiU6 promoter GUS activity detection of different tissues of Arabidopsis; Note: Duncan's test was used to determine the significant difference between samples (p < 0.01).
[0020] Figure 6 GUS staining analysis of MiU6 promoter in tobacco and mango leaves.
[0021] Figure 7 Construction of MiU6 promoter GUS fusion expression vector. DETAILED DESCRIPTION
[0022] The technical solutions of the present application will be further limited in combination with specific embodiments, but the scope of protection is not only limited to the description. If not specifically indicated, the technical means used in the following examples is the conventional means known to those skilled in the art; the experimental methods used are conventional methods, and the materials, reagents, etc. used can be obtained from commercial channels.
[0023] Screening and activity analysis of mango U6 promoter 1RESULTS 1.1MiU6 promoter sequence analysis (1) MiU6 promoter sequence analysis Based on the BLAST analysis of the 'Four Seasons Honey Mango' genome, 7 MiU6 promoter sequences were screened, and the full length of the conserved region was 102 bp. By comparing the sequences of these MiU6 promoters with AtU6 from Arabidopsis, MdU6 from apple, proHbU6 from corn and SlU6 promoter from tomato, Figure 1), it was found that MiU6 and other species U6 promoters all have a TATA-like Box (5'-CTTATATA-3') at -30 site and a USE (5'-GTCCCACATCG-3') at -60 site, and the distance between the two elements is fixed at about 30 bp. The transcription start site of all U6 promoters is 'G' base, in which the TATA-like Box is responsible for the recognition and binding of RNA polymerase III, and the USE directly affects the transcription activity of the promoter. In this study, the first base at the 5' end of each mango U6 snRNA and the sequence of about 350 bases upstream of the transcription start site were used as candidate promoter sequences for MiU6 promoter, and the seven promoter sequences were named MiU6-2P-1, MiU6-4P-1, MiU6-6P-1, MiU6-6P-2, MiU6-6P-3, MiU6-7P-1 and MiU6-7P-2, respectively.
[0024] >MinU6-02-1 (348 bp) CAATTTTTTACTATTTCTTTTATCAAAGATGTCACCTCAAAGTCAACATCAATCCTAGACACCAGCCTTAAAATGGGTTCCAATGACGTTATCAAATCGATAATAACGGGAAATTTCAAAAGAAATAAAATTAATGTAGTATAGATTCGTTGGGCTCTGGGCCCAAAATTAACAATGGAGGGTACGGCGAGTCGTATTCAATCATATAACTGAATTCAATTCAAATTCAAGCAGGTCTAGTGACGTGTTGAATGAGTGATCTAGTTGGAAACCAACTATCCCTCCCACATCGTTCAGTTGAGAAATAGCTAAACATTTTATAAGCGAAGGCAAGGCCACTAGTGGTCG >MinU6-04-1 (344 bp) CAAATGAGTCTTGTAACTTAAGAATTGGTTATTCCAGAATAATCTGTTGTGTTCCCTCTTGCAGATTATATCAATTATCTGTGAACATATTTAAAAAAGGTGCTGGCAGAATGTTCTAATAATAATCAAAGGCAAATAATAGAAAAAATTACGCCTTAATTATGATGAGTGGAAAACGGGCCGAACTGGTTGAGCCCTCGTACGTGTCAAAGCCAACAAACCCAAAGTGGCATTGGCATGACGTGTCAACTATTAAGTTTCATCGAATTGCATCTTAGTCCCACACTGCTCAGGTAGAAGTCAGAGTAGCCTCTTATAATAATGGTGCGACTTCTAGGTGATGG >MinU6-06-1 (327 bp) GATAAACTTAGTATTATACCATCCTTTGTGGTATAAGATGAAGCAGGCGAGAGAAATAAAAGTAATAAGCTATGGCAGAGTTTACAGTGACGTTTGCCATGGAAACCAAGTTGTTGATAAGTGTAAAGAAAGAAGTTGAAAGCATTAAAAATAGAATTCAAGATCATCAAATATTTCCTTAGGGATGCAGATAAAAAGGTATCAACCAAGAACATTGGATTGGTTTCAATGAAGGTCATCTGGTTAGAAATCAATTATTCCTCCCACATCGTTCAATTGAGACACAATACAATATTATATAACTAAAGACAAGGCAGCAGGTAGTCG >MinU6-06-2 (345 bp) CATTTGGTCTGTAATTCGACTCTAAACTGCTTTTAGCGATTAATGACTCCATTAAATCTATTTAACTACAGAGTATTAAATTAATATATTTCATTTTAAAAAGAAAAAAAAATCAATGAATATGGGCTGATACACTAGGCCCATGGTGTAGATTTGTCGGGCTCCGAGCCCAACCTTAGGATACGACTGTCGTATCCAACCAAATTCCTCAAATTCATTTAAAGTGAGCAGATCACGTGACGTGTAGAATGAGTCATCTAGTTAGAAACCAACTATGCATCCCACATCGTCCGGTTGAGAAACAGATGAATATATTATAAGCAAAGACAAGAGAAGGGGCATTTG >MinU6-06-3 (350 bp) AATAAAATAATGATTTTATTCCTGCTTTTAACAAAAATTTTAACTTAAATTAATTAATGAATAAAATTTTGAATTTTTTAAACTCTGTAAATATGAGTTTGAAAACATATTAAAACTTGGGCCTTTGGCCAAAAATTAATAATAAGAATGGTCACATTATTACCTGGCTTTGGTGTAGATTTGTCGGGCTTCGAGCCCAACATCAGGACTACTCAAATTCATTTAAAAGTGAACAGATCACGTGACGTGTAGAATGAGTCATCTAGTTAGAAACCAACTATGCCTCCCACATCGTCCAGTTGAGAAACAGATGAATATATTATAAGCAAAGACAAGGTATCAGGTTACTG >MinU6-07-1 (351 bp) GGAAATTATTACTTTTTAAACTTTGAGTAAGGGAAAATTACGGGATTTTTAAAATATAATAAAACTTTAATTTTTAAAATTTTTTTAATAAATAACAGTATTACCTTTAATCTTAATTGATATTTTTAAAAGAATTTTGTTTTATGGATAGATATTTGAGTTTTTGAAAATTGAA AAATAATATTTTAGAATTTTACTATACCTTGGATGGGAAGCCATTCATTTAAAAGTCAAAAGGTCACATTACATGAAAAATGAATGAGTCATTTAGTTGGAAACGAACTATCCCACGTTGTTCAGTTAAGAAACAGTGCAATGGTTTATAAACAAAGGCGATGCAGCAAGAGAATG MinU6-07-2 (346bp) ATTACGGGAGATGTGATAACATTATTTTCCATTTTCGGTTACCGGAAAGCAAGAAAATTGGAGGAAATGACGTTCGCCGTGGGATTGCCGTGGGATGTGGGAAAAATTCGAATTTTCTTTGAATTGCACAGTGAAAATTTGCTGTGTTTATATATGGGGGCAGTTTCGTCATT TCACAAAACATGGGCATTTTTGCTTTAACCTGAATTTTTTGGGCAATTTCGCTTAGACCCCTTTAATTTTGTCTAGTTTTTAAAATTTCCCAGTTAGAAACGAACTATCCCACATTGTTTAGTTGCGAAACAGTGCGATGGTTTATAAACAAAGAAGAAGCGGCAGGAGAGTG (2) Analysis of cis-components of the MiU6 starter Promoter analysis results show that ( Figure 2 The MiU6 promoter contains two conserved core sequence elements, as well as other promoter cis-elements. These mainly include promoter CAAT frames (for enhancing transcriptional activity), light-related elements, and elements responding to drought, abscisic acid, anaerobic conditions, gibberellin, and low temperature. Among these, the CAAT frames related to promoter-enhanced transcriptional activity are the most numerous.
[0025] 1.2 Analysis of transcriptional activity of the MiU6 promoter at different developmental stages in Arabidopsis thaliana Seven fusion expression vectors of MiU6 promoter driving GUS genes were constructed, and the positive plants were obtained by transforming Arabidopsis thaliana through floral dip method, antibiotic screening culture and PCR identification. The homozygous T3 generation of transgenic Arabidopsis thaliana plants obtained were used for subsequent analysis.
[0026] GUS staining analysis of MiU6 promoter in different development stages of transgenic Arabidopsis thaliana, wild type Arabidopsis thaliana (WT) and empty vector transgenic Arabidopsis thaliana (35S:GUS) plants was carried out, and the results are shown in Figure 3 . Wild type Arabidopsis thaliana did not show blue, while the positive control showed blue in the tissues at each period. In 7-day-old seedlings, except for the positive control 35S:GUS, other plants were not stained, indicating that the MiU6 promoter had no activity in 7-day-old seedlings. In 14-day-old and 21-day-old seedling leaves, MiU6-6P-3 showed blue throughout the plant, indicating the highest transcriptional activity; followed by MiU6-7P-2, which showed light blue. MiU6-2P-1 showed blue in the petiole of seedling leaves, and MiU6-6P-1 showed activity in the growth point. In contrast, MiU6-4P-1, MiU6-6P-2 and MiU6-7P-1 had the lowest activity, and no blue was observed.
[0027] 1.3 Analysis of transcriptional activity of MiU6 promoter in different tissue organs of Arabidopsis thaliana during flowering GUS staining was carried out during the flowering period of Arabidopsis thaliana, and the experimental results are shown in Figure 4 . The tissue organs of the positive control strain showed blue, while the negative control group did not show blue. MiU6-6P-3 showed the deepest staining in leaves, stems, seed pods, flowers and roots, showing the highest transcriptional activity; followed by MiU6-7P-2, which showed light blue in each tissue organ. MiU6-2P-1 only showed blue in the petiole of leaves, but no staining was observed in stems, seed pods, flowers and roots. MiU6-6P-1 only showed blue in flowers, similar to MiU6-7P-2, but did not stain in leaves, stems, seed pods and roots. In contrast, MiU6-4P-1, MiU6-6P-2 and MiU6-7P-1 did not show blue staining in all detected tissue organs.
[0028] 1.4 GUS activity detection The leaves, inflorescences and roots of positive promoter transgenic Arabidopsis thaliana were selected as three groups of measurement samples, and the 4-MU (4-Methylumbelliferone, 4-MU) fluorescence quantitative detection method was used to quantitatively analyze the expression intensity of GUS genes in transgenic Arabidopsis thaliana.
[0029] GUS The results of protein activity detection are shown in Table 2. Figure 5 The activities of MiU6 promoters in different organs of Arabidopsis were significantly different. The highest GUS activity was detected in MiU6-6P-3, followed by MiU6-2P-1, both of which were higher than the positive control (35S:GUS). Lower activities were detected in MiU6-4P-1, MiU6-6P-2 and MiU6-7P-2, while no activity was detected in the negative control (WT), MiU6-6P-1 and MiU6-7P-1. In Arabidopsis siliques, the highest GUS activity was detected in the positive control (35S:GUS), followed by MiU6-6P-3, then MiU6-7P-2. Lower activities were detected in MiU6-6P-2, MiU6-2P-1, MiU6-7P-1 and MiU6-6P-1, while no activity was detected in the negative control (WT) and MiU6-4P-1. In Arabidopsis roots, the highest GUS activity was detected in the positive control (35S:GUS), followed by MiU6-6P-3, then MiU6-2P-1. Lower activities were detected in MiU6-7P-2, MiU6-6P-2, MiU6-7P-1 and MiU6-6P-1, while no activity was detected in the negative control (WT) and MiU6-4P-1. Overall, MiU6-2P-1, MiU6-6P-3 and MiU6-7P-2 promoters showed higher activities in Arabidopsis leaves, siliques and roots, with significant differences.
[0030] 1.5 Transcriptional activity analysis of MiU6 promoters in tobacco and mango leaves Previous studies have shown that MiU6-6P-3, MiU6-7P-2 and MiU6-2P-1 have high transcriptional activity in transgenic Arabidopsis. To further verify the function of these promoters, these promoters were introduced into tobacco and mango leaves by transient expression technology. The results of GUS staining analysis are shown in Table 3. Figure 6 The tobacco and mango leaves in the negative control group did not show blue staining, while the positive control group showed blue staining in all tested tissues. The leaves of MiU6-6P-3 showed the deepest staining, indicating the highest transcriptional activity. The staining of MiU6-7P-2 was lighter and smaller in area, while MiU6-2P-1 showed almost no staining, indicating the lowest activity.
[0031] 2 Discussion and conclusion In this study, the transcriptional activity of the mango U6 promoter was analyzed after being truncated to about 350 bp. The results showed that MiU6-2P-1, MiU6-6P-1, MiU6-6P-3 and MiU6-7P-2 successfully drove the expression of GUS in Arabidopsis leaves, siliques and roots, with significant differences. GUSThe expression of the reporter gene, in which MiU6-6P-3 has the highest transcriptional activity. This study further confirmed that MiU6-6P-3 promoter exhibited extremely high transcriptional activity through quantitative detection, which was highly similar to the results of GUS staining.
[0032] In summary, this study screened 7 mango U6 promoters, and truncated the MiU6 promoter to about 350 bp in length, and then analyzed the transcriptional activity of the promoter. The results showed that MiU6-2P-1, MiU6-6P-1, MiU6-6P-3 and MiU6-7P-2 could successfully drive GUS The expression of the reporter gene, in which MiU6-6P-3 has the highest transcriptional activity. Through verification experiments in mango and tobacco, the functionality and applicability of these promoters were further confirmed. The research results provide important promoter resources for the optimization of the mango CRISPR / Cas9 genome editing system, especially MiU6-6P-3 can be used as an ideal candidate for driving sgRNA transcription. Future research can further verify the applicability of these promoters in different mango varieties and tissues, and combine molecular breeding techniques to promote mango functional genomics research and genetic improvement.
[0033] 3 MATERIALS AND METHODS 3.1 Experimental materials and reagents The Columbia-type Arabidopsis thaliana and Nicotiana benthamiana used in this study were preserved in the laboratory, and ‘Sijimeng’ was cultivated in the mango resource garden of Guangxi University. Competent EHA105 was purchased from Shanghai Angyuan Company; Potassium ferrocyanide, potassium ferricyanide, and Triton X-100 were purchased from Guangxi Nankemeng Biological Technology Co., Ltd. in Nanning; Bradford protein concentration determination kit, X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucoside), and GUS The reporter gene quantitative detection kit was from Coolai Bio Technology Co., Ltd.
[0034] 3.2 U6 promoter sequence analysis Using the conserved 102 bp U6 snRNA sequence of Arabidopsis thaliana (He Yukun et al., 2023) and the genome data of ‘Sijimeng’ in this research group for homologous alignment, 7 mango MiU6 promoter sequences were screened. Using DNAMAN software, the 7 MiU6 promoter sequences retrieved were aligned with the promoter sequences of Arabidopsis thaliana (AtU6), apple (MdU6), corn (proHbU6), and tomato (SlU6). The upstream about 350 bp fragment and the 1 bp U6 snRNA at the 5' end were extracted as candidate promoters.
[0035] PlantCARE software was used for predictive analysis of the MiU6 promoter sequence, and Tbtools was used for intuitive visualization. Primers were designed using Primer 5.0, and detailed primer information is shown in Table 1.
[0036] Table 1 Primer sequences used in this experiment Table 1 The primer sequences used in the experiments
[0037] 3.3 Construction of MiU6 promoter expression vector Build a bootloader based on MiU6 GUS Reporter gene fusion expression vectors, through detection GUS Gene expression levels can indirectly reflect promoter transcriptional activity. The construction of the fusion expression vectors pBI121-MiU6-2P-1::GUS-pBI121-MiU6-7P-2::GUS involves inserting the MiU6 promoter sequence into the pBI121 vector. Figure 7 The structure of this fusion expression vector was shown in detail, and then Agrobacterium-mediated transformation was performed according to the instructions for EHA105 competent cells.
[0038] 3.4 Arabidopsis genetic transformation Wild-type Arabidopsis thaliana was transformed using the inflorescence infection method. After bolting, Arabidopsis thaliana genomic DNA was extracted using the CTAB method (Yu et al., 2016). Promoter-specific upstream primers and... GUS PCR detection was performed using downstream primers for the gene; the PCR primer sequences are shown in Table 1. After detecting the target band, the samples were sent to Qingke Biotechnology Co., Ltd. for sequencing. Positive seedlings were then screened and harvested individually until the T3 generation.
[0039] 3.5 Transient expression in tobacco and mango leaves The fusion expression vector pBI12-MiU6::GUS was used as the experimental group, pBI12-35S::GUS as the positive control, and the empty vector as the negative control. These vectors were transformed into Agrobacterium EHA105 competent cells. The Agrobacterium cells carrying the expression vectors were then propagated and cultured overnight at 28°C with shaking in LB broth. The bacterial cells were collected by centrifugation at 8000 r / min for 10 min at room temperature and resuspended in a resuspending solution (0.01 mol / L MgCl2, 0.01 mol / L MES, 10⁻⁻¹). 4 mol / L AS) resuspended to OD 600For 0.8. 28℃ in the dark for 2-3 h after injection to normal growth of 4 weeks of N. benthamiana leaves. Each liquid injection of 3 strains, each injection of 3-4 leaves. 25℃ dark culture for 12 h, after 48 h in the light cycle 16 / 8 h dark sampling detection.
[0040] The method of transient expression of mango leaves is similar to tobacco, and the corresponding bacterial liquid is resuspended with resuspension solution and injected into 1-year-old mango leaves. After 25℃ dark culture for 12 h, sampling detection is carried out after 60 h culture under the light cycle 16 / 8 h dark.
[0041] 3.6 GUS staining GUS histochemical staining method is used to detect the activity of MiU6 promoter in transgenic Arabidopsis, transient expression tobacco and mango leaves. The darker the staining, the stronger the promoter activity. The staining solution (0.1 mol / L NaH2PO2, 0.1 mol / L Na2HPO2, 0.01 mol / L EDTA, 1% Triton X-100, 0.005 mol / L K4Fe(CN)6·3H2O, 0.005 mol / L K3 / 4Fe(CN)6, 1000 mg / L X-Gluc) is prepared and used immediately, and stored in the dark. Fresh plant tissue is immersed in GUS staining solution, vacuum pumping for 20 min, incubated in 37℃ oven overnight, and then decolorized with 75% ethanol until chlorophyll is removed. After decolorization, super-zoom 3D microscope is used for photography and observation.
[0042] 3.7 GUS activity quantitative detection The endogenous GUS activity in plants is very low, so this study uses GUS The reporter gene detects the expression of MiU6 promoter in Arabidopsis. The samples are divided into three groups of leaves, inflorescences and roots, each group is measured three times, and each group sets three parallel samples. GUS activity is determined by GUS fluorescence quantitative analysis, and Bradford protein concentration determination kit (Coolaber, China) and GUS gene quantitative kit (Coolaber, China) are used for detection. The calculation of GUS enzyme activity is based on the amount of fluorescent product generated per unit mass of total plant protein per unit time. Each sample is repeated 3 times.
[0043] Finally, it should be pointed out that the above examples are only more representative examples of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, but can also have many variations. All variations that can be directly derived or inferred by those skilled in the art from the disclosed content shall be considered as the protection scope of the present application.
Claims
1. A mango U6 promoter, characterized in that, The mango U6 promoter is one of MiU6-6P-3 and MiU6-7P-2, and the specific sequence is as follows: MiU6-6P-3: AATAAAATAATGATTTTATTCCTGCTTTTAACAAAAATTTTAACTTAAATTAATTAATGAATAAAATTTTGAATTTTTTAAACTCTGTAAATATGAGTTTGAAAACATATTAAAACTTGGGCCTTTGGCCAAAAATTAATAATAAGAATGGTCACATTATTACCTGGCTTTGGTGTAGATTTGTCGGGCTTCGAGCCCAACATCAGGACTACTCAAATTCATTTAAAAGTGAACAGATCACGTGACGTGTAGAATGAGTCATCTAGTTAGAAACCAACTATGCCTCCCACATCGTCCAGTTGAGAAACAGATGAATATATTATAAGCAAAGACAAGGTATCAGGTTACTG; MiU6-7P-2: ATTACGGGAGATGTGATAACATTATTTTCCATTTTCGGTTACCGGAAAGCAAGAAAATTGGAGGAAATGACGTTCGCCGTGGGATTGCCGTGGGATGTGGGAAAAATTCGAATTTTCTTTGAATTGCACAGTGAAAATTTGCTGTGTTTATATATGGGGGCAGTTTCGTCATTTCACAAAACATGGGCATTTTTGCTTTAACCTGAATTTTTTGGGCAATTTCGCTTAGACCCCTTTAATTTTGTCTAGTTTTTAAAATTTCCCAGTTAGAAACGAACTATCCCACATTGTTTAGTTGCGAAACAGTGCGATGGTTTATAAACAAAGAAGAAGCGGCAGGAGAGTG.
2. The gene fragment of claim 1, wherein, The mango U6 promoter is MiU6-6P-3.
3. The mango U6 promoter of claim 1 is used in CRISPR / Cas9 genome editing.
4. Use according to claim 3, wherein the compound is ###0002### The application in CRISPR / Cas9 genome editing is specifically driving the transcription of sgRNA.
5. The use according to claim 3, wherein the compound is ###0002### The CRISPR / Cas9 genome editing is mango CRISPR / Cas9 genome editing.
6. A mango CRISPR / Cas9 genome editing vector, characterized in that, The MiU6 promoter sequence is constructed into the pBI121 vector to obtain the MiU6 promoter expression vector.
7. The mango CRISPR / Cas9 genome editing vector of claim 6 is used in mango gene editing.
8. A method of mango CRISPR / Cas9 genome editing, characterized in that, The promoter sequence of claim 1 is introduced into a mango CRISPR / Cas9 genome editing vector, which is then transformed into Agrobacterium, and finally introduced into a mango plant through immersion.
9. The method of claim 8, wherein, The mango CRISPR / Cas9 genome editing vector is the vector of claim 6.