MYB transcription factor FngMYB10 gene for regulating and controlling color of yellow-hair strawberry fruit and application of MYB transcription factor FngMYB10 gene
By studying and regulating the MYB transcription factor FngMYB10 in yellow-haired strawberries, the problem of unclear fruit color regulation mechanism was solved, the change of fruit color and the improvement of anthocyanin content was achieved, and high-quality breeding of strawberries was promoted.
Patent Information
- Application Number
- CN202311858701.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-30
- Publication Date
- 2025-04-15
AI Technical Summary
At present, the molecular regulation mechanism of the white color of yellow-haired strawberry fruits is unclear, which affects its breeding and appearance quality improvement.
By studying the single-base mutation of the MYB transcription factor FngMYB10, the color and anthocyanin content of yellow-haired strawberry fruit were regulated, and the overexpression vector was constructed for transient transformation and stable genetic transformation, and the fruit color and anthocyanin synthesis pathway were changed.
The color of yellow-haired strawberry fruits has been successfully changed, from white to red, and the expression levels of anthocyanin synthesis genes and transport genes have been significantly improved, and the content of anthocyanin has been increased.
Smart Images
Figure CN119823998A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of high-quality strawberry breeding, and particularly relates to a MYB transcription factor FngMYB10 gene for regulating the fruit color of yellow-haired strawberry and an application thereof. Background Art
[0002] Fruit color is a core indicator that affects the appearance quality and commercial value of strawberry fruit. Fragarianilgerrensis Schlecht. is a wild diploid strawberry with white fruit and a strong peach aroma. It is also an important small berry with medicinal and edible properties and has extremely high development value. The unique fruit color and aroma make Fragarianilgerrensis Schlecht. an important resource for genetic improvement of cultivated strawberry fruit. The currently popular decaploid cultivated strawberry variety "Taoxun" with peach flavor is bred by hybridization of diploid Fragarianilgerrensis Schlecht. and Kurume No. 1. The hybrid retains the characteristics of white fruit and peach aroma. However, the molecular regulatory mechanism for the white fruit color of Fragarianilgerrensis Schlecht. is not clear.
[0003] The color of strawberry fruit is closely related to the content of anthocyanins, and MYB transcription factors play an important regulatory role in the accumulation of anthocyanins. In order to improve the breeding and appearance quality of yellow-haired strawberry, the applicant used yellow-haired strawberry as the material and focused on studying the regulation of the FngMYB10 single base mutation on the fruit color of yellow-haired strawberry. By constructing an overexpression vector, transient transformation and stable genetic transformation were performed to identify its effects on the fruit color of yellow-haired strawberry and endogenous anthocyanin synthesis genes, providing a basis for further research on anthocyanin synthesis metabolic pathways and high-quality strawberry breeding, which has important theoretical significance and application value. Summary of the invention
[0004] The purpose of the present invention is to provide a MYB transcription factor FngMYB10 gene for regulating the fruit color of yellow-haired strawberry and its application, so as to solve the problems raised in the above background technology.
[0005] To achieve the above object, the present invention provides the following technical solutions:
[0006] The MYB transcription factor FngMYB10 gene that regulates the fruit color of yellow-haired strawberry includes:
[0007] S1. The nucleotide sequence of the FngMYB10 gene is shown in SEQ ID No: 1;
[0008] S2. The amino acid sequence of the FngMYB10 gene is shown in SEQ ID No: 2;
[0009] S3. The FngMYB10 nucleotide sequence shown in SEQ ID No: 3 was site-directedly mutated into FngMYB10 (MU) (c.246G>T, p.Arg82Ser) by homologous recombination.
[0010] Transient transformation and stable genetic transformation of S4 and FngMYB10(MU) genes can change the anthocyanin content of yellow-haired strawberry;
[0011] S5. The key genes in anthocyanin-related pathways were significantly upregulated after overexpression of the FngMYB10 (MU) gene.
[0012] Preferably, the yellow-haired strawberry MYB10 gene is obtained as follows:
[0013] The expression vector is pCAMBIA1301-EGFP-35S-NOS.
[0014] Preferably, the method for regulating the fruit color of yellow-haired strawberry by FngMYB10 gene comprises the following steps:
[0015] S1, amplification of mutant FngMYB10 using homologous recombination method;
[0016] S2, construct pCAMBIA1301-EGFP-35S-FngMYB10 overexpression vector;
[0017] S3, transform Agrobacterium and identify positive colonies;
[0018] S4, using the injection method (transient transformation) to infect yellow-haired strawberry fruits;
[0019] S5, using the leaf disc method to stably transform the callus of yellow-haired strawberry;
[0020] S6. Application of the stable transformation medium formula by the leaf disc method.
[0021] Application of FngMYB10 gene in regulating fruit color and anthocyanin content of yellow-haired strawberry.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] (1) The present invention injects Agrobacterium carrying an overexpression vector of the target gene into the yellow-haired strawberry fruit, and observes the color change of the fruit after 5-7 days. In the present invention, the empty vector was injected as the control group. After the vector overexpressing FngMYB10(MU) was injected, the color of the empty fruit did not change, while the color of the fruit overexpressing FngMYB10(MU) turned red, and the color was more obvious (see Figure 2). Figure 6 ), it can be seen that the mutation at position 246 in the FngMYB10 gene sequence plays an important role in the fruit color of yellow-haired strawberry.
[0024] (2) In the present invention, the stable overexpression of the FngMYB10(MU) gene by the leaf disc method can significantly increase the expression levels of anthocyanin synthesis genes and transporter genes in the callus of yellow-haired strawberry, thereby leading to an increase in the pigmentation of the callus and the content of anthocyanins (Appendix Figure 7 ); therefore, these results indicate that changing the single base from T to G at position 246 of the FngMYB10 sequence affects the function and activity of FngMYB10. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 A schematic diagram of single nucleotide mutation and sequencing verification of the present invention;
[0026] Figure 2 It is a schematic diagram of overlap extension PCR of the present invention;
[0027] Figure 3 This is a schematic diagram of the amplification results of the FngMYB10 (MU) gene of the present invention;
[0028] Figure 4 Schematic diagram of the expression vector pCAMBIA1301-EGFP-35S-NOS used in the present invention;
[0029] Figure 5 The three-dimensional structure diagram of the FngMYB10 (MU) protein of the present invention;
[0030] Figure 6 This is a diagram showing transient overexpression of the FngMYB10 (MU) gene of the present invention in yellow-haired strawberry fruit;
[0031] Figure 7 This is a schematic diagram of the stable transformation of the FngMYB10(MU) gene of the present invention in yellow-haired strawberry callus for 6 months.
[0032] Figure 8 This is a diagram showing the verification results of a positive callus batch after the stable transformation of the FngMYB10 (MU) gene of the present invention in yellow-haired strawberry.
[0033] Fig. 9 This is a diagram showing the results of real-time fluorescence quantitative analysis (qRT_PCR) of key genes and regulatory genes in the anthocyanin synthesis pathway after the FngMYB10 (MU) gene of the present invention was stably transformed into yellow-haired strawberry. DETAILED DESCRIPTION
[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0035] The experimental methods in the following examples, unless otherwise specified, are all conventional methods or conditions recommended by the manufacturer.
[0036] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.
[0037] Embodiment 1:
[0038] Obtaining the mutant FngMYB10(MU) gene of yellow-haired strawberry:
[0039] (1) Use ripe yellow-haired strawberry fruit as the material. Plant RNA Kit (omego) was used to extract RNA, and qualified RNA was tested using TaKaRa's PrimeScript TM RT reagent Kit with gDNA Eraser kit reverse transcribes RNA into cDNA;
[0040] (2) Using genomic information as a template, primers were designed and the mutant MYB10 was amplified using overlap extension PCR (see Appendix for the principle). Figure 2 As shown in the figure, primers FngMYB10-F and FngMYB10-Rm are used to amplify the first fragment, and primers FngMYB10-Fm and FngMYB10-R are used to amplify the second fragment by PCR. The two fragments are spliced together by homologous recombination to generate a site-directed mutant MYB10 gene (see Figure 2). Figure 3 ), the primers are shown in Table 1 below:
[0041] Primer Sequence(5'to 3') FngMYB10-F ATGGAGGGTTTCGGTGTGAG FngMYB10-Rm CAATTAAAGACCACCTGTTTCCTAGAAGCTTATGAAGC FngMYB10-Fm CTAGGAAACAGGTGGTCTTTAATTGCCG FngMYB10-R TCATACGTAGGAGATGTTGACTGGATC
[0042] Table 1
[0043] (3) PCR amplification: PCR amplification was performed according to the reaction system in Table 2 below:
[0044]
[0045]
[0046] Table 2
[0047] (4) The PCR reaction procedure was as follows: denaturation at 95°C for 2 min; denaturation at 95°C for 20 sec, annealing at 59°C for 20 sec, extension at 72°C for 1 sec, 40 cycles; 72°C for 5 min, and insulation at 4°C. The PCR products were detected by 1% agarose gel electrophoresis (see Appendix). Figure 3 ), and the target DNA bands were recovered by gel recovery using the Thermo Scientific DNA gel recovery kit; the two fragments were recovered and purified using Hieff Plus One Step Cloning Kit (Yisheng) was used for homologous recombination.
[0048] (5) Use Vazyme's 5min TMTA / Blunt-Zero Cloning Kit to connect the recombinant complete FngMYB10(MU) gene to the cloning vector.
[0049] (6) Transform the ligation product into Escherichia coli, and transform the ligation product into Escherichia coli according to the instructions of the full-form gold Trans1-T1 competent cells; finally, culture at 37°C on LB solid medium containing Kan antibiotics to screen positive clones, sequence and verify, and use Plasmid was extracted using Plasmid MiniPrep Kit to obtain TA-FngMYB10(MU) plasmid.
[0050] (7) The obtained TA-FngMYB10 vector plasmid was used as a template, and the primers P1301-FngMYB10-F and P1301-FngMYB10-R with BamH I and Xba I (Takara, QuickCut) restriction sites were used to amplify the MYB10 gene by PCR. The PCR system and reaction procedure were the same as shown in Table 2 above, and the FngMYB10 (MU) target gene product was obtained.
[0051] (8) The target gene product and pCAMBIA1301-EGFP-35S-NOS vector plasmid (see Appendix) were double-digested with BamH I and Xba I. Figure 4 ); 1% gel electrophoresis was followed by gel purification, and the target fragment of FngMYB10 (MU) and the expression vector pCAMBIA1301 were connected using T4 ligase (Takara) at 16°C overnight; the ligation product was transformed into Trans1-T1 competent cells, positive colonies were identified by PCR, and sequencing was performed (see Appendix Figure 1 ), and finally obtained the 35S:FngMYB10(MU) recombinant plasmid; 700 μL of 50% glycerol was added to every 300 μL of the correctly identified bacterial solution and stored at -80°C.
[0052] Embodiment 2:
[0053] Recombinant expression vector 35S:FngMYB10(MU) was transiently injected into strawberry fruit:
[0054] Referring to the experimental steps in the EHA105 Agrobacterium competence instruction manual, the overexpression vector 35S:FngMYB10(MU) was transferred into the Agrobacterium strain using the freeze-thaw method.
[0055] Specifically, the steps of Agrobacterium-mediated transient transformation of yellow-haired strawberry fruit are as follows:
[0056] (1) Single clones were selected from the screening plate. After the bacterial batch was verified to be positive, they were inoculated into 10 ml of liquid YEB medium containing resistance (YEB + Rif 20 μg / ml + Kan 50 μg / ml), and cultured overnight on a constant temperature shaker at 28°C until the OD value of the bacterial solution reached 0.8-1.0;
[0057] (2) Centrifugation at 5000 rpm for 10 min to collect bacteria;
[0058] (3) Resuspend the cells in MS injection buffer (10 mM MgCl2 + 10 mM MES, pH 5.6 + 200 μM acetosyringone) to dilute the bacterial solution to an OD value of 0.6; culture at 28°C for 2 h to activate the cells;
[0059] (4) Use a 1 ml syringe to draw up the infection solution and use a 32 g needle to inject it into the fruit from the fruit base, and continue to observe the development and ripening process of the strawberry fruit;
[0060] (6) Inject more than 10 fruits for each gene and collect the fruits for testing after 5-7 days.
[0061] Embodiment 3:
[0062] Genetic transformation of Fragaria tumefaciens using the Agrobacterium-mediated leaf disc method was performed as follows:
[0063] (1) Preparation of sterile seedlings: peel off the achenes (seeds) on the surface of strawberry fruits, wash and soak them in PE tubes at 4°C for 7 days for vernalization; before sowing, wash and sterilize them with 75% alcohol, 5% sodium hypochlorite and sterile water in sequence, then sow and germinate (see Table 3 for details), and place them in a culture room at 23°C for cultivation;
[0064] (2) Activation of bacterial solution: The bacterial solution stored at -80°C was streaked and activated in a liquid YEB medium containing resistance, and a single clone was selected for culture; the bacterial solution was centrifuged (5000 rpm, 10 minutes), and the supernatant was discarded; the bacterial solution was resuspended to OD600 = 0.5 using the pre-prepared MS resuspension solution;
[0065] (3) Preparation of explant materials: In a clean bench, use sterile scissors to cut young, shiny leaves from sterile seedlings, remove the leaf tips and petioles, and cut the central part of the leaves into pieces of approximately 0.5 × 0.5 cm. 2 small pieces; placed in the pre-prepared liquid co-culture medium MS1 (see Table 3 for details) for co-cultivation;
[0066] (4) Agrobacterium transformation: Transfer the prepared explants to the resuspension solution, shake and soak for infection for about 10 min, then transfer to sterile filter paper, absorb the surface bacterial liquid and transfer to solid culture medium MS2, and place in an incubator at 23-25°C in the dark for 3 days;
[0067] (5) Induction of callus tissue: After dark culture, the explants were transferred to the liquid co-culture medium MS3 for washing, then transferred to the regeneration solid medium MS4 for dark culture for 4 days, and finally transferred to the screening medium MS5 for dark culture; after 10 days, the explants were transferred to the tissue culture room and cultured at 23°C, 16 h light and 8 h dark; the selected callus was subcultured with MS5 medium about every 15 days, and the callus condition was continuously observed during this period;
[0068] Type of culture medium
[0069] Medium type culture medium formula
[0070] Composition
[0071] Germination medium 1 / 2MS + Sucrose 30g / L + Agar 7g / L, PH = 5.8
[0072] MS resuspension: MS+30g / L Sucrose+5g / L Glucose+200μM AS, PH=5.6
[0073] Liquid culture medium MS1 MS+2.0mg / L TDZ+0.2mg / L IBA+25g / L Sucrose+5g / L Glucose+0.5g / L CH, PH=5.6
[0074] Solid co-culture MS2 MS+2.0mg / L TDZ+0.2mg / L IBA+25g / L Sucrose+5g / L Glucose+0.5g / L CH+7g / L Agar, PH=5.8
[0075] Liquid co-culture MS3 MS+2.0mg / L TDZ+0.2mg / L IBA+25g / L Sucrose+5g / L Glucose+250mg / L Carb+250mg / L Tim+0.5g / L CH, PH=5.6
[0076] Regeneration solid medium MS4 MS+2.0mg / L TDZ+0.2mg / L IBA+30g / L Sucrose+250mg / LCarb+250mg / L Tim+7g / L Agar, PH=5.8
[0077] Screening medium MS5 MS+2.0mg / L TDZ+0.2mg / L IBA+25g / L Sucrose+5g / L Glucose+250mg / L Carb+250mg / L Tim+5mg / L Hyg+7g / L Agar, PH=5.8
[0078] Table 3
[0079] (6) Identification of positive transgenic materials: The positive calli selected by the resistance culture medium were used for DNA extraction. Sterile water and untreated calli (CK) were used as negative controls. PCR detection was performed using hygromycin resistance gene and EGFP gene specific primers (see Table 4). The primers used for detection are shown in Table 4:
[0080] Primer Sequence(5'to 3') EGFP-F ATGGTGAGCAAGGGCGAGGAGCTG EGFP-R CCAGCAGGACCATGTGATCGCGCTTC Hyg-F TACTTCTACACAGCCATCGGTCCAG Hyg-R AAGGAATCGGTCAATACACTACATGGC
[0081] Table 4
[0082] Experimental results:
[0083] The recombinant 35S:FngMYB10(MU) plasmid was sequenced and the results showed (Appendix Figure 1 ), the mutant FngMYB10(MU) was compared with the coding region of the genome sequence, and it was found that the 246th position changed from T to G, and the rest of the sequence was exactly the same as the mutant. After translation into amino acid sequence, the results are as shown in the attached figure. Figure 4 As shown, position 82 changed from Ser (S) to Arg (R). The results were the same as expected, which laid the foundation for the functional study of the FngMYB10 gene.
[0084] Transient injection of 35S:FngMYB10(MU) overexpression plasmid showed that compared with the blank control and empty vector control, the fruit of FngMYB10(MU) overexpressing yellow-haired strawberry changed from white to red (see Appendix Figure 6). Furthermore, Swissmodel was used to predict the protein three-dimensional structure, and Pymol, missense3d, etc. were used to evaluate the protein structure after amino acid mutation. The results show (Appendix Figure 5 ), the 82nd amino acid mutated from arginine Arg to tryptophan Ser, and this replacement led to protein structural conflict (the local conflict score of the mutant FngMYB10 (MU) was 15.61, while the local conflict score of the control FngMYB10 was 42.55), indicating that single-base mutations may affect the stability and function of the protein.
[0085] After testing the transient transformation system in strawberry fruit, the leaf disc transformation method was used for stable genetic transformation of yellow-haired strawberry. The calli of the wild-type control and the empty control were yellow-green, while the calli overexpressing the FngMYB10(MU) gene underwent pigmentation during growth, changing from green to purple-red, indicating that there may be significant accumulation of anthocyanins (see Appendix Figure 7 ). In order to identify transgenic callus, sterile water and untreated callus (CK) were used as negative controls, and PCR detection was performed using hygromycin resistance gene and EGFP gene specific primers. The results showed ( Figure 8 ), the negative control did not amplify the target band, while the transgenic calli amplified the band of corresponding length, indicating that the transgenic calli were positive.
[0086] In the present invention, transient overexpression with injection of MS infection solution as blank control and injection of empty vector as control group (CK) showed that the fruit color of the vector injected with overexpression FngMYB10 (MU) changed significantly, while the fruit color of the empty vector did not change (see Appendix Figure 7 ). In addition, the leaf disc method was used for stable genetic transformation, such as Figure 8 and 9 As shown in the figure, in the callus of yellow-haired strawberry overexpressing the FngMYB10 (MU) gene, the expression levels of anthocyanin synthesis-related genes (FngPAL1, FngCHS, FngF3H, FngDFR, FngUFGT, FngANS, FngANR), regulatory genes (FngBHLH, FngWD40, FngMYB10) and anthocyanin transport gene FngGST were significantly higher than those in the control group callus. These results indicate that changing the single base at position 246 of the FngMYB10 sequence from T to G can promote the synthesis of anthocyanins in yellow-haired strawberry.
[0087] >FngMYB10(702bp)
[0088] ATGGAGGGTTTCGGTGTGAGAAAAGGTGCATGGACTAAAGAGGAAGATGAACTTCTGAAACAGTTCATCGAAATCCATGGAGAAGGCAAATGGCATCATGTTCCTCTCAAATCAGGCTTAAACAGATGCAGGAAGAGCTGTAGACTGAGGTGGCTGAATTATTTGAAGCCGAATA TCAAGAGAGGACAGTTTGCAGAGGATGAAGTTGATTTGATCATCAGGCTTCATAAGCTTCTAGGAAACAGTTGGTCTTTAATTGCCGGAAGATTGCCAGGAAGAACTGCCAATGATGTGAAGAACTATTGGAATACTTATCAAAGGAAAGAGATCAAAAGACGGCTTCATACGCA AAGAAACTGAAAGTTAAATCCCAAGAAAATACAAAAGCCACCACAATTGTAAGACCTTCGACCACGAACCTTCATCAAAAGGTTCAATTTTACAGAGAGATATGAAAATATAGAGCATAATCATTCAGAAATGAGTTATACCAGTTCTTTACCAATAGCACCACCACAGACTCTACAATTAGAAAATGTAACTGATTGGTGGAAAGATTTCGCAGAAGACAGTACAGAGAGGATTGATAGAACAATGTGTTCTGGTCTTGGTTTGGAGGATCATGACTTCTTCACAAACTTTTGGGTTGAGGATATGGTACAATCGGCAAGCAATGATCCAGTCAACATCTCCTACGTATGA
[0089] >FngMYB10(1-234)
[0090] MEGFGVRKGAWTKEEDELLKQFIEIHGEGKWHHVPLKSGLNRCRKSCRLRWLNYLKPNIKRGQFAEDEVDLIIRLHKLLGNSWSLIAGRLPGRTANDVKNYWNTYQRKKDQKTASY AKKLKVKSQENTKATTIVRPRPRTFIKRFNFTERYENIEHNHSEMSYTSSLPIAPPQTLQLENVTDWWKDFAEDSTERIDRTMCSGLGLEDHDFFTNFWVEDMVQSASNDPVNISYV
[0091] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. The MYB transcription factor FngMYB10 gene that regulates the fruit color of yellow-haired strawberry is characterized by: include: S1. The nucleotide sequence of the FngMYB10 gene is shown in SEQ ID No: 1; S2. The amino acid sequence of the FngMYB10 gene is shown in SEQ ID No: 2; S3. The FngMYB10 nucleotide sequence shown in SEQ ID No: 3 was site-directedly mutated into FngMYB10 (MU) (c.246G>T, p.Arg82Ser) by homologous recombination. Transient transformation and stable genetic transformation of S4 and FngMYB10(MU) genes can change the anthocyanin content of yellow-haired strawberry; S5. The key genes in anthocyanin-related pathways were significantly upregulated after overexpression of the FngMYB10 (MU) gene.
2. The MYB transcription factor FngMYB10 gene for regulating the fruit color of yellow-haired strawberry according to claim 1, characterized in that: The yellow-haired strawberry MYB10 gene is obtained as follows: The expression vector is pCAMBIA1301-EGFP-35S-NOS.
3. The MYB transcription factor FngMYB10 gene for regulating the fruit color of yellow-haired strawberry according to claim 1, characterized in that: The method for regulating the fruit color of yellow-haired strawberry by using the FngMYB10 gene comprises the following steps: S1, amplification of mutant FngMYB10 using homologous recombination method; S2, construction of pCAMBIA1301-EGFP-35S-FngMYB10 overexpression vector; S3, transform Agrobacterium and identify positive colonies; S4, using the injection method (transient transformation) to infect yellow-haired strawberry fruits; S5, using the leaf disc method to stably transform the callus of yellow-haired strawberry; S6. Application of the stable transformation medium formula by the leaf disc method.
4. Use of the FngMYB10 gene according to any one of claims 1 to 3 in regulating the fruit color and anthocyanin content of yellow-haired strawberry.