Application of apple MdMYB41L gene in regulation and control of seed size
By overexpressing the apple MdMYB41L gene in Arabidopsis thaliana, the technical bottleneck of seed size improvement in apple breeding has been solved, resulting in a significant increase in seed size. This provides a new tool for breeding and shortens the breeding cycle.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LIAOCHENG UNIV
- Filing Date
- 2026-01-29
- Publication Date
- 2026-04-24
AI Technical Summary
Traditional apple breeding faces significant technical bottlenecks in the genetic improvement of seed size traits, and apples are perennial woody plants with long breeding cycles.
By overexpressing the apple MdMYB41L gene, a recombinant vector was constructed and transformed into Arabidopsis thaliana to achieve overexpression of the MdMYB41L protein to regulate seed size.
It significantly increases the width, length, and surface area of Arabidopsis seeds, providing genetic resources for improving plant seed size, shortening the breeding cycle, and improving seed propagation efficiency.
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Figure CN121914239A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of the apple MdMYB41L gene in seed regulation and breeding. Background Technology
[0002] Seed size is one of the important agronomic traits of fruit trees. It not only directly affects the seed propagation efficiency and seedling cost, but is also closely related to fruit size, yield and quality.
[0003] Among them, MYB transcription factors are widely present in plants and participate in multiple processes such as plant growth and development, secondary metabolism and stress response.
[0004] However, in traditional apple breeding practices, there are significant technical bottlenecks in the genetic improvement of seed size traits.
[0005] Apples are perennial woody plants with a long juvenile period, taking several years from hybridization and sowing to flowering and fruiting.
[0006] Therefore, identifying key genes that regulate apple seed size and developing stable and efficient molecular marker tools will provide technical support for achieving efficient molecular design breeding of plant seed size.
[0007] Therefore, the invention described in this application is needed. Summary of the Invention
[0008] The purpose of this invention is to provide a method for regulating seed size using the apple MdMYB41L gene, thereby increasing seed size through overexpression of MdMYB41L.
[0009] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an apple MdMYB41L protein, characterized in that its amino acid sequence is as shown in SEQ ID NO: 2.
[0010] In a second aspect, the present invention provides an MdMYB41L gene, characterized in that it encodes the MdMYB41L protein as described in claim 1.
[0011] Preferably, the CDS nucleotide sequence is as shown in SEQ ID NO: 1.
[0012] Thirdly, the present invention provides the application of a recombinant vector containing the MdMYB41L gene in regulating plant seed size.
[0013] As a preferred method, the plant is Arabidopsis thaliana.
[0014] Furthermore, the specific manifestations of the regulation of plant seeds are as follows: compared with wild-type Arabidopsis, the width, length and surface area of Arabidopsis seeds of the overexpression line are significantly increased; the cotyledon size of the overexpression line is significantly larger than that of the wild type.
[0015] Fourthly, the present invention provides a plant breeding method, characterized in that the method includes the following steps: (1) Vector construction: Based on the gene sequence, corresponding primers were designed. Using apple GL-3 cDNA as a template, PCR reaction was performed, and the results were detected and recovered by gel extraction. The target gene fragment was ligated with the cloning vector, and after ligation, it was transformed and screened for bacteria by PCR. After successful sequencing, the plasmid and expression vector pZP211 were digested with enzymes, and the corresponding fragments were recovered. T4 ligation reaction was performed. Bacterial screening and sequencing were performed, and the plasmid was extracted. The recombinant plasmid was transferred into Agrobacterium GV3101. (2) Genetic transformation of Arabidopsis thaliana: After disinfection, Arabidopsis thaliana seeds were vernalized at 4℃ for 3 days and grown on culture medium for 7 days before being transplanted into nutrient pots; Agrobacterium was activated and Agrobacterium precipitate was collected; the bacterial cells were suspended in the infection solution, and the inflorescence of Arabidopsis thaliana was soaked in the infection solution for 15 seconds and then left overnight in the dark; after 3 infections, the seeds were harvested after they matured for use. (3) Screening of positive plants: After disinfecting the Arabidopsis seeds, they were spread on a medium containing kanamycin for screening to obtain resistant seedlings; the selected transgenic Arabidopsis were tested at the DNA and transcription levels.
[0016] Preferred primer sequences for PCR amplification are: MdMYB41L-F: GGATCCATGGTTAGAGCCCCTTGTTG; MdMYB41L-R:GTCGACATCTGCAAGAACATACTCTAGAA.
[0017] The beneficial effects of this invention are: it identifies and verifies the positive regulatory effect of the MdMYB41L gene on apple seed size, which can provide new gene resources for improving plant seed size and has important application value in plant breeding. Attached Figure Description
[0018] Figure 1 DNA level identification of MdMYB41L transgenic Arabidopsis thaliana; Figure 2 Expression levels of MdMYB41L in wild-type and MdMYB41L transgenic Arabidopsis thaliana; Figure 3Seed size, note: (A) Phenotype of wild-type and MdMYB41L transgenic Arabidopsis seeds; (BD) Weight (B), width (C), and length (D) of wild-type and MdMYB41L transgenic Arabidopsis seeds. Figure 4 Cotyledon size, Note: (A) Arabidopsis cotyledon phenotype after 5 and 9 days of growth on 1 / 2 MS medium; (BC) Arabidopsis cotyledon surface area after 5 days (B) and 9 days (C) of growth on 1 / 2 MS medium. Detailed Implementation
[0019] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description.
[0020] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.
[0021] Unless otherwise specified, the test methods in the following examples are conventional methods.
[0022] Unless otherwise specified, all reagents and materials used can be purchased from the market.
[0023] Example 1: Cloning of the MdMYB41L gene and construction of the overexpression vector Design appropriate primers based on the gene sequence and add suitable restriction enzyme sites (F: GGATCCATGGTTAGAGCCCCTTGTTG; R: GTCGACATCTGCAAGAACATACTCTAGAA).
[0024] Using apple GL-3 cDNA as a template, PCR was performed. After the reaction, 1.0% agarose gel electrophoresis was used to detect whether the amplified band was the target band.
[0025] The rubber is then cut and recycled.
[0026] The target gene fragment was ligated to the cloning vector at 25°C, and the ligation product was transformed into E. coli and cultured overnight at 37°C.
[0027] Positive clones were selected, and sequencing was performed after shaking the culture.
[0028] After confirming that the sequencing results were correct, plasmid DNA was extracted using a plasmid mini-prep kit.
[0029] The plasmid and expression vector pZP211 were digested with enzymes at BamH1 and Sal1 sites, and the corresponding fragments were recovered after detection by agarose gel electrophoresis. The target gene fragment was then ligated with the digested pZP211 vector using a T4 ligation reaction.
[0030] The ligation product was transformed into E. coli, positive clones were picked, and sequencing was performed after shaking.
[0031] Analyze the sequencing results and extract plasmid DNA.
[0032] The plasmid was transformed into Agrobacterium GV3101, and single colonies were picked to screen for positive clones.
[0033] 1) PCR reaction system (50 μL): After mixing, place in PCR and run under the following conditions: 95℃, 3 min; 95℃, 15 s; 52℃, 15 s; 72℃, 60 s; 35 cycles, 72℃, 5 min.
[0034] 2) Connection reaction Reaction system (5 μL): After mixing, connect at 25 ℃ for 5 min.
[0035] 3) Enzyme digestion of plasmid DNA Reaction system (40 µL): Enzyme digestion was performed in a metal bath at 37°C for 30 min.
[0036] 4) T4 connection reaction: 16°C overnight connection.
[0037] The CDS nucleotide sequence of the apple MdMYB41L gene is shown in SEQ ID NO: 1 below:
[0038] The amino acid sequence of the apple MdMYB41L protein, SEQ ID NO: 2, is as follows: MVRAPCCDEESSLKKGPWTTEEDAKLMDYISRNGHGSWRALPKLAGLNRCGKSCRLRWTNYLRPDIKRGKFSEEEERVIINLHSVLGNKWSKIATHLPGRTDNEIKNYWNTQLRKKLLHMGIDPNTHRPRTDLNQLLDLSRLLSVALVGNSNKMTSPWDNALKLKAAAGAAELTKMQLL QHLYMMPVVRTSNSSVPNNYNVDLMNPSSSLFGSHILNPFGGGHVSGASTMLSGQEWNQQADIVDGFHAISNSLEGFEGGFGAQGVISRNSPDQENMSSSCCYDDIYVQTKNHPLPALVSSSSSVYPGTSPSAANQMEISMSGPAAPARTAIFDAWEKLMDDETSESYWKDILEYVLAD.
[0039] Example 2: Genetic transformation of Arabidopsis thaliana (1) Arabidopsis seeds were disinfected with 75% ethanol for 2 min, 2% NaClO for 10 min, washed 4 times with sterile water, vernalized at 4℃ for 3 days, spread on 1 / 2 MS medium, and cultured under long-day conditions for 7 days before being transplanted into nutrient pots.
[0040] (2) Select wild-type Arabidopsis thaliana with good growth status at 4 weeks old.
[0041] (3) Activate Agrobacterium, centrifuge at 5000 rpm for 5 min, and collect the Agrobacterium precipitate; suspend the bacterial cells in the infection solution to make the final concentration OD600 value about 0.8; Infection solution formula: 5% sucrose, 0.05% Silwet L-77.
[0042] (4) The inflorescences of Arabidopsis thaliana were soaked in the infection solution for 15 seconds and then placed horizontally in the dark overnight.
[0043] Continue culturing in long-day conditions.
[0044] It was infected once every 7 days, for a total of 3 times.
[0045] Harvest the seeds after they mature for later use.
[0046] Identification of transgenic Arabidopsis (1) After disinfection, Arabidopsis seeds were spread on 1 / 2 MS medium containing 30 mg / L kanamycin for screening to obtain resistant seedlings.
[0047] DNA was extracted from leaves of wild-type and transgenic Arabidopsis thaliana according to the instructions of the plant DNA extraction kit, and plants containing positive clones were screened.
[0048] Primers for the target gene were designed (F: ATGGTTAGAGCCCCTTGTTG; R: ATCTGCAAGAACATACTCTAGAA), and PCR reactions were performed using wild-type and transgenic Arabidopsis DNA as templates, respectively.
[0049] After the reaction was completed, 1.0% agarose gel electrophoresis was used to detect whether the target band was present.
[0050] The results showed that positive clones were detected in all three transgenic lines (OE1, OE2, and OE3), and the band size of the positive clones was consistent with that of the target gene band, while no band was observed in the wild type. Figure 1 ).
[0051] T2 generation seeds containing positive clones were collected and screened on a resistant (30 mg / L kanamycin) medium to obtain homozygous transgenic plants.
[0052] (2) RNA was extracted from the leaves of wild-type and transgenic Arabidopsis thaliana according to the instructions of the plant RNA extraction kit.
[0053] RNA is reverse transcribed into cDNA.
[0054] The expression of MdMYB41L in transgenic Arabidopsis thaliana was detected by real-time fluorescence quantitative PCR.
[0055] The results showed that the expression level of MdMYB41L in transgenic Arabidopsis thaliana was significantly higher than that in wild-type ( Figure 2 ).
[0056] Ultimately, three MdMYB41L overexpression lines were obtained.
[0057] Arabidopsis seed size Weigh the mature dry seeds.
[0058] Each replicate contained 500 seeds and was weighed using an electronic analytical balance.
[0059] MdMYB41L overexpressing and wild-type Arabidopsis seeds were observed under an SZX16 microscope (Olympus, Tokyo, Japan), and the length, width, and surface area of the seeds of wild-type and overexpressing plants were measured using ImageJ.
[0060] The results showed that, compared with the wild type, the MdMYB41L overexpression plants produced significantly larger seeds. Figure 3 A).
[0061] Further analysis showed that MdMYB41L overexpression significantly increased seed width, length, and surface area compared to the wild type. Figure 3 BD).
[0062] The weight of overexpressed seeds was also significantly higher than that of wild-type seeds.
[0063] In addition, we also observed Arabidopsis thaliana grown on 1 / 2 MS medium for 5 and 9 days and found that the cotyledon size of the overexpression lines was significantly larger than that of the wild type. Figure 4 ).
[0064] The above research results indicate that the apple MYB transcription factor MdMYB41L positively regulates plant seed size.
[0065] Seed size is one of the important agronomic traits of fruit trees, affecting seed propagation efficiency and seedling costs, and is closely related to fruit size, yield and quality.
[0066] The discovery that apple MdMYB41L regulates seed size provides an important tool for modern molecular design breeding.
[0067] Developing functional molecular markers based on MdMYB41L can enable precise genotypic selection during the seedling stage, shortening the breeding cycle.
[0068] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.
Claims
1. Apple MdMYB41L protein, characterized in that, The amino acid sequence is shown in SEQ ID NO:
2.
2. The apple MdMYB41L gene, characterized by, Encoding the MdMYB41L protein as described in claim 1.
3. The MdMYB41L gene according to claim 2, characterized in that, The CDS nucleotide sequence is shown in SEQ ID NO:
1.
4. The application of the recombinant vector containing the MdMYB41L gene as described in claim 2 in regulating plant seed size.
5. The application according to claim 4, characterized in that, The plant in question is Arabidopsis thaliana.
6. The application according to claim 5, characterized in that, The specific manifestations of the regulation of plant seeds are as follows: compared with wild-type Arabidopsis, the width, length and surface area of Arabidopsis seeds of the overexpression line are significantly increased; the cotyledon size of the overexpression line is significantly larger than that of the wild type.
7. A plant breeding method, characterized in that, The method includes the following steps: (1) Vector construction: Based on the gene sequence, corresponding primers were designed. Using apple GL-3 cDNA as a template, PCR reaction was performed, and the results were detected and recovered by gel extraction. The target gene fragment was ligated with the cloning vector, and after ligation, it was transformed and screened for bacteria by PCR. After successful sequencing, the plasmid and expression vector pZP211 were digested with enzymes, and the corresponding fragments were recovered. T4 ligation reaction was performed. Bacterial screening and sequencing were performed, and the plasmid was extracted. The recombinant plasmid was transferred into Agrobacterium GV3101. (2) Genetic transformation of Arabidopsis thaliana: After disinfection, Arabidopsis thaliana seeds were vernalized at 4℃ for 3 days and grown on culture medium for 7 days before being transplanted into nutrient pots; Agrobacterium was activated and Agrobacterium precipitate was collected; the bacterial cells were suspended in the infection solution, and the inflorescence of Arabidopsis thaliana was soaked in the infection solution for 15 seconds and then left overnight in the dark; after 3 infections, the seeds were harvested after they matured for use. (3) Screening of positive plants: After disinfecting the Arabidopsis seeds, they were spread on a medium containing kanamycin for screening to obtain resistant seedlings; the selected transgenic Arabidopsis were tested at the DNA and transcription levels.
8. The plant breeding method according to claim 7, characterized in that, The primer sequences for PCR amplification are as follows: MdMYB41L-F: GGATCCATGGTTAGAGCCCCTTGTTG; MdMYB41L-R:GTCGACATCTGCAAGAACATACTCTAGAA.