A MsCBR3 gene of alfalfa, its encoded protein, and its applications.

Abstract

This invention discloses a type of alfalfa MsCBR3 This invention relates to genes, their encoded proteins, and their applications, falling under the field of plant genetic engineering technology. This invention discovers an alfalfa gene. MsCBR3 By constructing alfalfa MsCBR3 Gene overexpression plants were found in alfalfa MsCBR3 Compared with control plants, overexpressing plants showed significantly increased numbers and total length of secondary branches, aboveground fresh weight, and the number of branches and leaves after cutting. This led to the determination of... MsCBR3 Genes play a regulatory role in the biomass of alfalfa. Therefore, this invention provides a basis for breeding high-yield alfalfa plants and related breeding work, and has great application value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to an alfalfa species. MsCBR3 Genes and their encoded proteins and their applications. Background Technology

[0002] alfalfa ( Medicago sativa Alfalfa (L.) is an important perennial leguminous forage crop and one of the world's most important temperate leguminous forage crops. Alfalfa has good forage production potential; for alfalfa with root nodules, high dry matter content can be obtained without nitrogen fertilizer. Furthermore, alfalfa is an important multifunctional crop, rich in minerals such as calcium, magnesium, potassium, iron, and zinc, as well as vitamins and other nutrients. Alfalfa also plays an important role in ecosystems such as crop rotation, significantly improving soil fertility, soil structure, and soil ecology. my country cultivates approximately 70 million mu of alfalfa, but the alfalfa industry lags far behind staple crops like rice and wheat, with significant room for improvement in both yield and quality per unit area. Meanwhile, with changing dietary habits, the demand for alfalfa in the domestic forage industry is increasing daily. Therefore, identifying genes that regulate biomass in alfalfa and conducting research on their regulatory networks is of significant application value and guiding significance for creating high-yielding new germplasm and breeding new alfalfa varieties. Summary of the Invention

[0003] To address the aforementioned shortcomings of existing technologies, the objective of this invention is to provide an alfalfa... MsCBR3 Genes and their encoded proteins and applications to increase alfalfa biomass.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A type of alfalfa is provided. MsCBR3 Gene, MsCBR3 The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.1.

[0005] This invention provides the above-mentioned alfalfa MsCBR3 The protein encoded by the gene MsCBR3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0006] This invention provides the above-mentioned alfalfa MsCBR3 Application of genes in regulating alfalfa biomass.

[0007] Furthermore, regulating alfalfa yield was achieved through overexpression. MsCBR3 After gene modification, the number of secondary branches in alfalfa increases.

[0008] Furthermore, regulating alfalfa yield is achieved through overexpression. MsCBR3 After gene modification, the total length of secondary branches in alfalfa increases.

[0009] Furthermore, regulating alfalfa yield was achieved through overexpression. MsCBR3 After gene therapy, the fresh weight of the aboveground parts of alfalfa increased.

[0010] Furthermore, regulating alfalfa yield was achieved through overexpression. MsCBR3 After gene therapy, the number of branches after cutting alfalfa increases.

[0011] Furthermore, regulating alfalfa yield was achieved through overexpression. MsCBR3 After gene therapy, the number of leaves increased after alfalfa was cut.

[0012] The present invention also provides a formulation for increasing alfalfa yield, the formulation comprising the above-mentioned... MsCBR3 Genes or the above MsCBR3 The protein encoded by the gene.

[0013] This invention has the following beneficial effects: This invention discovers an alfalfa gene. MsCBR3 By constructing alfalfa MsCBR3 Gene overexpression plants were found in alfalfa MsCBR3 Compared with control plants, overexpressing plants showed significantly increased numbers and total length of secondary branches, aboveground fresh weight, and the number of branches and leaves after cutting. This led to the determination... MsCBR3 Genes play a regulatory role in the biomass of alfalfa. Therefore, this invention provides a basis for breeding high-yield alfalfa plants and related breeding work, and has great application value. Attached Figure Description

[0014] Figure 1 for MsCBR3 Diagram of in situ hybridization of genes;

[0015] Figure 2 for MsCBR3 Positive identification image of transgenic alfalfa with overexpressed gene;

[0016] Figure 3 for MsCBR3 Expression level of transgenic alfalfa with gene overexpression;

[0017] Figure 4 for MsCBR3 Phenotypic analysis of transgenic plants with gene overexpression; Figure A shows the phenotype of the transgenic plants with gene overexpression; Figure B shows the number of secondary branches of the transgenic plants with gene overexpression; Figure C shows the total length of secondary branches; Figure D shows the fresh weight of the transgenic plants with gene overexpression.

[0018] Figure 5 for MsCBR3Overexpression of the number of leaves and branches of the plant after cutting. Detailed Implementation

[0019] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0020] In the following examples, alfalfa transformed into Gongnong No. 1 plants, which were preserved in our laboratory. Experimental reagents included an RNA extraction kit (Omega), a reverse transcription kit (TAKARA), a quantitative PCR kit (TAKARA), a gel extraction kit (Polymerex), a plasmid extraction kit (Quanshijin), a homologous recombination ligase (Quanshijin), NSH medium (Coollab), and MS medium (Phyto Tech). Other commonly used biochemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. All quantitative experiments in the following examples were performed in at least three replicates, and the results were averaged.

[0021] Example 1: Alfalfa MsCBR3 Cloning of genes

[0022] (1) Alfalfa MsCBR3 Gene sequence information and phylogenetic analysis: Based on the analysis of the transcriptome at different stages of alfalfa shoot formation, a gene encoding the AP2 transcription factor family was found to be specifically expressed at different stages of shoot initiation. Therefore, we named this gene... CROWN BUD REGULATOR 3 ( MsCBR3 Based on the genomes provided by the MODMS alfalfa database, the genomes of the alfalfa varieties Xinjiang Daye and Zhongmu No. 1 were obtained. MsCBR3 Gene sequence information.

[0023] (2) MsCBR3 Gene cloning: RNA was extracted from the lateral bud tissue of the transformed variety Gongnong 1 and reverse transcribed to obtain cDNA from the lateral bud tissue of Gongnong 1. Using this cDNA as a template, PCR amplification was performed using primer pairs composed of F1 and R1. After amplification, the products were recovered and sequenced for comparison with alfalfa Gongnong 1. MsCBR3 The cDNA sequence of the gene. Among them, MsCBR3 The CDS nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; the nucleotide sequences of F1 and R1 are shown below:

[0024] F1: 5'-ATGAGTAACTGGTTAGGATTT-3' (SEQ ID NO.3);

[0025] R1: 5'-TCATTCATTCCACAAAGCA-3' (SEQ ID NO. 4).

[0026] The PCR reaction system is shown in Table 1.

[0027] Table 1 PCR reaction system (50 μL)

[0028]

[0029] The PCR reaction procedure is shown in Table 2.

[0030] Table 2 PCR reaction procedure

[0031]

[0032] Example 2: Alfalfa MsCBR3 In situ hybridization expression localization of genes in lateral buds

[0033] (1) Alfalfa MsCBR3 Synthesis of gene probes: Based on the obtained alfalfa... MsCBR3 Gene sequence information (SEQ ID NO.1), and specific primer pairs F2 and R2 with a length of 300bp to 400bp were designed. PCR amplification was performed using cDNA from alfalfa lateral bud tissue as a template. The PCR product was recovered and ligated into the pEASY-blunt vector (Quanshijin) to obtain a plasmid containing the target fragment. Using this plasmid as a template, the target fragment was amplified by PCR using T7 (promoter) and F2 and R2, respectively, and then purified. The purified target fragment was transcribed, and the quality of the positive and negative probes was finally detected by agarose gel electrophoresis. The nucleotide sequences of T7, F2, and R2 are shown below:

[0034] F2: 5'-AGAACTTCTCAATACCGTGGTG-3' (SEQ ID NO.5);

[0035] R2: 5'-GAATTGAAGTCCCTCGGCTGT-3' (SEQ ID NO. 6);

[0036] T7: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO. 7).

[0037] (2) Sample preparation: Stem nodes and cotyledon nodes of alfalfa seedlings at different growth stages after germination were collected, fixed overnight in 4wt% paraformaldehyde at 4℃, dehydrated by gradient ethanol, cleared with xylene, and embedded in paraffin. The section thickness was set to 8-10μm, attached to poly-L-lysine slides, dried and ready for use.

[0038] (3) Sample tissue pretreatment: After dewaxing and rehydration to 1×PBS solution using xylene and gradient ethanol in sequence, the sections were immersed in proteinase K solution and incubated at 37°C for 10 minutes, and the reaction was terminated with 1×PBS. Subsequently, the sections were immersed in 4wt% paraformaldehyde to fix RNA, and after dehydration by gradient ethanol, the sections were removed and dried at room temperature.

[0039] (4) In situ hybridization: The hybridization buffer containing the probe is evenly covered on the slide, and the slide is placed in a humidified box and hybridized overnight in an oven at 55°C.

[0040] (5) Elution and blocking: Two rounds of elution were repeated in a 65°C water bath using 2×SSC buffer and 50wt% formamide. Then, RNase A digestion solution was used to remove unhybridized single-stranded RNA probes. Antibody was then blocked, and anti-DIG-AP (Anti-Digoxigenin-Alkaline Phosphatase Conjugate) antibody was added to the blocking solution for incubation.

[0041] (6) Antibody staining: Add nitroblue tetrazolium / 5-bromo-4-chloro-3-indole phosphate (NBT / BCIP) chromogenic solution in the dark and incubate at a suitable temperature for 24 hours. Observe the progress of color development. When the signal appears, terminate the reaction with TE (Tris-EDTA Buffer) solution and mount the slide.

[0042] In situ hybridization results as follows Figure 1 As shown. By Figure 1 It can be seen that, MsCBR3 Gene signals are concentrated in axillary buds and meristems. Therefore, MsCBR3 The gene is mainly expressed in axillary buds and meristems.

[0043] Example 4 MsCBR3 Obtaining transgenic alfalfa with gene overexpression

[0044] (1) Overexpression MsCBR3 Transgenic vector construction: The plant overexpression vector pCAMBIA3301 (purchased from Zhuangmeng) was digested with restriction endonucleases NcoⅠ and BglⅡ, and the F3 and R3 cells were amplified using primer pairs. MsCBR3 The CDS region of the gene is used to obtain the vector arm. MsCBR3 The CDS sequence of the gene was obtained. The vector and target fragment were recombined via seamless cloning, and the recombinant product was transformed into DH5α competent cells. Overexpression was confirmed by positive detection and sequencing. MsCBR3 The recombinant plasmid p35S::MsCBR3 was used. The nucleotide sequences of F3 and R3 are shown below:

[0045] F3: 5'-ACGGGGGACTCTTGACCATGGATGAGTAACTGGTTAGGATTT-3' (SEQ ID NO. 8);

[0046] R3: 5'-AGAAATTTACCCTCAGATCTTCATTCATTCCACAAAGCA-3' (SEQ ID NO. 9).

[0047] (2) MsCBR3 Obtaining transgenic alfalfa with overexpression: The recombinant plasmid was transformed into Agrobacterium EHA105 competent cells to obtain positive recombinant Agrobacterium, which was then frozen at -80℃ and stored in glycerol. The alfalfa variety Gongnong 1 was transformed using the Agrobacterium-mediated leaf disc method. The specific operational steps are as follows:

[0048] ① Streak Agrobacterium EHA105 containing the target vector on solid LB medium and incubate at 28°C for 2 days. Pick positive single clones and inoculate them into 3-5 mL of LB liquid medium containing rifampicin (50 μg / mL) and kanamycin (10 μg / mL) and incubate overnight at 28°C in a shaker.

[0049] ② Inoculate the activated bacterial culture into 50 mL of LB liquid medium containing the above-mentioned concentration of antibiotics and culture until the OD600 value is about 0.3;

[0050] ③ Centrifuge the bacterial culture at 2,800g for 10 min and discard the supernatant. Resuspend the bacterial culture precipitate in NSDK liquid medium containing auxin, cytokinin and acetylsuccinone (see Table 3);

[0051] ④ Select tender, disease-free leaves of Gongnong No. 1, disinfect the leaves with a 10% sodium hypochlorite solution (containing 0.1% surfactant Triton X-100) for 10 minutes, rinse them 5 times with sterile water, and then place them on a clean bench to dry.

[0052] ⑤ After drying the leaves, place them in the resuspension solution, vacuum for 15 minutes, sonicate for 2 minutes, and after they have all settled to the bottom, take out the leaves in the ultra-clean workbench and use sterile filter paper to absorb the excess bacterial liquid on the surface of the leaves.

[0053] ⑥ Use sterilized forceps to transfer the explants to the NSDK co-culture medium and incubate in the dark at 22°C for 2 days; the NSDK co-culture medium has the same composition as the liquid medium in step ③, but with the addition of Phytagel.

[0054] ⑦ Two days later, the explants were transferred to callus induction medium (NSDKB) containing herbicide to induce callus formation in the dark, and subcultured every two weeks; the formulation of NSDKB callus induction medium is shown in Table 4.

[0055] ⑧ Transfer the callus tissue to shoot regeneration medium (MSBK) and place it in a 22℃ constant temperature and light incubator (16h light / 8h dark). Subculture every 2 weeks until shoot regeneration occurs. The formula of MSBK shoot regeneration medium is shown in Table 5.

[0056] ⑨ Transfer the regenerated buds to 1 / 2 MS medium and place them in a 22℃ constant temperature and light incubator (16h light / 8h darkness) to induce rooting. Once the plants have grown a little, transplant them into the soil.

[0057] ⑩ DNA was extracted from the young leaves of the regenerated plants, and primer pairs F4 and R4 were used to identify the DNA level of the regenerated plants, resulting in 8 positive plants (see...). Figure 2 P is the positive control recombinant plasmid p35S::MsCBR3, CK is the negative control, and 1-10 are the transgenic plant numbers. The PCR reaction system is shown in Table 6, and the PCR reaction procedure is shown in Table 7. The nucleotide sequences of F4 and R4 are shown below:

[0058] F4: 5'-CATTTCATTTGGAGAGAACAC-3' (SEQ ID NO. 10);

[0059] R4: 5'-AAAACTAGAAATTTACCCTCAGAT-3' (SEQ ID NO. 11).

[0060] Table 3 NSDK co-culture medium formulation

[0061]

[0062] Table 4 NSDKB callus induction medium formulation

[0063]

[0064] Table 5 MSBK bud regeneration medium formulation

[0065]

[0066] Table 6 PCR reaction system (50 μL)

[0067]

[0068] Table 7 PCR reaction procedure

[0069]

[0070] (3) Genetically modified alfalfa MsCBR3Expression level identification: Total RNA was extracted from leaves of regenerated negative control plants (CK) and positive plants (OE4, OE8, and OE10), and cDNA was reverse transcribed. Quantitative PCR amplification was performed using specific primer pairs with cDNA as a template; the primer pair composed of F5 and R5 was used for identification. MsCBR3 Gene expression levels; primer pairs composed of F6 and R6 were used to identify the internal reference gene. The nucleotide sequences of F5 and R5, and F6 and R6 are shown below:

[0071] F5: 5'-GTAATTTAGCACACAAGAGGAGGC-3' (SEQ ID NO. 12);

[0072] R5: 5'-GAATTGAAGTCCCTCGGCTGT-3' (SEQ ID NO. 13);

[0073] F6: 5'-GGCTCCACCAGAGAGAAAGTACAGT-3' (SEQ ID NO. 14);

[0074] R6: 5'-GCCAGACTCGTCATATTCACCCTTG-3' (SEQ ID NO. 15).

[0075] Depend on Figure 3 It can be seen that, MsCBR3 Gene expression levels were significantly upregulated in transgenic plants.

[0076] Example 5 MsCBR3 Phenotypic analysis of transgenic plants with overexpressed genes

[0077] Select negative control plants (CK) and alfalfa MsCBR3 After overexpressing plants (OE4, OE8, and OE10) were propagated by cuttings, plants with uniform growth were selected for unified management. Figure 4 AC indicates that... MsCBR3 The overexpressing plants had significantly more secondary branches (i.e., lateral branches) than the negative control plants; at the same time, MsCBR3 The secondary branches of overexpressing plants are significantly longer. Figure 4 D indicates that... MsCBR3 The fresh weight of the overexpressing plants was significantly higher than that of the negative control plants. After harvesting, MsCBR3 The overexpressing plants also had significantly more branches and leaves than the negative control plants (see...). Figure 5 These all indicate MsCBR3 The gene has the potential to increase yield both before and after alfalfa harvesting. Therefore, it is evident that... MsCBR3 Genes involved in regulating alfalfa biomass, overexpression MsCBR3Following gene modification, alfalfa biomass increased significantly. Therefore, this invention provides a basis for breeding high-yield alfalfa plants and related breeding work, and has great application value.

[0078] In this invention MsCBR3 The nucleotide sequence of the gene's CDS region and the amino acid sequence of its encoded protein are as follows:

[0079]

[0080] (2) The amino acid sequence encoding the protein: (SEQ ID NO.2).

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Overexpression of alfalfa MsCBR3 The application of genes in increasing alfalfa biomass is characterized by, The MsCBR3 The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.1; MsCBR3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The increase in alfalfa biomass is achieved through overexpression. MsCBR3 After gene modification, the number of secondary branches in alfalfa increases.

3. The application according to claim 1, characterized in that, The increase in alfalfa biomass is achieved through overexpression. MsCBR3 After gene modification, the total length of secondary branches in alfalfa increases.

4. The application according to claim 1, characterized in that, The increase in alfalfa biomass is achieved through overexpression. MsCBR3 After gene therapy, the fresh weight of the aboveground parts of alfalfa increased.

5. The application according to claim 1, characterized in that, The increase in alfalfa biomass is achieved through overexpression. MsCBR3 After gene therapy, the number of branches after cutting alfalfa increases.

6. The application according to claim 1, characterized in that, The increase in alfalfa biomass is achieved through overexpression. MsCBR3 After gene therapy, the number of leaves increased after alfalfa was cut.

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