Medicago sativa MsCBR3 gene as well as encoding protein and application thereof

Abstract

The invention discloses a medicago sativa MsCBR3 gene as well as an encoding protein and application thereof, and belongs to the technical field of plant genetic engineering. According to the invention, a medicago sativa gene MsCBR3 is found, and through construction of a medicago sativa MsCBR3 gene overexpression plant, it is found that compared with a control plant, the number and total length of secondary branches of the plant, the fresh weight of an overground part, and the number of branches and the number of leaves after cutting are significantly increased. Therefore, it is determined that the MsCBR3 gene has a regulation effect on the biomass of medicago sativa. Therefore, the invention provides a basis for cultivating high-yield alfalfa plants and breeding the alfalfa plants, and has great application value.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly relates to a Medicago sativa MsCBR3 gene, its encoded protein and application. Background Art

[0002] Medicago sativa ( Medicago sativa L.) is an important perennial leguminous forage crop and is one of the important temperate leguminous forage crops in the world. Medicago sativa has good forage production potential. For Medicago sativa with established root nodules, high dry matter of forage can be obtained without applying nitrogen fertilizer. In addition, Medicago sativa is also an important multi-functional crop, which contains rich minerals such as calcium, magnesium, potassium, iron, zinc and nutrients such as vitamins. Medicago sativa also plays an important role in ecological systems such as farmland rotation, and can significantly improve soil fertility, soil structure and soil ecology. In China, the planting area of Medicago sativa is about more than 70 million mu, but the development of the Medicago sativa industry lags far behind that of staple crops such as rice and wheat, and there is still great room for improvement in the yield per unit area and quality. At the same time, with the change of people's dietary structure, the demand for Medicago sativa in the domestic forage industry is increasing day by day. Therefore, exploring the genes regulating biomass in Medicago sativa and then studying the regulatory network of these genes have important application value and guiding significance for creating new high-yield germplasms of Medicago sativa and cultivating new varieties of alfalfa. Summary of the Invention

[0003] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present invention is to provide a Medicago sativa MsCBR3 gene, its encoded protein and application to improve the biomass of Medicago sativa.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: Provide a Medicago sativa MsCBR3 gene, MsCBR3 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1.

[0005] The present invention provides an encoded protein of the above Medicago sativa MsCBR3 gene, MsCBR3 The amino acid sequence of the encoded protein of the gene is shown in SEQ ID NO.2.

[0006] The present invention provides an application of the above Medicago sativa MsCBR3 gene in regulating the biomass of Medicago sativa.

[0007] Furthermore, the regulation of the yield of Medicago sativa is overexpression MsCBR3 After the gene, the number of secondary branches of Medicago sativa increases.

[0008] Furthermore, the regulation of the yield of Medicago sativa is overexpressionMsCBR3 After the gene, the total length of the secondary branches of alfalfa increases.

[0009] Furthermore, when regulating the yield of alfalfa by overexpressing MsCBR3 the gene, the fresh weight of the above-ground part of alfalfa increases.

[0010] Furthermore, when regulating the yield of alfalfa by overexpressing MsCBR3 the gene, the number of branches after alfalfa cutting increases.

[0011] Furthermore, when regulating the yield of alfalfa by overexpressing MsCBR3 the gene, the number of leaves after alfalfa cutting increases.

[0012] The present invention also provides a preparation for increasing the yield of alfalfa, and the preparation includes the above MsCBR3 gene or the encoded protein of the above MsCBR3 gene.

[0013] The present invention has the following beneficial effects: The present invention discovers an alfalfa gene MsCBR3 , by constructing an alfalfa MsCBR3 gene overexpression plant, it is found that compared with the control plant, the number and total length of the secondary branches, the fresh weight of the above-ground part, and the number of branches and leaves after cutting of the alfalfa MsCBR3 overexpression plant are all significantly increased. Thus, it is determined that MsCBR3 the gene has a regulatory effect on the biomass of alfalfa. Therefore, the present invention provides a basis for cultivating high-yield alfalfa plants and their breeding work, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is MsCBR3 the in-situ hybridization map of the gene; Figure 2 is MsCBR3 the positive identification map of the gene overexpression transgenic alfalfa; Figure 3 is MsCBR3 the expression level map of the gene overexpression transgenic alfalfa; Figure 4 is MsCBR3 the phenotypic analysis map of the gene overexpression transgenic plant; wherein Figure A is the phenotypic map of the overexpression transgenic plant; Figure B is the map of the number of secondary branches of the overexpression transgenic plant; Figure C is the map of the total length of the secondary branches; Figure D is the map of the fresh weight of the overexpression transgenic plant; Figure 5 is MsCBR3 the map of the number of leaves and branches after cutting of the overexpression plant. DETAILED DESCRIPTION OF THE INVENTION

[0015] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0016] In the following examples, the alfalfa transformed single plant is Gongnong No. 1, which is preserved in our laboratory. The experimental reagents include RNA extraction kit (Omega), reverse transcription kit (TAKARA), quantitative PCR reagent (TAKARA), gel extraction kit (Juhemei), plasmid extraction kit (Quanshijin), homologous recombination ligase (Quanshijin), NSH medium (Kulaibo) and MS medium (Phyto Tech). Other commonly used biochemical reagents are purchased from Sinopharm Chemical Reagent Co., Ltd. In the following examples, for the quantitative tests, at least three repeated experiments are set, and the results are averaged.

[0017] Example 1 Alfalfa MsCBR3 Gene Cloning (1) Alfalfa MsCBR3 Gene sequence information and phylogenetic analysis: Based on the analysis of the transcriptome at different stages of alfalfa bud formation, a gene encoding the AP2 transcription factor family was found to be specifically expressed at different stages of bud initiation. Therefore, we named this gene CROWN BUD REGULATOR 3 ( MsCBR3 ) According to the genome provided by the alfalfa database MODMS, the MsCBR3 gene sequence information of alfalfa varieties Xinjiang Daye and Zhongmu No. 1 was obtained.

[0018] (2) MsCBR3 Gene cloning: After extracting RNA from the lateral bud tissue of the transformed variety Gongnong No. 1 and performing reverse transcription, cDNA of the lateral bud tissue of Gongnong No. 1 was obtained. Using this cDNA as a template, PCR amplification was performed with the primer pair composed of F1 and R1 respectively. After amplification, the product was recovered and sequenced to compare the cDNA sequence of the MsCBR3 gene in alfalfa Gongnong No. 1. Among them, MsCBR3 the CDS nucleotide sequence of is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2; the nucleotide sequences of F1 and R1 are shown as follows: F1: 5'-ATGAGTAACTGGTTAGGATTT-3' (SEQ ID NO.3);

[0019] The PCR reaction system is shown in Table 1.

[0020] Table 1 PCR reaction system (50 μL)

[0021] The PCR reaction program is shown in Table 2.

[0022] Table 2 PCR reaction procedure

[0023] Example 2 Alfalfa MsCBR3 In situ hybridization localization of gene expression in lateral buds (1) Alfalfa MsCBR3 Synthesis of gene probe: Based on the obtained alfalfa MsCBR3 Gene sequence information (SEQ ID NO.1), design specific primer pairs F2 and R2, with a length of 300bp to 400bp. PCR amplification was performed using cDNA from alfalfa lateral bud tissue as a template, and the PCR product was recovered and connected to the pEASY-blunt vector (Quanshi Gold) to obtain a plasmid containing the target fragment. Using this plasmid as a template, PCR amplification of the target fragment was performed using T7 (promoter) and F2 and R2, respectively, and then purified. The purified target fragment was subjected to transcription reaction, and finally the quality of the forward and antisense probes was detected by agarose gel electrophoresis. Among them, the nucleotide sequences of T7, F2, and R2 are shown below: F2: 5'-AGAACTTCTCAATACCGTGGTG-3' (SEQ ID NO.5); R2: 5'-GAATTGAAGTCCCTCGGCTGT-3' (SEQ ID NO. 6); T7: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO. 7).

[0024] (2) Sample preparation: The stem nodes and cotyledon nodes of alfalfa seedlings at different growth stages after germination were collected and fixed in 4wt% paraformaldehyde at 4°C overnight. They were then dehydrated by gradient ethanol, transparentized by xylene, and embedded in paraffin. The slices were set to 8-10 μm thick, attached to poly-lysine slides, and dried for later use.

[0025] (3) Sample tissue pretreatment: After dewaxing and rehydrating to 1× PBS solution using xylene and gradient ethanol, the sections were immersed in proteinase K solution and incubated at 37°C for 10 minutes, and the reaction was terminated with 1× PBS. The sections were then immersed in 4wt% paraformaldehyde to fix RNA, and then dehydrated with gradient ethanol and removed and dried at room temperature.

[0026] (4)In situ hybridization: Evenly cover the section with the hybridization buffer containing the probe, place the glass slide in a humid chamber and hybridize overnight in an oven at 55 °C.

[0027] (5)Washing and blocking: Repeat two rounds of washing in a water bath at 65 °C using 2×SSC buffer and 50 wt% formamide, then digest with RNase A digestion solution to remove unhybridized single-stranded RNA probes. Subsequently, block the antibodies and add anti-DIG-AP (Anti-Digoxigenin-Alkaline Phosphatase Conjugate) antibody to the blocking solution for incubation.

[0028] (6)Antibody staining: Dropwise add nitro blue tetrazolium / 5-bromo-4-chloro-3-indolyl phosphate (NBT / BCIP) chromogenic solution in the dark, incubate at an appropriate temperature in the dark for 24 hours, observe the chromogenic progress. When the signal appears, terminate the reaction with TE (Tris-EDTA Buffer) solution and mount the slide.

[0029] The results of in situ hybridization are as Figure 1 shown. It can be seen from Figure 1 that MsCBR3 the signals of the gene are concentrated in the axillary buds and meristems. Thus, it can be seen that MsCBR3 the gene is mainly expressed in axillary buds and meristems.

[0030] Example 4 MsCBR3 Obtaining of transgenic alfalfa with overexpression of the gene (1)Overexpression MsCBR3 Construction of transgenic vector: Double digest the plant overexpression vector pCAMBIA3301 (purchased from Zhuangmeng) with restriction endonucleases NcoⅠ and BglⅡ. At the same time, amplify the CDS region of the MsCBR3 gene using primer pairs F3 and R3 to obtain the CDS sequence of the MsCBR3 gene containing the vector arms. Recombine the vector and the target fragment by seamless cloning and transform the recombinant product into DH5a Escherichia coli competent cells. After positive detection and sequencing verification, the recombinant plasmid p35S::MsCBR3 with overexpression of MsCBR3 was obtained. Among them, the nucleotide sequences of F3 and R3 are shown as follows: F3: 5'-ACGGGGGACTCTTGACCATGGATGAGTAACTGGTTAGGATTT-3' (SEQ ID NO.8); R3: 5'-AGAAATTTACCCTCAGATCTTCATTCATTCCACAAAGCA-3' (SEQ ID NO.9).

[0031] (2) MsCBR3Obtaining of transgenic alfalfa with gene overexpression: The recombinant plasmid was transformed into competent Agrobacterium tumefaciens EHA105 to obtain positive recombinant Agrobacterium, which was cryopreserved at -80°C with glycerol preservation. The alfalfa variety Gongnong No. 1 was transformed by the Agrobacterium-mediated leaf disc method. The specific operation steps are as follows: ① Streak the Agrobacterium tumefaciens EHA105 containing the target vector on solid LB medium and culture it in an incubator at 28°C for 2 days. Pick positive monoclonal colonies and inoculate them into 3 - 5 mL of LB liquid medium containing rifampicin (50 μg / mL) and kanamycin (10 μg / mL), and culture them overnight on a shaker at 28°C; ② Inoculate the activated bacterial liquid into 50 mL of LB liquid medium containing the above - mentioned concentrations of antibiotics for culture until the OD600 value reaches about 0.3; ③ Centrifuge the bacterial liquid at 2,800 g for 10 min, discard the supernatant. Resuspend the bacterial liquid precipitate with NSDK liquid medium containing auxin, cytokinin and acetosyringone (see Table 3); ④ Select young and disease - free leaves of Gongnong No. 1, disinfect the leaves with 10% sodium hypochlorite solution (containing 0.1% surfactant TritonX - 100) for 10 min, then wash them 5 times with sterile water, and then place them in a clean bench to dry; ⑤ Place the dried leaves in the resuspension solution, evacuate for 15 min and sonicate for 2 min. After all the leaves sink to the bottom, take out the leaves in a clean bench and dry the excess bacterial liquid on the leaf surface with sterile filter paper; ⑥ Transfer the explants to the co - culture medium (NSDK) with sterilized forceps and culture them in the dark at 22°C for 2 days; among them, the formula of the NSDK co - culture medium is the same as the liquid medium components in step ③, and phytagel is added; ⑦ After 2 days, transfer the explants to the callus induction medium (NSDKB) containing herbicide and induce callus in the dark, and subculture every 2 weeks; among them, the formula of the NSDKB callus induction medium is shown in Table 4; ⑧ Transfer the callus to the shoot regeneration medium (MSBK), place it in an incubator at 22°C with constant temperature and light (16 h light / 8 h dark), and subculture every 2 weeks until shoot regeneration; among them, the formula of the MSBK shoot regeneration medium is shown in Table 5; ⑨ Transfer the regenerated shoots to 1 / 2MS medium, place it in an incubator at 22°C with constant temperature and light (16 h light / 8 h dark) to induce rooting, and transplant the plants to the soil when they grow a little larger; ⑩ Extract DNA from the young leaves of the regenerated plants, and use primer pairs F4 and R4 to identify the regenerated plants at the DNA level to obtain 8 positive plants (see Figure 2, where P is the positive control recombinant plasmid p35S::MsCBR3, CK is the negative control, and 1-10 are the transgenic plant numbers). Among them, the PCR reaction system is shown in Table 6, and the PCR reaction program is shown in Table 7; the nucleotide sequences of F4 and R4 are shown as follows: F4: 5'-CATTTCATTTGGAGAGAACAC-3' (SEQ ID NO.10); R4: 5'-AAAACTAGAAATTTACCCTCAGAT-3' (SEQ ID NO.11).

[0032] Table 3 NSDK co-culture medium formula

[0033] Table 4 NSDKB callus induction medium formula

[0034] Table 5 MSBK bud regeneration medium formula

[0035] Table 6 PCR reaction system (50 μL)

[0036] Table 7 PCR reaction program

[0037] (3) Transgenic alfalfa MsCBR3 Expression level identification: Total RNA was extracted from the leaves of the regenerated negative control plants (CK) and positive plants (OE4, OE8, and OE10) and reverse transcribed into cDNA. Using cDNA as a template, quantitative PCR amplification was performed with specific primer pairs; among them, the primer pair composed of F5 and R5 was used to identify MsCBR3 the expression level of the gene; the primer pair composed of F6 and R6 was used to identify the internal reference gene. The nucleotide sequences of F5 and R5, F6 and R6 are shown as follows: F5: 5'-GTACATTTAGCACACAAGAGGAGGC-3' (SEQ ID NO.12); R5: 5'-GAATTGAAGTCCTCGGCTGT-3' (SEQ ID NO.13); F6: 5'-GGCTCCACCAGAGAGAAAGTACAGT-3' (SEQ ID NO.14); R6: 5'-GCCAGACTCGTCATATTCACCCTTG-3' (SEQ ID NO.15).

[0038] It can be seen from Figure 3 that MsCBR3 the expression level of the gene is significantly up-regulated in transgenic plants.

[0039] Example 5 MsCBR3 Phenotypic analysis of transgenic plants overexpressing the gene After selecting negative control plants (CK) and alfalfa MsCBR3 overexpressing plants OE4, OE8 and OE10 were cut, and plants with consistent growth were selected for unified management. The results are shown in Figure 5 . It can be seen from Figure 4 A-C that MsCBR3 the number of secondary branches (i.e., lateral branches) of overexpressing plants is significantly more than that of negative control plants; at the same time, MsCBR3 the length of secondary branches of overexpressing plants is significantly longer. It can be seen from Figure 4 D that MsCBR3 the fresh weight of overexpressing plants is significantly higher than that of negative control plants. After mowing, MsCBR3 the number of branches and the number of leaves of overexpressing plants are also significantly more than those of negative control plants (see Figure 5 ), all of which indicate that MsCBR3 the gene has the potential to increase yield before and after alfalfa mowing. Thus, MsCBR3 the gene is involved in regulating the biomass of alfalfa. After overexpressing MsCBR3 the gene, the biomass of alfalfa increased significantly. Therefore, the present invention provides a basis for cultivating high-yield alfalfa plants and its breeding work, and has great application value.

[0040] In the present invention MsCBR3 the nucleotide sequence of the CDS region of the gene and the amino acid sequence of its encoded protein are as follows: (2) Amino acid sequence of the encoded protein: MSNWLGFSLTPHLRIDEEFGTENQNQNQNQNHVAEASEIGRNYVPTSSHPHPHHLSIMPLRSDGSLCVSDSFTPQEWRYEHAITDGNSNEEGPKLEDFLGCYSNQNQNSTTTSTMSKINVNVSPSFCTNNNPEIEAGENLTNQSLIHSFHAYNDNNNNHHALIHDNSMYKSWMAQTQFSSEGKTTSSSDGNGFQSLNLTMSPSVQNGVGVGGGSAISNVQVNEDPRKRSLSKSNAREPVPRKSIDTFGQRTSQYRGVTRHRWTGRYEAHLWDNSCRKEGQTRKGRQVYLGGYDKEEKAAKAYDLAALKYWGPTTHINFPLSTYDKELEEMKHMTRQEFVANLRRKSSGFSRGASVYRGVTRHHQHGRWQARIGRVAGNKDLYLGTFSTQEEAAEAYDIAAIKFRGTSAVTNFDISRYDVKRICSSSTLITGDLAKRSPKDSTPPATTAEDFNSCGSSSTLSQPPPLTITDGEQHSDELSNMVWNSNNDEQKPQNDTNITESSQHGSPSNKNEMNPQSPKCSLGLPNEFGVSGADYGHGYFTLHGPKFDDGSNENDHMNNNRLGNLGLVNQVPMFALWNE (SEQ ID NO.2).

[0041] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An alfalfa MsCBR3 gene, characterized in that The said MsCBR3 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.

1.

2. The alfalfa described in claim 1 MsCBR3 The encoded protein of the gene, characterized in that The said MsCBR3 The amino acid sequence of the encoded protein of the gene is shown in SEQ ID NO.

2.

3. The application of the alfalfa described in claim 1 MsCBR3 in regulating the biomass of alfalfa.

4. The application according to claim 3, characterized in that, The regulation of alfalfa biomass is overexpression MsCBR3 After the gene, the number of secondary branches of alfalfa increases.

5. The application according to claim 3, wherein The regulation of alfalfa biomass is overexpression MsCBR3 After the gene, the total length of the secondary branches of alfalfa increases.

6. The application according to claim 3, wherein The regulation of alfalfa biomass is overexpression MsCBR3 After the gene, the fresh weight of the above-ground part of alfalfa increases.

7. The application according to claim 3, characterized in that The regulation of alfalfa biomass is overexpression MsCBR3 After the gene, the number of branches of alfalfa after mowing increases.

8. The application according to claim 3, characterized in that, The regulation of alfalfa biomass is overexpression MsCBR3 After the gene, the number of leaves of alfalfa after cutting increases.

9. A preparation for increasing the yield of alfalfa, characterized in that, The preparation comprises the MsCBR3 gene as claimed in claim 1 or the MsCBR3 encoded protein of the gene as claimed in claim 2.

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