Medicago sativa MsCBR1 gene as well as encoding protein and application thereof

Abstract

The invention discloses a medicago sativa MsCBR1 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 MsCBR1 is found, and a medicago sativa mscbr1 mutant is constructed by knocking out the MsCBR1 gene. After cutting, compared with a control plant, the alfalfa mscbr1 mutant has the advantages that the number of regenerated branches, the number of leaves and the number of crown buds are reduced. Therefore, it is determined that the MsCBR1 gene has a regulation effect on regeneration of plants after alfalfa is cut. Therefore, the invention provides new theoretical support for cultivating multi-branch and high-yield medicago sativa materials.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and specifically relates to a Medicago sativa MsCBR1 gene, its encoded protein and applications. Background Art

[0002] Medicago sativa ( Medicago sativa L.) is a perennial herbaceous plant of the genus Medicago in the legume family. Because of its wide adaptability, high forage value, rich nutrition, strong regeneration ability and tolerance to mowing, it has become an important high-quality forage for herbivores such as dairy cows and enjoys the reputation of "king of forages". At present, about more than 70 million mu of Medicago sativa is planted in China, but the development of the Medicago sativa industry lags far behind that of staple crops such as rice and wheat. Therefore, it is an urgent problem to improve the breeding and production levels of domestic Medicago sativa and increase the supply of high-quality and high-yield alfalfa.

[0003] Mowing is a key link in the production and utilization of Medicago sativa. Reasonable mowing can significantly promote its regeneration performance and subsequent yield. Timely mowing can stimulate the elongation of crown buds from the root neck and the base of the stem of Medicago sativa, achieving multiple regenerations, thereby increasing the total forage yield per unit area and ensuring the continuous supply of feed. The number of branches after mowing and regeneration is a key index determining its regeneration ability and production performance. Therefore, excavating genes regulating mowing and regeneration and developing and utilizing them will help increase the yield of Medicago sativa. Summary of the Invention

[0004] To solve the above-mentioned deficiencies existing in the prior art, the purpose of the present invention is to provide a Medicago sativa MsCBR1 gene, its encoded protein and applications to increase the yield of Medicago sativa after mowing.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: Provide a Medicago sativa MsCBR1 gene, MsCBR1 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1.

[0006] The present invention provides an encoded protein of the above Medicago sativa MsCBR1 gene, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0007] The present invention provides an application of the above Medicago sativa MsCBR1 gene in regulating the regeneration ability of Medicago sativa after mowing.

[0008] Furthermore, regulating the regeneration ability of Medicago sativa after mowing is to knock out the MsCBR1 gene, and the number of branches of Medicago sativa decreases after mowing.

[0009] Furthermore, regulating the regeneration ability of Medicago sativa after mowing is to knock outMsCBR1 The gene causes a decrease in the number of leaves of alfalfa after cutting.

[0010] Furthermore, regulating the regeneration ability of alfalfa after cutting is achieved by knocking out MsCBR1 the gene, which results in a decrease in the number of crown buds of alfalfa after cutting.

[0011] The present invention also provides a preparation for regulating the regeneration ability of alfalfa after cutting, which includes the above MsCBR1 gene or the encoded protein of the above MsCBR1 gene.

[0012] The present invention has the following beneficial effects: The present invention discovers an alfalfa gene MsCBR1 , and constructs an alfalfa MsCBR1 mutant by knocking out mscbr1 the gene. After cutting, it is found that compared with the control plants, the number of regenerated branches, leaves and crown buds of the alfalfa mscbr1 mutant are all reduced. Thus, it is determined that MsCBR1 the gene has a regulatory effect on the regeneration of alfalfa plants after cutting. Therefore, the present invention provides new theoretical support for cultivating alfalfa materials with multiple branches and high yields. Description of the Drawings

[0013] Figure 1 is MsCBR1 the change diagram of the expression level of the gene in the root collar and crown bud parts; Figure 2 is MsCBR1 the periodic expression trend diagram of the gene during crown bud development; Figure 3 is MsCBR1 the in situ hybridization diagram of the gene; Figure 4 is the vector map of pRGEB31-MsCBR1; Figure 5 is mscbr1 the positive identification diagram of the alfalfa Figure 6 is mscbr1 the sequencing analysis diagram of 24 positive monoclonal of the mutant; Figure 7 is mscbr1 the diagram of the number of branches and crown buds of mutants No. 4, 7 and 8 and negative control seedlings before cutting; Figure 8 is mscbr1 the diagram of the number of leaves and branches of alfalfa mutants No. 4, 7 and 8 and control plants at 2 days, 2 weeks and 5 weeks after cutting; Figure 9 is mscbr1Base crown bud diagrams and quantity diagrams of mutants and control plants; wherein, the white triangles indicate crown buds and the numbers represent the number of crown buds. Detailed implementation mode

[0014] The following examples are only used to explain the present invention and are not used to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The experimental reagents in the following examples include RNA extraction kit (Omega), reverse transcription kit (TAKARA), quantitative PCR reagent (TAKARA), gel extraction kit (Polymerase), plasmid extraction kit (GenScript), homologous recombination ligase (GenScript), NSH medium (Coolaber) and MS medium (Phyto Tech). Other commonly used biochemical reagents are purchased from Sinopharm Chemical Reagent Co., Ltd. In the following examples, all quantitative tests are set with at least three repeated experiments, and the results are averaged.

[0015] Example 1: Alfalfa MsCBR1 Cloning of genes (1) Alfalfa MsCBR1 Sequence information and phylogenetic analysis of genes: Based on the transcriptome analysis of different stages of crown bud formation in alfalfa, it was found that MS.gene036441 genes had significant differential expression at different stages of bud formation (see Figure 1 , where FPKM (Fragments Per Kilobase of transcript per Million mapped fragments) is a standardized index for measuring gene expression level), and it was named CROWN BUD REGULATOR 1 ( MsCBR1 ) According to the genome provided by the alfalfa database MODMS, the sequence information of the MsCBR1 gene of alfalfa varieties Xinjiang Daye and Zhongmu No. 1 was obtained.

[0016] (2) MsCBR1 Cloning of genes: After extracting RNA from the crown bud tissue of the transformed single plant Zhongmu No. 1 and performing reverse transcription, cDNA of the crown bud tissue of Zhongmu No. 1 was obtained. Then, using this cDNA as a template, PCR amplification was carried out with the primer pair composed of F1 and R1. After amplification, the product was recovered and sequenced and compared with the CDS sequence of the MsCBR1 gene of alfalfa Zhongmu No. 1; wherein, the CDS sequence is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.2; the nucleotide sequences of F1 and R1 are as follows: F1: 5'-ATGCAGCAGCACCTGATGCAG-3' (SEQ ID NO.3); R1: 5'-CTAATTCCCATCACCAGAAGA-3' (SEQ ID NO.4).

[0017] The PCR reaction system is shown in Table 1.

[0018] Table 1 PCR reaction system (50 μL)

[0019] The PCR reaction procedure is shown in Table 2.

[0020] Table 2 PCR reaction procedure

[0021] Example 2: Alfalfa MsCBR1 Analysis of gene spatio-temporal expression pattern (1) Obtaining cDNA of crown bud tissue samples at different developmental stages of alfalfa: RNA of crown bud tissue at 12 developmental stages from 2 - 90 DAG (Days After Germination) was extracted and reverse transcribed to obtain cDNA of crown bud tissue at different developmental stages.

[0022] (2) Analysis of the expression pattern of the MsCBR1 gene in crown bud tissue at different developmental stages of alfalfa: According to the obtained gene sequence information of alfalfa, specific fluorescence quantitative primer pairs F2 and R2 were designed to identify the expression level of the MsCBR1 gene, and F3 and R3 were used to identify the expression level of the internal reference gene ( MsCBR1 gene expression level). Real-time fluorescence quantitative PCR amplification was performed using the cDNA extracted in step (1) as a template; the nucleotide sequences of F2 and R2, F3 and R3 are shown as follows: ACTIN3 F2: 5'-CATAGTGAAGGGGCTAATGTTGG-3' (SEQ ID NO.5); R2: 5'-GCACTTCCAATGTCTTGCTTACC-3' (SEQ ID NO.6); R2: 5'-GCACTTCCAATGTCTTGCTTACC-3' (SEQ ID NO.6); F3: 5'-GGCTCCACCAGAGAGAAAGTACAGT-3' (SEQ ID NO.7); R3: 5'-GCCAGACTCGTCATATTCACCCTTG-3' (SEQ ID NO.8).

[0023] The quantitative PCR reaction system is shown in Table 3.

[0024] Table 3 Quantitative PCR reaction system (10 μL)

[0025] The quantitative PCR amplification conditions are shown in Table 4.

[0026] Table 4 Quantitative PCR amplification conditions

[0027] The results are as Figure 2 shown, MsCBR1 the gene showed a periodic expression trend during the development of alfalfa crown buds. Specifically, from 2 DAG to 20 DAG, MsCBR1 the gene expression level decreased; however, since 20 DAG, its expression level began to increase; after 35 DAG, the expression level turned down again; subsequently, it showed an upward trend again around 60 DAG.

[0028] Example 3: Alfalfa MsCBR1 In-situ hybridization expression localization of the gene in alfalfa crown buds MsCBR1 (1) Synthesis of the alfalfa gene probe: Using the cDNA of alfalfa crown bud tissue as a template for PCR amplification, recovering the PCR product and ligating it to the pEASY-blunt vector (Quanshijin) to obtain a plasmid containing the target fragment. Using this plasmid as a template, PCR amplify the target fragment with T7 (vector T7 promoter) and F4, R4 primers respectively and purify. Perform a transcription reaction on the purified target fragment, and finally detect the quality of the sense and antisense probes by agarose gel electrophoresis; among them, the nucleotide sequences of T7 and F4, R4 are shown as follows: F4: 5'-TCTCAACCCCAACCACCTACTA-3' (SEQ ID NO.9); R4: 5'-GCACTTCCAATGTCTTGCTTACC-3' (SEQ ID NO.10);

[0029] T7: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO.11).

[0030] (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.

[0031] (4) In situ hybridization: Evenly cover the section with hybridization buffer containing the probe, place the slide in a wet box and hybridize overnight in an oven at 55°C.

[0032] (5) Elution and blocking: Repeat two rounds of elution using 2×SSC buffer and 50 wt% formamide in a 65°C water bath, followed by RNase A digestion to remove unhybridized single-stranded RNA probes, followed by blocking antibodies, and adding anti-DIG-AP antibodies to the blocking solution for incubation.

[0033] (6) Antibody staining: Add NBT / BCIP colorimetric solution in a dark place and incubate at an appropriate temperature for 24 hours. Observe the color development progress. When the signal appears, terminate the reaction with TE solution and seal the slide.

[0034] Depend on Figure 3 It can be seen that MsCBR1 The gene signal is concentrated in the crown bud meristem, which indicates MsCBR1 The gene is mainly expressed in the crown bud meristem.

[0035] Embodiment 4: MsCBR1 Obtaining gene knockout transgenic alfalfa (1) MsCBR1 Construction of gene knockout transgenic vector: Based on the obtained MsCBR1 The CDS sequence of the gene was compared with the published Xinjiang Daye genome and Zhongmu No. 1 genome, and the CRISPR-P online tool was used to select highly conservative sequences on exons to design targets. Finally, four targets were designed. The above four target sequences were introduced into the alfalfa CRISPR / Cas9 gene editing tool to construct the vector pRGEB31-MsCBR1 (see the vector map for details). Figure 4 , construction method reference: Multiplex CRISPR / Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUSTa1 (MsFTa1) leads to delayed flowering time with improved forage biomassyield and quality, DOI: 10.1111 / pbi.14042). Among them, the sequences of the four target sites are as follows: gRNA1: 5'-CAACCACCTACTATGCCTGG-3' (SEQ ID NO.12); gRNA2: 5'-TCAAGTGGAATGATGCAGCA-3' (SEQ ID NO.13); gRNA3: 5'-GATGACACAACAACAATTAA-3' (SEQ ID NO.14); gRNA4: 5'-AGTGGTCACTCTGGTGATGG-3' (SEQ ID NO.15).

[0036] (2) Alfalfa mscbr1 Obtaining and identification of mutants: The recombinant plasmid was transformed into competent Agrobacterium tumefaciens EHA105 to obtain positive recombinant Agrobacterium, which was stored at 80°C in glycerol. The single plant of alfalfa Zhongmu No. 1 was transformed by the Agrobacterium-mediated leaf disc method. The specific operation steps are as follows: ① The Agrobacterium containing the target vector EHA105 was streaked on solid LB medium and cultured in an incubator at 28°C for 2 days. Positive monoclonal colonies were picked and inoculated into 5 mL of LB liquid medium containing rifampicin (50 μg / mL) and kanamycin (10 μg / mL), and cultured overnight on a shaker at 28°C; ② The activated bacterial solution was inoculated into 50 mL of LB liquid medium containing the above concentrations of antibiotics for amplification culture until OD600 = 0.3; ③ The bacterial solution was centrifuged at 2800 g for 10 min, and the supernatant was discarded. The bacterial solution precipitate was resuspended with NSDK liquid medium containing auxin, cytokinin and acetosyringone (see Table 5); ④ Young and disease-free leaves of Zhongmu No. 1 were selected, disinfected with 10% sodium hypochlorite solution (containing 0.1% surfactant TritonX-100) for 10 min, washed 5 times with sterile water, and then placed in a laminar flow bench to dry; ⑤ The dried leaves were placed in the resuspension solution, vacuumed for 15 min and sonicated for 2 min. After all the leaves sank to the bottom, the leaves were taken out from the laminar flow bench, and the excess bacterial solution on the surface of the leaves was blotted dry with sterile filter paper; ⑥ The explants were transferred to the co-culture medium (NSDK) with sterilized forceps and cultured in the dark at 22°C for 2 days; among them, the formula of the NSDK co-culture medium added phytagel more than the liquid medium in step ③, and the other components were the same; ⑦ After 2 days, the explants were transferred to the callus induction medium (NSDKB) containing herbicide for dark induction of callus, and subcultured every 2 weeks; among them, the formula of the NSDKB callus induction medium is shown in Table 6; ⑧ Transfer the callus onto the shoot regeneration medium (MSBK), place it in a constant temperature light incubator at 22 °C (16 h light / 8 h dark), and subculture it every 2 weeks until shoot regeneration; among them, the formula of the MSBK shoot regeneration medium is shown in Table 7; ⑨ Transfer the regenerated shoots onto the 1 / 2MS medium and place them in a constant temperature light incubator at 22 °C (16 h light / 8 h dark) to induce root formation. After the plants grow slightly larger, transfer them to the soil; ⑩ Extract DNA from the young leaves of the regenerated plants and use Cas9 Gene primer pairs F5 and R5 to identify the transgenic regenerated plants. Among them, the PCR reaction system is shown in Table 8, and the PCR reaction procedure is shown in Table 9; The nucleotide sequences of F5 and R5 are as follows: F5: 5'-GAAATTCAAGGTGCTGGGCAAC-3' (SEQ ID NO.16); R5: 5'-AACAGGCCATTCTTCTTCTCGC-3' (SEQ ID NO.17).

[0037] Table 5 Formulation of NSDK co-culture medium

[0038] Table 6 Formulation of NSDKB callus induction medium

[0039] Table 7 Formulation of MSBK shoot regeneration medium

[0040] Table 8 PCR reaction system (50 μL)

[0041] Table 9 PCR reaction procedure

[0042] The positive identification results are shown in Figure 5 (where M is Marker; CK is the negative control). To further confirm mscbr1 the genotype of the mutant, use primer pairs F6 and R6 to clone the fragment near the target point and ligate it into the pEASY-blunt vector. Pick 24 positive monoclonal colonies for sequencing and alignment analysis, and the results are as Figure 6 shown. Among them, the nucleotide sequences of F6 and R6 are as follows: F6: 5'-TTGCCTAATCCTCCTAACTAAA-3' (SEQ ID NO.18); R6: 5'-TGCTTACTTGGCTTGAGATAGA-3' (SEQ ID NO.19).

[0043] It can be seen from Figure 6 that all four alleles of mutants 4, 7, and 8 of alfalfa mscbr1- have mutated, among which three alleles are homozygous mutations and another allele is a heterozygous mutation.

[0044] Example 5: Phenotypic analysis of alfalfa mutants mscbr1 For mutants 4, 7, and 8 of alfalfa and negative control seedlings (CK), unified cutting management was carried out. After the cuttings budding, the relevant traits such as the number of branches after mowing were counted. It can be seen from that there was no significant difference in the number of branches and the number of crown buds before mowing between mutants 4, 7, and 8 and negative control seedlings. However, after mowing, the mutant plants showed a weakened regeneration ability, specifically manifested as a decrease in the number of regenerated branches and the number of leaves after mowing (see mscbr1- ). By observing and counting the crown buds at the base of mutants 4, 7, and 8 of alfalfa, it was found that the number of crown buds of mutants 4, 7, and 8 of alfalfa was significantly less than that of negative control plants (see Figure 7 ), indicating that mscbr1- the gene has a regulatory effect on the regeneration of alfalfa plants after mowing. Figure 8 For mutants 4, 7, and 8 of alfalfa mscbr1- The nucleotide sequence of the CDS region of the gene and the amino acid sequence of its encoded protein in the present invention are shown as follows: mscbr1- (1) Figure 9 MsCBR1

[0045] MsCBR1 In the present invention MsCBR1 the nucleotide sequence of the CDS region of the gene and the amino acid sequence of its encoded protein are shown as follows: (1) MsCBR1CDS nucleotide sequence of the gene: ATGCAGCAGCACCTGATGCAGATGCAGCCCATGATGGCAGCTTACTATCCTAACAACGTCACTACTGATCATATTCAACAGTATCTTGATGAGAACAAGTCCTTGATTCTCAAGATTGTTGAAAGCCAGAACTCTGGCAAGCTCACCGAGTGTGCTGAGAACCAATCAAGGCTGCAGAGAAATCTCATGTACCTAGCTGCAATAGCTGATTCTCAACCCCAACCACCTACTATGCCTGGCCAGTACCCTTCAAGTGGAATGATGCAGCAGGGAGGACACTACATGCAGGCTCAACAAGCTCAGCAGATGACACAACAACAATTAATGGCTGCACGTTCCTCTCTTATGTATGCTCAACAGCTTCAACAGCAGCAAGCCTTGCAAAGCCAACTCGGTATGAATTCGAGTGGAAGTCAAGGCCTTCACATGTTGCATAGTGAAGGGGCTAACGTTGGAGGCAATTCATCGTTAGGTACTGGAGGTTTTCCTGATTTTGGTCGTAGCTCGGCTGGTGATGGTTTGCACGGTAGTGGCAAGCAAGACATTGGAAGTGCTGATGGCCGCGGTGGAAGCTCTAGTGGTCACTCTGGTGATGGCGGCGAAACCCTTTACCTAAAATCTTCTGGTGATGGGAATTAG (SEQ ID NO.1); (2) MsCBR1 Encoded protein amino acid sequence: MQQHLMQMQPMMAAYYPNNVTTDHIQQYLDENKSLILKIVESQNSGKLTECAENQSRLQRNLMYLAAIADSQPQPPTMPGQYPSSGMMQQGGHYMQAQQAQQMTQQQLMAARSSLMYAQQLQQQQALQSQLGMNSSGSQGLHMLHSEGANVGGNSSLGTGGFPDFGRSSAGDGLHGSGKQDIGSADGRGGSSSGHSGDGGETLYLKSSGDGN (SEQ ID NO.2) 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 principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. An alfalfa MsCBR1 gene, characterized in that The said MsCBR1 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.

1.

2. The alfalfa described in claim 1 MsCBR1 The encoded protein of the gene, characterized in that The MsCBR1 amino acid sequence of the encoded protein of the gene is as shown in SEQ ID NO.

2.

3. The application of the alfalfa described in claim 1 MsCBR1 in regulating the regeneration ability of alfalfa after cutting.

4. The application according to claim 3, characterized in that, The regulation of the regeneration ability of alfalfa after cutting is achieved by knocking out MsCBR1 gene, and the number of branches of alfalfa decreases after cutting.

5. The application according to claim 3, characterized in that, The regulation of the regeneration ability of alfalfa after cutting is achieved by knocking out MsCBR1 genes, resulting in a decrease in the number of leaves of alfalfa after cutting.

6. The application according to claim 3, wherein The regulation of the regeneration ability of alfalfa after cutting is achieved by knocking out MsCBR1 genes, resulting in a decrease in the number of crown buds of alfalfa after cutting.

7. A preparation for regulating the regeneration ability of alfalfa after mowing, characterized in that, The preparation comprises the MsCBR1 gene as claimed in claim 1 or the MsCBR1 encoded protein of the gene as claimed in claim 2.

Images