Application of MsMYB-ZD gene in regulating leaf size and yield of alfalfa

Abstract

This invention discloses a MsMYB-ZD The application of genes in regulating alfalfa leaf size and yield falls under the field of plant genetic engineering. This invention utilizes transcriptome analysis to clone a gene that regulates alfalfa leaf size. MsMYB-ZD Transcription factors; experimental data show that, MsMYB-ZD The gene exhibits a gradually upregulated expression pattern during leaf development; compared to wild-type alfalfa Gongnong 1 (1-16) individual plants, overexpression... MsMYB-ZD MsMYB-ZD The leaf area of ​​the transgenic plants increased by an average of 77%, and the expression level of the transgenic lines showed a significant positive correlation with the leaf area. The fresh grass yield of the transgenic plants increased by 158.7%, and the dry grass yield increased by 207.5%. These results indicate that this gene is of great significance for improving alfalfa yield.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a... MsMYB-ZD Application of genes in regulating leaf size and yield in alfalfa. Background Technology

[0002] With the upgrading of residents' dietary structure, my country's agricultural food system faces problems such as resource scarcity, rigidly increasing demand for imported dairy, meat, and forage, and a prominent domestic supply shortage. As a major cost in livestock production, the stability and quality of feed supply play a decisive role in the industry's development. Alfalfa, known as the "King of Forage," is an indispensable high-quality feed source for dairy and meat production due to its rich protein content, good palatability, and high digestibility. However, due to the late start and long-term insufficient investment in alfalfa breeding in my country, there is a severe shortage of alfalfa varieties with independent intellectual property rights. In the past few decades, new varieties developed through traditional breeding methods have made limited contributions to increasing alfalfa yields, failing to keep pace with the rapid development of the livestock industry and meet the ever-growing production demands. Statistics show that my country currently relies heavily on imports for high-quality alfalfa, with a self-sufficiency rate of only 64%. Furthermore, with the continuous expansion of the livestock industry, the domestic forage supply gap is widening, which not only increases livestock production costs but also poses a potential threat to industry security. Increasing alfalfa production and cultivating high-yield, domestically developed varieties have become urgent priorities for alleviating the shortage of feed resources and ensuring the sustainable development of animal husbandry in my country.

[0003] The protein in alfalfa plants is mainly concentrated in the leaf tissue, making the leaves a key site for nutrient accumulation and directly affecting the overall yield of alfalfa. Among the components of alfalfa yield, leaf size is closely related to the final yield; larger leaves mean more biomass accumulation, directly affecting the yield level of alfalfa. From the perspective of the entire growth and development cycle of alfalfa, leaf size plays a crucial role at different stages. In the seedling stage, relatively large leaves can accumulate more photosynthetic assimilates for the plant, providing energy and material support for the robust development of the root system, enhancing the seedling's ability to absorb soil nutrients, and improving the seedling's resistance and survival rate. Entering the vigorous growth stage, the continuous and stable expansion of leaf area can continuously provide the material basis for the growth of stems and leaves and the development of branching in the above-ground parts of the plant, directly supporting the linear growth of biomass and playing a key role in promoting alfalfa yield. Comparing different alfalfa varieties, high-yielding alfalfa varieties usually exhibit the typical characteristics of large, well-developed leaves. Extensive field trial data have shown a significant positive correlation between leaf area per alfalfa plant and fresh forage yield. Therefore, leaf size is one of the core traits determining alfalfa yield, and its scientific and precise regulation holds promise as a key breakthrough in overcoming bottlenecks in alfalfa yield improvement.

[0004] Although leaf size significantly impacts alfalfa yield, effective techniques for controlling leaf size are extremely limited in actual breeding practices. Alfalfa's characteristics, such as being autotetraploid, highly heterozygous, and self-incompatible, make it difficult to grasp the segregation patterns of genetic traits during hybridization, resulting in significant challenges in selecting target traits. Furthermore, traditional hybridization breeding requires multiple generations of hybridization, backcrossing, and selection, leading to a lengthy breeding cycle—typically 8-10 years or even longer—to develop a relatively stable new variety, which is insufficient to meet the rapidly changing market demands and the pace of industrial development. While modern molecular biology techniques are increasingly being applied to alfalfa genetic improvement, key gene resources that can precisely target leaf size and are suitable for genetic engineering remain extremely scarce. Therefore, there is an urgent need to discover key genes that can regulate alfalfa leaf size, overcome existing breeding technology bottlenecks, achieve a leapfrog increase in alfalfa yield, and provide a solid forage guarantee for the sustainable development of my country's livestock industry. Summary of the Invention

[0005] To address the aforementioned shortcomings of the existing technology, the objective of this invention is to provide a... MsMYB-ZD The application of genes in regulating alfalfa leaf size and yield to increase alfalfa production.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method is provided. MsMYB-ZD Application of genes in regulating alfalfa leaf size.

[0007] This invention provides MsMYB-ZD Application of genes in regulating alfalfa yield.

[0008] further, MsMYB-ZD The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.1.

[0009] further, MsMYB-ZD The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0010] Furthermore, regulating alfalfa leaf size was achieved through overexpression. MsMYB-ZD Genes that increase leaf area in transgenic alfalfa.

[0011] Furthermore, regulating alfalfa yield is achieved through overexpression. MsMYB-ZD Genetic modification increased both fresh and hay yields of individual transgenic alfalfa plants.

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

[0013] This invention also provides a method for preparing a high-yield alfalfa variety, through overexpression MsMYB-ZD Genes were used to develop high-yield alfalfa varieties.

[0014] This invention has the following beneficial effects: This invention utilizes transcriptome analysis to clone a regulator that controls alfalfa leaf size. MsMYB-ZD Genes, experimental data show, MsMYB-ZD The gene exhibits a gradually upregulated expression pattern during leaf development; compared to wild-type alfalfa Gongnong 1 (plants 1-16), overexpression... MsMYB-ZD The leaf area of ​​the transgenic single plant increased by an average of 77%, and its expression level showed a significant positive correlation with the leaf area of ​​alfalfa; the fresh grass yield of the transgenic single plant increased by 158.7%, and the dry grass yield of the single plant increased by 207.5%; the above results indicate that this gene is of great significance for improving alfalfa yield. Attached Figure Description

[0015] Figure 1 Transcriptome analysis of alfalfa leaf size mutants and MsMYB-ZD A diagram illustrating the developmental process involved in regulating alfalfa leaf size;

[0016] Figure 2 Genes regulating leaf size in alfalfa MsMYB-ZD Domain and homologous gene clustering analysis diagram;

[0017] Figure 3 for MsMYB-ZD Identification of overexpressing transgenic plants and detection of expression levels;

[0018] Figure 4 for MsMYB-ZD Phenotypic image of transgenic alfalfa plants overexpressing the gene showing enlarged leaves. 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] Example 1: Alfalfa MsMYB-ZD Gene screening and identification

[0021] 1. Genes regulating alfalfa leaf size MsMYB-ZD Identification and domain analysis

[0022] This embodiment uses transcriptome analysis to identify differentially expressed genes in the leaf development process between the large-leaf mutant and the wild type of alfalfa cultivar WL366. The large-leaf mutant DAYE and the wild type of alfalfa cultivar WL366, obtained through previous screening, were used as materials. Leaf development was analyzed in the early stage (young leaf, stage 1) and middle stage (rapid elongation stage, stage 2), respectively (see...). Figure 1 A) RNA-Seq transcriptome sequencing was performed. Using the Xinjiang Daye genome as a reference genome, differentially expressed genes were analyzed using DESeq2 software. The screening criteria were |log2FC|≥1 and FDR<0.05, and a total of 11560 differentially expressed genes (DEGs) were identified (see A). Figure 1 B). Among them. MsMYB-ZD ( Ms.gene58556 The transcriptional level of the gene was significantly higher in the large-leaf mutant than in the wild type at all developmental stages. Its expression level in mature leaves of the mutant was 100 times that of the wild type, and the expression level showed a gradual upward trend with leaf development (see...). Figure 1 C), based on its most significant expression difference among transcription factors and its high correlation with leaf development process, MsMYB-ZD It was given priority in being identified as a candidate gene.

[0023] Using SMART (Simple Modular Architecture Research Tool) software, for MsMYB-ZD Protein domain analysis of the gene revealed that its full-length coding sequence is 948 bp, encoding a transcription factor containing 316 amino acids. Domain prediction analysis indicated that the protein contains a conserved SANT domain (see...). Figure 2 A), belongs to the MYB structural domain superfamily.

[0024] 2. MsMYB-ZD Homologous gene identification and evolutionary analysis

[0025] Using HMMER software, MsMYB-ZD The protein sequence is the search sequence, found in Arabidopsis thaliana ( Arabidopsis thaliana ), Tribulus terrestris ( Medicago truncatula ), Xinjiang large-leaf varieties of alfalfa, soybeans ( Glycine max L.), rice ( Oryza sativa L.), corn ( Zea mays L.), wheat ( Triticum aestivum L.), basal angiosperms without oil camphor ( Amborella trichopoda ) and small bowl moss ( Physcomitrella patens Homologous sequence searches were performed in genomic databases, and phylogenetic trees were constructed. Phylogenetic tree analysis results showed that... MsMYB-ZD with ArabidopsisAt1g74840 Soybeans GLYMA_02G026300 Clustered in the same evolutionary branch (see Figure 2 B). It is worth noting that, At1g74840 and GLYMA_02G026300 Although they are the same MYB These are transcription factors, but their biological functions remain unexplained. Therefore, MsMYB-ZD As with At1g74840 The highly homologous gene is a novel functional gene that has never been discovered or studied before in the mechanism of alfalfa leaf size regulation.

[0026] Example 2: MsMYB-ZD Gene cloning and construction of overexpression vectors

[0027] 1. MsMYB-ZD Cloning of genes

[0028] Total RNA was extracted from leaf tissues of the WL366 large-leaf mutant DAYE during early leaf development using the EZNA® Plant RNA Kit (OMEGA Bio-TEK). cDNA was synthesized via reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). The Xinjiang large-leaf genome was then analyzed. MsMYB-ZD Specific primers F1 and R1 (nucleotide sequences other than the underlined portions of SEQ ID NO.3 and SEQ ID NO.4) were designed at the start and stop codons of the gene sequence. pCAMBIA3301 overexpression vectors (purchased from Zhuangmeng) were designed upstream and downstream of the specific primers, respectively. Sma I and Hind The homologous base sequence at the III restriction site (the underlined nucleotide sequence in SEQ ID NO.3 and SEQ ID NO.4). Using the above alfalfa cDNA as a template, PCR amplification was performed according to the following reaction system (polymerase mix purchased from TaKaRa), and the amplification program is shown in the table below.

[0029] F1:5'- AACAGAGCTCGGTACCCGGG ATGTCTTTGTCTCGCACG-3' (SEQ ID NO.3);

[0030] R1:5'- ACTCTAGAGGATCCCCC TCAACGCCTCGGGCGCACTTG-3' (SEQ ID NO. 4).

[0031] Table 1 is used for MsMYB-ZD Amplification PCR reaction system (50 μL)

[0032]

[0033] Table 2 is used for MsMYB-ZD Amplification PCR reaction procedure

[0034]

[0035] 2. MsMYB-ZD Construction of gene overexpression vectors

[0036] The amplified fragments were subjected to 1.5% agarose gel electrophoresis and then recovered by gel extraction (gel extraction kit purchased from Polymermax). The gel-extracted products were ligated into the pCAMBIA3301 linear vector using homologous recombinase (purchased from Zhuangmeng). Sma I and Hind Double digestion with III endonuclease). The reaction product was transformed into *E. coli* (…). E.coli Trans1-T1 competent cells (purchased from TransGen Biotech, Beijing). Positive clones were screened on kanamycin-added LB agar plates and sent to Sangon Biotech (Beijing) for sequencing verification. Positive recombinant plasmids with correct sequencing results were named... 35S: MsMYB-ZD The above plasmid was transformed into Agrobacterium tumefaciens EHA105 competent cells (prepared and preserved in the laboratory).

[0037] Example 3: MsMYB-ZD Obtaining and identifying transgenic alfalfa plants overexpressing the gene

[0038] 1. Alfalfa MsMYB-ZD Transformation

[0039] Referencing the alfalfa leaf disc conversion method (Jiang Q, Fu C, Wang ZY. Aunified Agrobacterium -mediated transformation protocol for alfalfa ( Medicago sativa L.) and Medicago truncatula Methods Mol. Biol. 2019;1864:153-163.) Transform the regenerable single plants 1-16 of Gongnong No. 1 obtained in the previous screening in our laboratory.

[0040] 2. MsMYB-ZD Identification of overexpression transgenic plants and detection of expression levels

[0041] based on 35S: MsMYB-ZD On the carrier MsMYB-ZDSpecific primers F2 and R2 for the pCAMBIA3301 vector were designed upstream and downstream of the start and stop codons of the gene sequence. Using gDNA from wild-type single plants of Gongnong 1 (1-16) and transgenic seedlings selected for Basta resistance as templates, PCR amplification was performed according to the following reaction system (polymerase mix purchased from Vazyme). Positive plants were obtained as follows: Figure 3 As shown in Figure A.

[0042] F2:5'-AACAGAGCTCGGTACCCGGG-3' (SEQ ID NO.5);

[0043] R2: 5'-ACTCTAGAGGATCCCCC-3' (SEQ ID NO. 6).

[0044] Table 3 is used for MsMYB-ZD PCR reaction system for identifying overexpressing plants (20 μL)

[0045]

[0046] Table 4 is used for MsMYB-ZD PCR reaction procedure for identifying overexpressing plants

[0047]

[0048] Total RNA extraction and cDNA synthesis from the transgenic plants were performed in the same manner as in Example 2. According to... 35S: MsMYB-ZD Vector sequence design for qRT-PCR primers F3 and R3 (primer positions as follows) Figure 3 (As shown in B). Genes constitutively expressed in alfalfa. MsActin For internal reference, using LightCycler ® A 96 Real-Time PCR instrument (TB green, purchased from TaKaRa) was used to perform real-time quantitative PCR analysis using cDNA from the aforementioned transgenic plants as a template. The experiment was performed in triplicate. Results are as follows: Figure 3 As shown in B, the three overexpression lines MsMYB-ZD Gene expression levels were significantly higher than those of wild-type Gongnong 1-16 alfalfa.

[0049] F3:5'-TCGTCGTCATCGGCTTCATCATC-3' (SEQ ID NO.7);

[0050] R3:5'-CCTCGCCCTTGCTCACCATA-3' (SEQ ID NO.8);

[0051] MsActin-F:5'-GACAATGGAACTGGAATGG-3' (SEQ ID NO. 9);

[0052] MsActin -R:5'-CAATACCGTGCTCAATGG-3' (SEQ ID NO. 10).

[0053] Table 5. Plants used for overexpression MsMYB-ZD Gene expression level detection qRT-PCR reaction system (10 μL)

[0054]

[0055] Table 6. Plants used for overexpression MsMYB-ZD Gene expression level detection qRT-PCR reaction procedure

[0056]

[0057] Example 4: MsMYB-ZD Leaf phenotypic analysis of transgenic alfalfa plants overexpressing the gene

[0058] The transgenic material was further cultured until the budding stage (culture conditions: 16 h light, 8 h dark, 22℃). Leaves from the middle part of the transgenic lines and wild-type plants in the late developmental stage were photographed and analyzed. Leaf phenotype is as follows. Figure 4 As shown in Figure A. The results showed that the leaf area of ​​the #2 overexpression line was significantly increased, averaging 3.46 cm², which was 77.77% larger than that of the wild type (1.94 cm²); the yield of fresh grass per plant increased by 158.7%, and the yield of hay per plant increased by 207.5% (see Table 7). Furthermore, the leaf area and biomass of all three overexpression transgenic lines were significantly better than those of the wild type. This verified the positive regulatory effect of this gene on leaf size and yield.

[0059] Table 7 Overexpression MSMYB-ZD Significantly increases alfalfa biomass

[0060]

[0061] The above embodiments show that, MsMYB-ZD As a newly discovered gene that positively regulates leaf size, it can serve as an important molecular target for improving the yield trait of alfalfa. Its coding sequence, protein structure, and regulatory methods can be applied to high-yield molecular breeding of alfalfa, and have significant industrial application value.

[0062] In this invention MsMYB-ZD The nucleotide sequence of the gene's CDS region and the amino acid sequence of its encoded protein are shown below:

[0063] (1)CDS region: ATGTCTTTGTCTCGCACGTGCTCACAGTGCGGCAACAACGGCCACAACTCGAGAACATGCAACGACGGTGGAGAAGAAAAAGGCATCATGATTTTTGGTGTTCGACTCACCGGAGGAAACAACAACCCCAACACCTCCACCACCACCACCACCAATAACAATCCTTTCAGAAAAAGTGCTAGCATGACCAACCTTTCTCAATATGAACAACCACCTCCACAAGATTCCAACCCCGCGGATGCCGGTTATGTATCCGATGACATCGTTCACGCCTCTGGTCGATCCAGAGAACGCAAACGAGGTGTTCCTTGGACAGAGGAAGAACACAAGCTCTTCTTGTTGGGGTTGCAACAGGTTGGTAAAGGTGATTGGAGAGGAATTTCTAGAAACTTTGTGAAAACAAGGACACCAACTCAGGTTGCAAGTCATGCTCAGAAGTATTTCCTCCGCCGTCATAACCAGAATCGCCGCCGCCGGAGATCCAGTCTCTTTGATATTACCACCGATACGGTGATGGAACCTTCAACTATAATGGAAGATGATCAATTTCAGCAAGAAACAGTGGCGCCACTGCCACCACCAACCCCCGCCGCATATCCATCCTCACATTACGGCGGCATCCCCGGGACACCTTTTCCGATGGGTCTTGGTCCGATAACATTGCCGGTAATGAGTGCTGAAAGAGTGGCCAAGCCAATTAGGCCAACACCAATGCTGCCTCCTTCTTCTAAGATGGCTAATCTGAACTTGAAAGACAAAGCTTCTTCTTCTTCTTCCATTGAGCCTTTTCCATTGTCATTGAAGCTGCAAACTTCGGATCCATCGGAAGATCATTCACCGGAAAGTAGCAGCCATTCGTCGTCATCGGCTTCATCATCGGCTTTTAAAACCATGGCTGCAGGGAAGTATAATGGTGGGGGTGGGGATAGCATTATCAGTGTTGCTTGA (SEQ ID NO.1);

[0064] (2) Encoded protein: MSLSRTCSQCGNNGHNSRTCNDGGEEKGIMIFGVRLTGGNNNPNTSTTTTTNNNPFRKSASMTNLSQYEQPPPQDSNPADAGYVSDDIVHASGRSRERKRGVPWTEEEHKLFLLGLQQVGKGDWRGISRNFVKTRTPTQVASHAQKYFLRRHNQN RRRRRSSLFDITTDTVMEPSTIMEDDQFQQETVAPLPPPTPAAYPSSHYGGIPGTPFPMGLGPITLPVMSAERVAKPIRPTPMLPPSSKMANLNLKDKASSSSSIEPFPLSLKLQTSDPSEDHSPESSSHSSSSASSSAFKTMAAGKYNGGGGDSIISVA (SEQ ID NO.2).

[0065] 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. MsMYB-ZD The application of genes in regulating alfalfa leaf size is characterized by, The regulation of alfalfa leaf size was achieved through overexpression. MsMYB-ZD The gene, transgenic alfalfa, increases leaf area; wherein, the... MsMYB-ZD The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.

1.

2. MsMYB-ZD The application of genes in regulating alfalfa yield is characterized by, The regulation of alfalfa yield is achieved through overexpression. MsMYB-ZD Gene-modified alfalfa plants showed increased fresh and dry hay yields; among which, the aforementioned MsMYB- ZD The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.

1.

3. The application according to claim 1 or 2, characterized in that, The MsMYB-ZD The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

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