Application of MsGDSL1 gene in regulating plant leaf size and yield

Abstract

The application discloses a kind of MsGDSL1 The application discloses application of a gene in regulating plant leaf size and yield, and belongs to the technical field of plant genetic engineering. MsGDSL1 The application clones a gene involved in regulating alfalfa leaf size by using transcriptome analysis. MsGDSL1 Experimental data show that, compared with wild type control plants, the leaf area of alfalfa MsGDSL1 gene overexpression plants is enlarged by an average of 60.54%, the fresh grass yield of transgenic plants is increased by an average of 58.97%, and the dry grass yield is increased by an average of 61.88%; and the leaf area of Arabidopsis overexpression lines is enlarged by an average of 4 times compared with wild type. MsGDSL1 MsGDSL1 MsGDSL1 MsGDSL1 MsGDSL1 MsGDSL1 MsG This result further indicates that the function of the gene in regulating plant leaf size is highly conserved across species, and proves the core value of the gene in improving the yield of alfalfa.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and particularly relates to a method for improving yield of alfalfa MsGDSL1 and application of the gene in regulating plant leaf size and yield. BACKGROUND

[0002] In the livestock breeding industry, feed costs account for as high as 65%-70%, and its supply stability and quality directly determine the development level of the industry. At present, China's livestock industry is in a critical stage of transformation and upgrading, but the problem of feed resource shortage is becoming increasingly prominent. Alfalfa, as a recognized high-quality forage grass, has become a core forage grass resource to ensure the efficient production of livestock industry due to its excellent feeding value. However, the development of China's alfalfa industry is facing multiple difficulties, such as weak market competitiveness of self-cultivated varieties, low efficiency of traditional breeding, and other problems, which cannot meet the urgent demand for high-quality forage grass. With the acceleration of the process of large-scale development of domestic livestock industry, the market demand for high-quality alfalfa continues to rise, and the supply gap of domestic forage products is gradually expanding. Therefore, through technological innovation to improve the yield of alfalfa and cultivate high-yield and high-quality varieties with independent intellectual property rights has become a key breakthrough and core task to alleviate the shortage of feed resources and ensure the sustainable development of the livestock industry.

[0003] Leaf, as the core organ of photosynthesis and nutrient synthesis of alfalfa, its morphological characteristics have a decisive influence on the overall productivity of the plant. Studies have shown that leaf size is closely related to the biomass accumulation of alfalfa. Large leaves not only can significantly improve the photosynthetic efficiency, but also can promote the absorption and utilization of nutrients by the plant. In the early growth stage of alfalfa, larger leaves help the seedling to quickly establish the root system and enhance its adaptability to adversity; while in the later growth stage, the developed leaf system is the key factor to realize high yield. In addition, leaf size is also closely related to the forage quality of alfalfa. Leaf is not only the main storage organ of crude protein in alfalfa, its size directly affects the synthesis and storage efficiency of protein. Generally, larger leaves can provide more space and resources for protein accumulation, thereby improving the feeding value of alfalfa. For example, multi-leaf alfalfa has excellent forage quality due to its high leaf contribution rate, rich leaf protein and mineral content, and large leaf-stem ratio.

[0004] Although leaf size, as one of the core traits of alfalfa, has a significant impact on its yield and quality. However, the genetic improvement of alfalfa leaf size still faces many technical difficulties. The complex genetic background and long breeding cycle make it difficult for traditional breeding methods to quickly breed ideal large-leaf varieties. With the development of modern molecular biology techniques, it has been gradually applied in the field of genetic improvement of alfalfa. However, in terms of leaf size regulation, the key gene resources that can precisely act on leaf size traits and can be used for genetic engineering operation are still extremely scarce. Therefore, in-depth research on the molecular mechanism of alfalfa leaf development, screening and identification of key regulatory genes, has important practical significance and application value for innovative breeding technology and improving the yield of alfalfa. SUMMARY

[0005] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide a kind of MsGDSL1 The application of the gene in regulating the size of plant leaves and yield to improve the size of plant leaves and yield.

[0006] The technical scheme for solving the above technical problems of the present application is as follows: to provide a kind of MsGDSL1 The application of the gene in regulating the size of plant leaves.

[0007] The present application provides a kind of MsGDSL1 The application of the gene in regulating the yield of plants.

[0008] Further,The nucleotide sequence of the CDS region of the gene is shown as SEQ ID NO. 1. MsGDSL1 Further,The amino acid sequence of the encoded protein of the gene is shown as SEQ ID NO. 2.

[0009] MsGDSL1 Further, the regulation of the size of plant leaves is overexpression The leaf area of the transgenic plant is increased.

[0010] MsGDSL1 Further, the regulation of the yield of plants is overexpression The fresh grass and dry grass yield of the transgenic plant per plant are increased.

[0011] MsGDSL1 Further, the plants include monocotyledonous plants and dicotyledonous plants.

[0012] Further, the dicotyledonous plants include alfalfa and Arabidopsis thaliana.

[0013] The present application also provides a preparation for improving the yield of alfalfa, which comprises the above-mentioned The gene or the encoded protein of the above-mentioned

[0014] Gene. MsGDSL1 MsGDSL1

[0015] This invention has the following beneficial effects: This invention utilizes transcriptome analysis to clone a gene involved in regulating alfalfa leaf size. MsGDSL1 Genes. Experimental data shows that... MsGDSL1 The gene was predominantly expressed in alfalfa leaves, exhibiting a progressively upregulated expression pattern during leaf development. The expression level of this gene in transgenic plants showed a significant positive correlation with leaf area. Compared to wild-type control plants, alfalfa… MsGDSL1 The leaf area of ​​the overexpressing gene plants increased by an average of 60.54%, and the fresh grass yield of the transgenic plants increased by an average of 58.97%, while the hay yield increased by an average of 61.88%. Furthermore, the leaf area of ​​the Arabidopsis overexpressing lines was on average four times larger than that of the wild type. This result further indicates... MsGDSL1 Genes exhibit high conservation across species in their function of regulating plant leaf size. The above experimental data strongly demonstrates this. MsGDSL1 Genes play a crucial role in improving alfalfa yield. Therefore, this invention provides a solid theoretical basis and a highly promising genetic resource for the targeted breeding of high-yielding alfalfa varieties using molecular breeding techniques. Attached Figure Description

[0016] Figure 1 Transcriptome analysis of alfalfa leaf size mutants and MsGDSL1 It participates in regulating the developmental process of alfalfa leaf size;

[0017] Figure 2 for MsGDSL1 Expression levels of the gene at different developmental stages of the L123 large-leaf mutant leaf;

[0018] Figure 3 Genes regulating leaf size in alfalfa MsGDSL1 Homologous gene clustering analysis;

[0019] Figure 4 Arabidopsis thaliana MsGDSL1 Detection of expression levels in overexpressing transgenic plants;

[0020] Figure 5 alfalfa MsGDSL1 Identification of overexpressing transgenic plants and detection of expression levels;

[0021] Figure 6 Arabidopsis thaliana MsGDSL1 Overexpression leads to an enlarged leaf phenotype in plants;

[0022] Figure 7 alfalfa MsGDSL1 Overexpression results in an enlarged leaf phenotype in plants. Detailed Implementation

[0023] 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.

[0024] Example 1: Alfalfa MsGDSL1 Gene screening and identification

[0025] (1) Alfalfa leaf size regulation genes MsGDSL1 Filtering

[0026] This embodiment uses transcriptome analysis to study the differentially expressed genes between the large-leaf mutant L123 and the wild type of alfalfa cultivar 'Zhongmu 3' during leaf development. Using the large-leaf mutant L123 obtained through previous mutagenesis screening in the laboratory and the control wild type of 'Zhongmu 3' as materials, leaf tissue samples were collected according to the leaf development sequence at the early stage (young leaves, S1), early-middle stage (rapid elongation period, S2), and middle stage (early leaf unfolding stage, S3) (see...). Figure 1 A), conduct RNA-Seq transcriptome sequencing.

[0027] Using the alfalfa 'Xinjiang Large Leaf' as the reference genome, differential gene analysis was performed using DESeq2 software. By setting the screening criteria of |log2FC|≥1 and FDR<0.05, a total of 7395 genes with significant expression differences were identified (see...). Figure 1 B). Cluster analysis of these differentially expressed genes revealed that several genes showed a gradually upregulated expression trend as leaf development progressed (Cluster IV, see...). Figure 1 C). Notably, during the early stages of leaf development (S1-S3), the expression levels of this gene in the L123 large-leaf mutant were significantly higher than those in the wild type (Cluster IV, see...). Figure 1 C).

[0028] Further research revealed that MsGDSL1 ( MS.gene002668 The transcriptional level of the gene in the L123 large-leaf mutant at all developmental stages of the leaves was significantly higher than that in the wild-type plant. Figure 1 D). This gene showed upregulated expression during the differentiation of young leaves, the rapid elongation of leaves, the leaf unfolding and maturation stage, and the terminal bud tissue of alfalfa, and its expression level was significantly positively correlated with the leaf development process. Figure 2 Based on the above results, MsGDSL1 It was given priority screening as an important candidate gene for regulating alfalfa leaf size.

[0029] (2) MsGDSL1Identification and evolutionary analysis of homologous genes

[0030] As can be seen from the Pfam structure field annotations, MS.gene002668 The gene encodes a GDSL family lipase (EC3.1.1.3), whose protein N' terminus contains a conserved GDSL amino acid sequence. Further analysis was conducted using HMMER software to... MsGDSL1 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.) and the basal angiosperm *Cinnamomum camphora* ( Amborella trichopoda Homologous sequence searches were performed in the genome database, and a phylogenetic tree was constructed. Phylogenetic analysis showed that MsGDSL1 clustered with Arabidopsis thaliana At5g45670, At1g29670, At1g29660, and *Cinnamomum camphora* W1Q0B8 in the same evolutionary branch (see...). Figure 3 This indicates MsGDSL1 Homologous genes existed early in the evolution of angiosperms. More than 100 members of the GDSL lipase family have been identified in Arabidopsis thaliana, but the functions of only a few members have been clearly elucidated. Among them, At5g45670 , At1g29670 and At1g29660 No studies have been reported on the functional role of these three genes in regulating plant leaf development (especially leaf size). Therefore, it is evident that... MsGDSL1 As with these three Arabidopsis thaliana GDSL 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.

[0031] Example 2: MsGDSL1 Gene cloning and construction of overexpression vectors

[0032] 1. MsGDSL1 Cloning of genes

[0033] Total RNA was extracted from leaf tissues of the large-leaf mutant L123 during early leaf development using the EZNA® Plant RNA Kit (OMEGA Bio-TEK). cDNA was synthesized by reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). MS.gene002668 Homologous genes in the 'Zhongmu No. 1' variety MsG0280006968.01For reference, specific primers F1 and R1 (nucleotide sequences of the non-underlined portions of SEQ ID NO.3 and SEQ ID NO.4) were designed at the start and stop codons of the sequence; simultaneously, the pSuper1300 overexpression vector (previously preserved in the laboratory) was introduced upstream and downstream of the specific primers, respectively. Xba I and Kpn I. Homologous base sequences matching the restriction enzyme sites (nucleotide sequences underlined in SEQ ID NO.3 and SEQ ID NO.4). Using the cDNA of the above-mentioned alfalfa 'Zhongmu 3' L123 mutant as a template, PCR amplification was performed according to the reaction system in Table 1 (polymerase mix purchased from TaKaRa), and the amplification program is shown in Table 2.

[0034] F1: 5'- ATACACCAAATCGACTCTAGA ATGGCATTTTGGGACCTCT

[0035] CT-3' (SEQ ID NO.3);

[0036] R1: 5'- GCCCTTGCTCACCATGGTACCTTA GATTTGAGCTAAACGA

[0037] TT-3' (SEQ ID NO.4).

[0038] Table 1 is used for MsGDSL1 Amplification PCR reaction system (50 μL)

[0039]

[0040] Table 2 is used for MsGDSL1 Amplification PCR reaction procedure

[0041]

[0042] (2) MsGDSL1 Construction of gene overexpression vectors

[0043] The PCR amplification fragments obtained in Example 1 were recovered by gel extraction after 1.5% agarose gel electrophoresis (gel extraction kit purchased from Polymermax). The gel extraction products were ligated into the pSuper1300 linear vector using homologous recombinase (purchased from Zhuangmeng). Xba I and Kpn Double digestion with I 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... Super: MsGDSL1 The above plasmid was transformed into Agrobacterium tumefaciens.EHA105 Competent cells (prepared and preserved in the laboratory).

[0044] Example 3: MsGDSL1 Obtaining and identifying transgenic Arabidopsis and alfalfa plants overexpressing the gene

[0045] (1) Arabidopsis thaliana Super: MsGDSL1 Transformation

[0046] Transformation of Arabidopsis Col-0 ecotype using the Floral Dip method (Reference: Clough SJ, Bent AF. Floral dip: a simplified method for...) Agrobacterium -mediated transformation of Arabidopsis thaliana . Plant J. 1998;16(6):735-43.).

[0047] (2) Alfalfa Super: MsGDSL1 Transformation

[0048] The regenerable single plants 1-16 of Gongnong No. 1 obtained in our laboratory in the previous screening were transformed using the alfalfa leaf disc transformation method (reference: Jiang Q, Fu C, Wang ZY. A unified Agrobacterium -mediatedtransformation protocol for alfalfa ( Medicago sativa L.) and Medicago truncatula . Methods Mol. Biol. 2019;1864:153-163.).

[0049] (3) MsGDSL1 Identification of overexpression transgenic plants and detection of expression levels

[0050] Homozygous transgenic Arabidopsis lines were obtained through hygromycin resistance screening. Total RNA extraction and cDNA synthesis methods for both transgenic lines and wild-type plants were the same as in Example 2. Super: MsGDSL1 Vector sequence design for qRT-PCR primers F2 and R2 (primer positions as follows) Figure 4 (As shown), constitutive gene expression in Arabidopsis thaliana. At-ACTIN This serves as an internal reference gene (primer sequences are shown below). Using the cDNA of the above-mentioned plants as a template, LightCycler was used... ® Real-time quantitative PCR was performed using a 96 Real-Time PCR instrument (reaction system shown in Table 3, TB green was purchased from TaKaRa; reaction procedure shown in Table 4). The experiment was performed in triplicate. Results are as follows:Figure 4 As shown, among the three overexpression lines MsGDSL1 Gene expression levels were significantly higher than in the wild type, indicating that MsGDSL1 The gene was efficiently overexpressed in transgenic Arabidopsis thaliana.

[0051] F2: 5'-GCTCAGTTCTCCTCCAGTCAACGA-3' (SEQ ID NO.5);

[0052] R2: 5'-TCCGTGAAGCAAACATCGGACCA-3' (SEQ ID NO. 6);

[0053] At-ACTIN -F: 5'-ATCAATTCGATCACTCAGAGC-3' (SEQ ID NO.7);

[0054] At-ACTIN -R: 5'-CTATGATGCACTTGTGTGTGA-3' (SEQ ID NO. 8).

[0055] Table 3. Plants used for overexpression MsGDSL1 qRT-PCR reaction system for gene expression level detection (10 μL)

[0056]

[0057] Table 4. Plants used for overexpression MsGDSL1 qRT-PCR reaction procedure for gene expression detection

[0058]

[0059] based on Super: MsGDSL1 On the carrier MsGDSL1 The gene sequence was analyzed, and pSuper1300 vector-specific primers F3 and R3 were designed in the upstream and downstream regions of its start and stop codons. The gene was obtained from wild-type single plants of Alfalfa Gongnong 1 (1-16) and after Basta resistance screening. Super: MsGDSL1 Using the gDNA of the transgenic seedlings as a template, PCR amplification was performed according to the following reaction system (reaction system shown in Table 5, where the polymerase mix was purchased from Vazyme; reaction procedure shown in Table 6), and positive plants were obtained as follows. Figure 5 As shown in Figure A.

[0060] F3: 5'-AGCAAGAACGGAATGCGCGT-3' (SEQ ID NO.9);

[0061] R3: 5'-GCCCTTGCTCACCATGGTACCTTA-3' (SEQ ID NO. 10).

[0062] Table 5 is used for MsGDSL1 PCR reaction system for identifying overexpressing plants (20 μL)

[0063]

[0064] Table 6 is used for MsGDSL1 PCR reaction procedure for identifying overexpressing plants

[0065]

[0066] Total RNA extraction and cDNA synthesis from alfalfa transgenic plants were performed in the same manner as in Example 2. Based on MsGDSL1 Gene qRT-PCR primers F2 and R2 were used to constitutively express the gene in alfalfa. MsACTIN For internal reference, using the cDNA of the above-mentioned plants as a template, LightCycler was used. ® Real-time quantitative PCR was performed using a 96 Real-Time PCR instrument (TB green purchased from TaKaRa). The experiment was repeated three times (reaction procedures and systems are shown in Tables 3-4). Results are as follows: Figure 5 As shown in B, among the three overexpression lines MsGDSL1 The gene expression level was significantly higher than that of wild-type Gongnong 1-16 alfalfa plants, fully verifying that... MsGDSL1 High-efficiency expression of genes in transgenic alfalfa.

[0067] MsACTIN -F: 5'-GACAATGGAACTGGAATGG-3' (SEQ ID NO. 11);

[0068] MsACTIN -R: 5'-CAATACCGTGCTCAATGG-3' (SEQ ID NO. 12).

[0069] Example 4: MsGDSL1 Leaf phenotypic analysis of overexpressing transgenic plants

[0070] (1) Phenotypic analysis of transgenic Arabidopsis plants

[0071] Arabidopsis thaliana MsGDSL1 The overexpression line and wild-type Col-0 were cultured on 1 / 2 MS medium for 7 days (culture conditions: 16 h photoperiod, 8 h dark, temperature 22℃). Their cotyledons were photographed and their phenotypes were measured. Figure 6As shown in Figure A. Statistical analysis showed that the cotyledon area of ​​the overexpressing lines was significantly increased compared to the wild type. Wild-type and transgenic Arabidopsis plants were cultured for 30 days and photographed for analysis (culture conditions: 16 h light, 8 h darkness, temperature 22℃). Leaf phenotypes are shown in Figure A. Figure 6 As shown in B. Mature leaves from wild-type and transgenic plants (see...) Figure 6 C (indicated by the white arrow) was used for quantitative statistics, and the results showed that: all MsGDSL1 The leaf area of ​​the overexpression lines was significantly increased, averaging greater than 3.11 cm² (compared to 0.65 cm² for wild-type leaves). 2 The leaf area of ​​the overexpression lines was on average 4 times larger than that of the wild type.

[0072] (2) Phenotypic analysis of transgenic alfalfa plants

[0073] Transgenic lines and wild-type alfalfa plants were transplanted and cultured under conditions of 16 hours of light, 8 hours of darkness, and 24°C. Leaves in the late developmental stage of the middle part of the plant were photographed and measured at three weeks of cultivation and at the budding stage. Observations and statistical analysis were performed (see...). Figure 7 The leaf area of ​​all three overexpressing transgenic lines was significantly larger than that of wild-type alfalfa plants. The average mature leaf area of ​​the overexpressing lines was 2.97 cm², an increase of 60.54% compared to the 1.85 cm² of the wild type. The transgenic plants showed an average increase of 58.97% in fresh grass yield and 61.88% in hay yield (see Table 7), with the leaf area and biomass of the three overexpressing transgenic lines significantly superior to the wild type. This verifies the positive regulatory effect of this gene on leaf size and yield.

[0074] Table 7 Overexpression MsGDSL1 Significantly increases alfalfa biomass

[0075]

[0076] Based on the phenotypic analysis results of the above Arabidopsis and alfalfa plants, it was confirmed that... MsGDSL1 This gene has a significant positive regulatory effect on plant leaf size. As a newly identified gene positively regulating leaf size, MsGDSL1 It can serve as a key molecular target for improving the yield trait of alfalfa. Its coding sequence, protein structure, and regulatory methods are applicable to high-yield molecular breeding of alfalfa, providing a novel gene resource and technical solution for high-yield molecular breeding of alfalfa, and possessing significant industrial application value and broad market prospects in the field of agricultural bio-breeding.

[0077] In this invention MsGDSL1 MsGDSL1 The nucleotide sequence of the gene's CDS region and the amino acid sequence of its encoded protein are shown below:

[0078] (1) CDS region:

[0079]

[0080] (2) Protein-encoding:

[0081] MAFWDLSMINIGIVVLVLSLWSGIGVAQQVPCYFIFGDSLVDNGNNNQLTSIAKANYLPY GIDFPGGPTGRFSNGKTTVDVIAEQLGFNGYIPSYASARGRQILRGVNYASAAAGIREE TGQQLGQRISFRGQVQNYQRTVSQLVNYFGDENTAANYLSKCIYTIGLGSNDYLNNYFMP TIYSTSRQFTPQQYANVLLQAYAQQLRILYNYGARKMALFGVGQIGCTPNALAQNSPDGR TCVARINSANQLFNNGLRSLVDQLNNQLPDARFIYVNVYGIFQDIITSPSSYGFRVTNAG CCGVGRNNGQITCLPFQPACRDRNGFLFWDAFHPTEAGNSVIGRRAYNAQSASDAYPID INRLAQI (SEQ ID NO. 2).

[0082] The above description is merely that of the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made without departing from the spirit and principle of the present application shall fall within the protection scope of the present application.

Claims

1. MsGDSL1 The application of genes in regulating plant leaf size is characterized by, The regulation of plant leaf size is achieved through overexpression. MsGDSL1 Genes that increase leaf area in transgenic plants; MsGDSL1 The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.1; the plants are Arabidopsis thaliana and alfalfa.

2. MsGDSL1 The application of genes in regulating plant yield is characterized by, The regulation of plant yield is achieved through overexpression. MsGDSL1 Genes that increase the yield of both fresh and dry grass per plant in transgenic plants; MsGDSL1 The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.1; the plants are Arabidopsis thaliana and alfalfa.

3. The application according to claim 1 or 2, characterized in that, The MsGDSL1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

4. A formulation for increasing alfalfa yield, characterized in that, The formulation comprises the one described in claim 2. MsGDSL1 Gene.

Images