CN115819540A - Application of alfalfa lateral organ boundary domain transcription factor MsLBD1 in regulation and control of plant growth and development - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to application of a transcription factor MsLBD1 of a lateral organ boundary region of alfalfa in regulation and control of plant growth and development. The invention clones the lateral organ boundary domain transcription factor MsLBD1 in alfalfa for the first time, overexpresses the MsLBD1 gene in alfalfa to obtain transgenic alfalfa overexpressing the MsLBD1 gene, researches the expression mode of the MsLBD1 gene in alfalfa and the function of the MsLBD1 gene in regulating and controlling the synthesis of secondary metabolites, the synthesis of sucrose and starch and the growth and development of plants, and proves that the MsLBD1 gene has the regulation and control function on plant crude protein, reducing sugar, neutral detergent fiber, acidic detergent fiber substances and the growth and development of plants through experiments.

Description

Application of alfalfa lateral organ boundary domain transcription factor MsLBD1 in regulation and control of plant growth and development

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of a transcription factor MsLBD1 of a lateral organ boundary region of alfalfa in regulation and control of plant growth and development.

Background

Alfalfa (Medicago sativa L.) is a perennial pasture with strong environmental adaptability, and enjoys the reputation of the king of pasture due to the characteristics of high protein content, good palatability and the like. The alfalfa is widely cultivated in the world, is one of the important perennial forage grass crops in the world, and the planting area of the alfalfa in the world reaches 3.2 multiplied by 10 ⁷ Hectare. The planting area of Chinese alfalfa is about 3.8 multiplied by 10 ⁶ Hectares, mainly distributed in northwest, north China and northeast. The alfalfa is rich in protein content, contains high active ingredients such as vitamins and flavonoids, and is very beneficial to livestock and human health.

Lateral organ boundary domain (LBD) genes are transcription factor gene families which are specific in plants, and the gene families have more members and are different in structure and function. According to the LOB domain, LBD family members fall into two main groups: the LOB domain of class I LBD genes have a complete zinc finger-like protein and a leucine-like zipper module, while class II LBD genes have only a zinc finger-like protein module. The LBD protein is characterized by containing an LOB domain, wherein the N terminal of the LOB domain has a CX2CX6CX3C type zinc finger structure (with DNA binding activity), the C terminal of the LOB domain has an LX6LX3LX6L type leucine zipper coiled coil motif and a glycine-alanine-serine module (GAS module). According to phylogenetic research and LBD protein function research, the I type LBD protein is mainly involved in plant development process, and the II type gene is mainly involved in metabolic process such as anthocyanin and nitrogen metabolism related process.

The establishment and maintenance of the meristem morphogenesis of plants plays an important role in plant morphogenesis, and the study of this process is still lacking. The research discovers from a transcription factor overexpression material that the overexpression of one MsLBD1 gene can cause the abnormality of the alfalfa plant type, and researches on the expression, the function and the biological function of the gene through a molecular biological means further lay a good foundation for clarifying a regulation mechanism and the function of an LBD family in the form of a plant meristem. In addition, the research on the action mechanism and the metabolic pathway of the MsLBD1 gene in the growth and development of alfalfa can also provide a new research idea for the high-yield and high-quality germplasm innovation of dicotyledonous plants. No study on the cloning, expression and application of the MsLBD1 gene in alfalfa and the regulation and control functions of the MsLBD1 on the plant growth and development in alfalfa are found in the existing study.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a transcription factor MsLBD1 of the alfalfa lateral organ boundary domain, a coding gene thereof and application thereof in regulating and controlling plant growth and development.

The technical scheme of the invention is as follows:

the application of the alfalfa lateral organ boundary domain transcription factor MsLBD1 in regulation and control of plant growth and development is disclosed, wherein the amino sequence of the alfalfa lateral organ boundary domain transcription factor MsLBD1 is shown in SEQ ID No. 1.

Preferably, the nucleotide sequence of CDS of the alfalfa lateral organ border domain transcription factor MsLBD1 gene is shown as SEQ ID NO. 2.

Preferably, said regulating the growth and development of a plant comprises regulating the plant height, leaf size and number of the plant.

The biological material containing the transcription factor MsLBD1 gene of the alfalfa lateral organ boundary region is obtained.

Furthermore, the biological material comprises an expression cassette, a vector, a transposon, an engineering bacterium, a host cell or a transgenic cell line.

The invention also aims to protect the application of the transcription factor MsLBD1 of the alfalfa lateral organ boundary domain or the biological material in regulating and controlling the plant feeding value.

Preferentially, the content of neutral detergent fibers and acidic detergent fibers of the alfalfa is reduced and the feeding value of the alfalfa is improved by improving the expression of the transcription factor MsLBD1 of the border domain of the lateral organs.

The transcription factor MsLBD1 of the alfalfa lateral organ boundary domain or the biological material is applied to construction of transgenic plants or plant genetic breeding.

The invention also provides a method for regulating the growth and development of the alfalfa or accumulating the metabolic substances, which is realized by regulating the expression of the transcription factor MsLBD1 of the alfalfa lateral organ boundary domain.

The invention discovers that the expression level of the transcription factor MsLBD1 of the alfalfa lateral organ boundary region influences the synthesis of secondary metabolites in plants, and the nutrition quality, feeding value and growth and development of alfalfa can be obviously improved by over-expressing the MsLBD 1. By improving the expression of a transcription factor MsLBD1 of a boundary region of a lateral organ, the growth and development of the alfalfa are influenced, or the accumulation of crude protein and reducing sugar in the alfalfa is improved, and the content of neutral detergent fiber and acidic detergent fiber substances is reduced.

Based on the function of the MsLBD1, the invention provides the alfalfa lateral organ boundary region transcription factor MsLBD1 or the gene for coding the alfalfa lateral organ boundary region transcription factor MsLBD1 or the application of biological materials containing the gene in regulation and control of plant growth and development.

The invention has the advantages of

The invention clones the lateral organ boundary domain transcription factor MsLBD1 in alfalfa for the first time, overexpresses the MsLBD1 gene in alfalfa to obtain transgenic alfalfa overexpressing the MsLBD1 gene, researches the expression mode of the MsLBD1 gene in alfalfa and the function of the MsLBD1 gene in regulating and controlling the synthesis of secondary metabolites, the synthesis of sucrose and starch and the growth and development of plants, and proves that the MsLBD1 gene has the regulation and control function on plant crude protein, reducing sugar, neutral detergent fiber, acidic detergent fiber substances and the growth and development of plants through experiments. The invention provides a theoretical basis and a new method for breeding high-quality new alfalfa varieties and breeding quality of other pasture.

Drawings

FIG. 1 is a diagram showing the result of electrophoresis of MsLBD1 gene clone in example 1 of the present invention, wherein M represents DNA marker with a size of 100-2000bp.

FIG. 2 shows the conserved domain structure analysis of MsLBD protein in example 1 of the present invention.

FIG. 3 is a phylogenetic tree analysis of the LBD proteins of alfalfa, chickpea, soybean, arabidopsis thaliana, rice, and brachypodium distachyon in example 1 of the present invention.

FIG. 4 shows the results of the analysis of the tissue expression characteristics of the MsLBD1 gene in example 1 of the present invention.

FIG. 5 is a diagram showing the PCR verification result of the recombinant plasmid constructed by the MsLBD1 gene overexpression vector in example 1 of the present invention, wherein M represents a DNA marker with a size of 100-2000bp.

FIG. 6 shows the procedure for obtaining transgenic alfalfa overexpressing MsLBD1 according to example 2 of the present invention.

FIG. 7 is a diagram showing the result of PCR verification of Hpt gene of MsLBD1 transgenic alfalfa in example 2 of the present invention.

FIG. 8 shows the results of the analysis of the relative expression amounts of the MsLBD1 genes in wild-type Medicago sativa and each transgenic plant in example 2 of the present invention, wherein WT represents wild-type Medicago sativa, and OE-13, OE-42, OE-64, OE-65, OE-73, OE-101, and OE-106 represent different transgenic alfalfa plants, respectively.

FIG. 9 shows phenotypic characteristics of leaf size and plant height of wild-type alfalfa and transgenic alfalfa plants in example 2, wherein WT represents wild-type alfalfa, and OE-64, OE-65 and OE-101 represent different transgenic alfalfa plants.

FIG. 10 shows the enrichment results of the gene KEGG differentially expressed from wild type and transgenic alfalfa in example 3 of the present invention.

Detailed Description

The invention clones the transcription factor MsLBD1 of the border domain of the lateral organ from alfalfa 'No. one', constructs an overexpression and gene interference vector of the MsLBD1 gene and carries out genetic transformation in an alfalfa explant to obtain an overexpression MsLBD1 transgenic alfalfa plant. The function of the MsLBD1 gene in regulating and controlling the growth and development of plants is analyzed by utilizing a wild type and an overexpression transgenic alfalfa. Experiments prove that the MsLBD1 gene influences the plant type and the leaf type of the alfalfa, improves the content of crude protein and reducing sugar of the plant, reduces neutral detergent fiber and acid detergent fiber, and improves the quality and the palatability of the alfalfa.

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 cloning of alfalfa lateral organ boundary Domain transcription factor MsLBD1

The cloning of the alfalfa lateral organ boundary domain transcription factor MsLBD1 comprises the following steps:

1. extraction of alfalfa leaf total RNA and cDNA synthesis

Taking about 100 mg of alfalfa seedling leaves of medium alfalfa I, grinding the leaves into powder in liquid nitrogen, and extracting the total RNA of the plant according to a Trizol method. Reverse transcription was performed using PrimeScript RT reagent Kit (TaKaRa) according to the instructions to obtain the reverse transcription product cDNA.

2. Design of alfalfa MsLBD1 gene cloning primer and gene cloning

(1) MtLBD1 gene sequence (Medtr 3g 077240) found in Medicago truncatula genome database Phytozome v12.1 (https:// Phytozome. Jgi. Doe. Gov/pz/portal. Html), cloning primers were designed using Primer 5.0 and NCBI Primer-BLAST (https:// www. NCBI. Nlm. Nih. Gov/tools/Primer-BLAST /) on-line tools, with the following Primer sequences (restriction sites underlined, determined from the gene sequence and the multiple cloning sites on the vector):

a forward primer: 5' -CGGGGTACCATGGCATCATCAAGCTCA -3'

(containing an enzyme cleavage site Kpn I);

reverse primer: 5' -CGAGCTCTCACAAATTACCTCCTCCTACACCT -3'

(including the enzyme cleavage site Sac I);

the primer is sent to Shanghai to be biosynthesized.

(2) The method comprises the following steps of carrying out PCR amplification by taking alfalfa cDNA obtained by reverse transcription as a template, selecting a Takara high-fidelity PCR enzyme PrimeSTAR Max reaction system for PCR, wherein the total volume of the system is 20 mu L, and the method comprises the following steps: 10. 1 muL of forward and reverse primers of muL PrimeSTAR Max,8 muL ddH2O and 10 muM and 1 muL of template cDNA. The PCR reaction program is: 95. 2 min at the temperature; 95. 30 s at 60 ℃, 30 s at 72 ℃, 45 s at 35 cycles; 10 min at 72 ℃. The reaction products were tested for specificity by agarose gel electrophoresis, and the amplified band of the MsLBD1 gene is shown in FIG. 1.

3. Construction of MsLBD1 gene cloning vector and gene sequencing

(1) The agarose gel with clear bands is about 600 bp in size, the MsLBD1 gene fragment is recovered by a gel recovery kit of TaKaRa according to the instruction, and the purity and the concentration of the recovered DNA fragment are measured by a NanoDrop2000 ultramicro ultraviolet spectrophotometer.

(2) The vector was ligated to pMD19-T vector by TaKaRa ligation kit according to the protocol as follows: 0.3 pmol of the fragment of interest (converted into volume according to concentration), 1. Mu.L of pMD19-T vector, 5. Mu.L of solution I, ddH ₂ And supplementing O to 10 mu L, uniformly mixing, and placing in a metal bath for reacting for 1 h at 16 ℃.

(3) The ligation product was gently added to the center of E.coli DH 5. Alpha. Competent cells, flicked 3-5, ice-cooled for 30 min, heat-shocked at 42 ℃ for 1 min, and allowed to stand on ice for 2 min.

(4) And adding 800 muL LB liquid culture medium into the transformed competent cells, inverting and uniformly mixing, placing on a shaker at 37 ℃, and carrying out shaking culture at 200rpm for 1 h.

(5) And uniformly coating 200 mu L of bacterial liquid on an LB solid culture medium plate containing 100 mg/L of ampicillin, and carrying out inverted culture in an incubator at 37 ℃ for 10-12 h. 10-20 single colonies were picked with a sterile pipette tip and added to 2 mL LB liquid medium (containing 100 mg/L Amp) separately, incubated at 37 ℃ with shaking at 200rpm for 10-12 h. Respectively taking 1 mu L of bacterial liquid, carrying out PCR identification on the bacterial liquid according to the method of the step 2 in the example 1, and checking the amplification specificity through agarose gel electrophoresis. 1 mL of the bacterial liquid containing the target fragment identified by PCR is taken and sent to Shanghai worker for sequencing (universal primer M13F is adopted for sequencing, and unidirectional sequencing is adopted).

4. Sequence and structural analysis and functional prediction of MsLBD1 gene

(1) The size of a coding region of the MsLBD1 gene sequence obtained by sequencing is 579 bp (the nucleotide sequence of the CDS of the MsLBD1 gene is shown as SEQ ID NO. 2), and the sequence comparison shows that the similarity with the medicago truncatula sequence is 97.93 percent and the total code is 192 amino acids (the amino acid sequence of the MsLBD1 is shown as SEQ ID NO. 1). The conserved domain of the amino acid sequence was analyzed on-line at the NCBI website (https:// www. NCBI. Nlm. Nih. Gov/Structure/cdd/wrpsb. Cgi), and as a result, as shown in FIG. 2, it was found to have a LOB domain identical to the amino acid conserved domain of the lateral organ border domain transcription factor LBD family member reported in other plants.

(2) The physical and chemical property prediction of the protein is carried out by using ExPASY (http:// web. ExPASy. Org/protparam /) online software, and the MsASMT protein is found to have the molecular weight of 20.768Kd, the theoretical isoelectric point of 6.81, the fat solubility coefficient of 71.15, the total average hydrophilic coefficient of-0.267 and the instability coefficient of 57.43, and belongs to stable and non-protein. Protein subcellular localization prediction was performed using CELLO (http:// CELLO. Life. Nctu. Edu. Tw /) online software to find that MsLBD1 localizes in the nucleus.

(3) According to the MsLBD1 amino acid sequence, the corresponding amino acid sequences of plants such as chickpea, soybean, brachypodium distachyon, rice, arabidopsis thaliana and the like are searched on a Phytozome website on line, MEGA 5.0 software is introduced to construct a phylogenetic tree, and the result is shown in figure 3, and the alfalfa MsLBD1 gene and the chick pea CaLBD1 gene have the highest homology.

5. MsLBD1 gene space-time expression characteristic analysis

(1) Taking about 100 mg of the roots, stems, young leaves and other parts of the alfalfa in the Chinese alfalfa I, extracting the total RNA of all parts according to a Trizol method, and synthesizing cDNA by using a PrimeScript RT reagent Kit reverse transcription Kit of TaKaRa company.

(2) According to the MsLBD1 gene sequence, a Primer 5.0 and an NCBI Primer-BLAST online tool are utilized to design a specific Primer and carry out real-time fluorescence quantitative PCR reaction, wherein the Primer sequence is as follows: msLBD1-F: 5 '-GCAGTGAATTCCTTGCTTATG-3', msLBD1-R: 5 '-GCCTCTCTGAACTTGTCTCTGTAG-3', and the sequence of the internal reference primer is as follows: 5' actin-F: 5' CAAAAGATGGCAGATGCTGAGGAT-3' actin-R: 5' CATGACA CCAGTATGACGAGGTCG-3'. As shown in FIG. 4, the expression level of the MsLBD1 gene was the highest in the stem and root, and the expression level was the lowest in the young leaf.

Example 2 MslBDD 1 overexpression and RNAi vector construction and transgenic alfalfa acquisition

The embodiment provides a method for constructing the overexpression of the transcription factor MsLBD1 of the lateral organ boundary region of alfalfa and obtaining transgenic alfalfa, which specifically comprises the following steps:

1. construction of MsLBD1 plant overexpression vector

(1) The Escherichia coli liquid containing pMD19T-MsLBD1 plasmid with correct sequencing is extracted with TaKaRa plasmid minification kit and the plasmid concentration is determined with NanoDrop 2000.

(2) Because the upstream and downstream primers of the gene clone respectively carry enzyme cutting recognition sites of Kpn I and Sac I, the two enzymes are selected to respectively carry out double enzyme cutting on a plasmid sample with correct sequencing and a plant overexpression vector pZH01, and the enzyme cutting system is as follows: plasmid DNA 1 mug (converted into volume according to concentration), kpn H I1 mug, sac I1 mug, cutsmart 5 mug and ddH ₂ Supplementing O to 50 mu L, uniformly mixing, and reacting at 37 ℃ for 1h. The cleavage result was verified by agarose gel electrophoresis.

(3) And recovering the enzyme-digested target gene fragment and the pZH01 vector fragment by using a TaKaRa gel recovery kit according to the instruction, and determining the concentration by using NanoDrop 2000. Connecting a target gene and a target vector fragment by using T4 DNA ligase, wherein the reaction system comprises the following steps: the target gene is as follows: vector fragment =10:1, 1 μ L of T4 DNA ligase, 2 μ L of Buffer, using ddH ₂ And supplementing O to 20 mu L, uniformly mixing, and connecting for more than 2 h at 16 ℃.

(4) The ligation product was transformed into E.coli DH 5. Alpha. Competent cells according to step 3 of example 1 and cultured. The PCR of the bacterial suspension was determined by referring to the method of step 2 in reference example 1, and the amplification product was detected by agarose gel electrophoresis as shown in FIG. 5.

(5) And (3) taking 1 mL of escherichia coli bacteria liquid with positive bacteria liquid PCR, sending the escherichia coli bacteria liquid to Shanghai engineering sequencing, extracting plasmids from the bacteria liquid with correct sequencing result, and obtaining the over-expression recombinant plasmids which are successfully constructed.

2. Agrobacterium-mediated genetic transformation of alfalfa and acquisition of transgenic plants

(1) Gently adding the recombinant plasmid into the center of EHA105 competent cell, gently flicking under 3-5, ice-bathing for 30 min, quickly freezing in liquid nitrogen for 5 min, heat-shocking at 37 deg.C for 5 min, and standing in ice for 2 min.

(2) And adding 500 mu L YEP liquid culture medium into the transformed competent cells, inverting and uniformly mixing, placing on a shaker at 28 ℃, and carrying out shaking culture at 200rpm for 2-4 h.

(3) And (3) coating 200 mu L of bacterial liquid on a YEP plate containing 100 mg/L kanamycin and 50 mg/L rifampicin, performing inversion culture at 28 ℃ in a dark place for 2 days, selecting a single colony for colony PCR detection, wherein the detection result is positive, and the result shows that the recombinant plasmid is successfully introduced into agrobacterium tumefaciens.

(4) And (4) selecting full alfalfa seeds of the medium alfalfa I into a 50 mL centrifuge tube, and cleaning and sterilizing the alfalfa seeds. The cleaned seeds were put on an MS solid medium (MS minimal medium: 4.43g/L, sucrose 30 g/L, agar 8 g/L, pH 5.8) for germination.

(5) Culturing 100 mu L of preserved agrobacterium liquid to 20 mL of YEP liquidCulturing in culture medium (50 mg/L Rif,100 mg/L Kan) at 28 deg.C and 200rpm for 14 h with shaking, adding As to 150 μ M/L, and shaking for 2 h to OD of bacterial liquid ₆₀₀ The value reaches 0.8-1.0, centrifuging at 3000 rpm for 10 min, collecting thalli, then re-suspending with 20 mL of resuspension liquid (3.908 g/L of N6 culture medium, 0.098 g/L of iron salt, 1 mL/L of organic components, 3 mg/L of 2,4-D,0.05 mg/L of KT,0.6 g/L of MES,20 g/L of cane sugar, pH 5.4) until the OD value of the bacterial liquid reaches 0.5-0.6, then slowly shaking at 28 ℃, 80 rpm for 2 h, and preparing the agrobacterium liquid for later use.

(6) Cutting the cotyledon of 10 d aseptic seedling into 3-4 mm ² Cutting hypocotyl into 2-3 mm segments, placing into Agrobacterium liquid, ice-cooling for 20 min, vacuumizing at-0.8 MPa for 10 min, and slowly shocking at 28 deg.C and 80 rpm for 30 min.

(7) Discarding the Agrobacterium liquid, transferring the explant to sterile filter paper, drying for 30-40 min, spreading on co-culture medium (N6 medium 3.908 g/L, iron salt 0.098 g/L, organic components 1 ml/L,3 mg/L2, 4-D,0.05 mg/L KT,0.6 g/L MES,20 g/L sucrose, 7 g/L agar, pH 5.8), and dark-culturing at 25 deg.C for 2-3D.

(8) The explants after the co-culture are washed 3-5 times by sterile water, are transferred to an induction medium after being dried in the air (3.908 g/L of N6 medium, 0.098 g/L of iron salt, 1 ml/L of organic components, 2 mg/L of 2,4-D,0.05 mg/L of KT,0.5 g/L of acid hydrolyzed casein, 20 g/L of sucrose, 200 mg/L of timentin, 5 mg/L of hygromycin, 7 g/L of agar, pH 5.8), and after being cultured in the dark at 25 ℃ for two weeks, the hygromycin concentration in the medium is increased to 10 mg/L, and the culture conditions are not changed.

(9) After 2 weeks, the viscous callus was inoculated onto a differentiation medium (N6 medium 3.908 g/L, iron salt 0.098 g/L, organic components 1 ml/L,0.6 mg/L KT,0.5 g/L acid hydrolyzed casein, 20 g/L sucrose, 200 mg/L timentin, 3 mg/L hygromycin, 7 g/L agar, pH 5.8), light-cultured at 25 ℃ for 2 weeks, then the differentiation medium was changed, and the torpedo-type embryos formed continuously were inoculated onto a rooting medium (MS basic medium 4.43g/L, sucrose 20 g/L, agar 7 g/L,200 mg/L timentin, 2 mg/L hygromycin, pH 5.8), and light-cultured at 25 ℃.

(10) Resistant seedlings grown to a height of about 10 cm in sterile rooting flasks were washed with root medium and transplanted into nutrient soil (see FIG. 6).

(11) Taking about 100 mg of resistant seedling leaves, grinding the resistant seedling leaves into powder in liquid nitrogen, and extracting the total DNA of the plants according to a CTAB method. Designing by using a Primer 5.0 and an NCBI Primer-BLAST online tool according to a screening marker gene Hpt sequence on the recombinant vector, wherein the Primer sequence is as follows:

Hpt-F:5’-TACTTCTACACAGCCATCGGTCCAG-3’

Hpt-R: 5’-CTTGACATT GGGGAGTTTAGCGAGA-3’。

the PCR reaction program of 2 (2) was followed to verify and obtain transgenic positive alfalfa (results are shown in FIG. 7).

(12) Total RNA of positive transgenic alfalfa leaves was extracted according to Trizol method, and RT-qPCR analysis was performed using cDNA synthesized by PrimeScript RT reagent Kit reverse transcription Kit of TaKaRa as a template with reference to the primers in step 2 of example 2, to determine the MsLBD1 gene expression level in positive transgenic alfalfa plants (the results are shown in FIG. 8).

Example 3 phenotypic and biological functional analysis of transgenic alfalfa

1. Phenotypic analysis of growth and development of transgenic alfalfa

After the transgenic alfalfa is transplanted into a greenhouse, phenotypic indexes such as plant height, stem thickness, leaf size and the like of the transgenic and wild-type plants are observed and measured (the results are shown in table 1), the plant height of the transgenic alfalfa over-expressing the MsLBD1 is shortened, the leaves are reduced, and the quantity of the leaves is increased (the results are shown in figure 9).

TABLE 1 transgenic alfalfa phenotype assay

2. Analysis of differentially expressed genes in transgenic alfalfa

About 1.5-2g of young leaves at the top ends of wild-type and over-expressed transgenic alfalfa are taken and sent to Wuhan Meiteweier biotechnology and Limited company for transcriptomics analysis of MsLBD transgenic alfalfa and wild-type alfalfa, the result is shown in figure 10, and the differential genes are mainly enriched to a secondary metabolic pathway, starch and sucrose metabolism, a plant pathogenic bacterium interaction pathway, a phenylpropanoid biosynthesis pathway and a glutathione metabolic pathway.

3. Determination of quality index in transgenic alfalfa

The overground parts of wild type alfalfa and over-expression transgenic alfalfa in the same growth period are taken, dried in a 65 ℃ oven, crushed and sieved, 0.5 g of powder is taken respectively, the Neutral Detergent Fiber (NDF) and Acid Detergent Fiber (ADF) contents are measured by a cellulose instrument, the crude protein content is measured by an element analyzer, and the soluble sugar content is measured by an anthrone colorimetric method. The results are shown in table 2, where the transgenic alfalfa overexpressing MsLBD1 had significantly reduced ADF and NDF content and significantly increased crude protein and soluble sugar content compared to the wild type.

TABLE 2 transgenic alfalfa quality index determination

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (9)

1. The application of the alfalfa lateral organ boundary domain transcription factor MsLBD1 in regulation and control of plant growth and development is characterized in that the amino sequence of the alfalfa lateral organ boundary domain transcription factor MsLBD1 is shown in SEQ ID No. 1.

2. The use according to claim 1, wherein the nucleotide sequence of CDS of the gene of alfalfa lateral organ border domain transcription factor MsLBD1 is shown as SEQ ID No. 2.

3. The use according to claim 1, wherein said modulating plant growth comprises modulating plant height, leaf size and number of plants.

4. Biological material containing the transcription factor MsLBD1 gene for the alfalfa lateral organ boundary domain.

5. The biomaterial of claim 4, comprising an expression cassette, a vector, a transposon, an engineered bacterium, a host cell or a transgenic cell line.

6. Use of the alfalfa lateral organ boundary domain transcription factor MsLBD1 of claim 1 or the biomaterial of claim 4 for modulating plant feed value.

7. The use of claim 6, wherein the feed value of alfalfa is increased by increasing the expression of lateral organ boundary region transcription factor MsLBD1, reducing the content of neutral detergent fiber and acidic detergent fiber of alfalfa.

8. Use of the alfalfa lateral organ border domain transcription factor MsLBD1 of claim 1 or the biological material of claim 4 for constructing a transgenic plant or for genetic breeding of plants.

9. A method for regulating and controlling the growth and development of alfalfa or the accumulation of metabolites is characterized in that the regulation and control of the expression of a transcription factor MsLBD1 of a lateral organ boundary domain of alfalfa is realized.

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