A gene related to the inhibition of lignin synthesis in alfalfa and its application

Abstract

This invention provides a gene related to the inhibition of lignin synthesis in alfalfa, named MsMYB17, which can be used in plant breeding. Overexpression of this gene in wild-type alfalfa reduces the lignin content by 30.4%–63.0%, and alfalfa overexpressing this gene can be used as germplasm material for forage breeding. This invention provides a new method for reducing the lignin content in alfalfa and improving forage quality.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and relates to the genetic engineering of alfalfa, specifically to a gene related to inhibiting lignin synthesis in alfalfa and its application. Background Technology

[0002] Alfalfa (Medicago sativa L.) is the world's most widely distributed perennial leguminous forage. Due to its rich nutrition, high palatability, wide cultivation area, and high protein content, it is known as the "King of Forages" and plays a vital role in livestock production. Lignin is an important component of plant cell walls, providing mechanical support and aiding in water transport in vascular tissues. However, because the lignin in alfalfa cannot be digested and absorbed by livestock, it reduces the digestibility of the forage and affects its quality. Therefore, current research on technologies to reduce the lignin content of alfalfa is of significant practical importance. However, breeding materials for low-lignin alfalfa are scarce, and selecting low-lignin varieties through conventional hybridization breeding is difficult.

[0003] In recent years, the continuous improvement of alfalfa genetic transformation technology and the continuous refinement of genomic information have provided a new technical approach for breeding new low-lignin alfalfa varieties. Using genetic engineering technology to reduce the lignin content in alfalfa, improve forage digestibility, and improve alfalfa quality, breeding new alfalfa varieties has significant ecological, social, and economic benefits.

[0004] Members of the MYB transcription factor family participate in multiple processes of plant growth and development. For example, they are involved in the biosynthesis of anthocyanins in maize; regulate the biosynthetic pathway of flavonoid pigments in Arabidopsis thaliana; and in maize and barley, members of this family are also associated with the differentiation of leaf hair cells. MsMYB17 belongs to the fourth subfamily of the MYB transcription factor family. Functional analyses of homologous genes of this transcription factor in plants such as Arabidopsis thaliana, Leucaena leucocephala, and maize have been reported, but functional analyses of this gene in alfalfa have not yet been reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a gene related to inhibiting lignin synthesis in alfalfa and its application, thereby solving the technical problem in existing technologies where the high lignin content in alfalfa affects the quality of forage.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A Medicago sativa lignin synthesis-inhibiting related gene, named MsMYB17, has the following nucleotide sequence: 5’-atgggaagatcaccttgttgtgaaaaagctcatacaaacaaaggagcatggacaaaagaagaagat gatagacttatatcatatattagggcacatggtgaaggttgttggagatctctccctaaagcagctggcttactccgatgtggtaaaagttgccgtctccggtggattaactatctcaggccagaccttaaacgtggtaacttcacagaagaagaagatgaactcatcatcaaactccatagtcttcttggtaacaaatggtctttgatagctggaagattacctggaagaacagataatgagataaagaattattggaacactcatataagaagaaagcttttgaatagaggaattgaccctgctactcatagacctttaaacgaagtttctcattctcattctcaatctcaatcacaatctcaaactcttcatcttcaaaatcaagaagctgttacaatagctgtagcagcatctacatcagctcctactgctacaaaaaccctaccaacgactatatcttttgcatcatccattaaacaagaacaatatcatcatcatcatcatcaagaaatgaacgcaaacatggttaaagggttggttttagaacgttgtcctgatttgaatcttgagctaacaattagtccaccacgtgttcaagaacatgatgaacaattcagaaacagagagaggaacaatctctgttttgtttgtagtttgggtttgcagaatagtaaggattgtacctgtgatgaaattgttggaaattctagcagtggaaatggttctactgcacctgcttatgatttcttgggtttgaaaggtggtgtttgggattacaaaggcttagaaatgaaatga-3’。

[0008] The full-length amino acid sequence encoded by the cds region of this gene is: 5'-MGRSPCCEKAHTNKGAWT KEEDDRLISYIRAHGEGCWRSLPKAAGLLRCGKSCRLRWINYLRPDLKRGNFTEEEDELIIKLHSLLGNKWSLIAGRLPGRTDNEIKNYWNTHIRRKLLNRGIDPATHRPLNEVSHSHSQSQSQSQTLHLQNQEAVTIAVAASTSAPTATKTLPTTISFASSIKQEQYHHHHHQEMNANMVKGLVLERCPDLNLELTISPPRVQEHDEQFRNRERNNLCFVCSLGLQNSKDCTCDEIVGNSSSGNGSTAPAYDFLGLKGGVWDYKGLEMK*-3'.

[0009] The present invention also has the following technical features:

[0010] This invention also protects the application of the alfalfa lignin inhibition-related genes as described above in plant breeding.

[0011] Specifically, the method of this application includes overexpressing genes related to the inhibition of lignin synthesis in wild-type alfalfa.

[0012] Compared with the prior art, the present invention has the following technical effects:

[0013] Overexpression of a gene related to lignin synthesis inhibition in wild-type alfalfa can reduce the lignin content of alfalfa by 30.4% to 63.0%, and alfalfa overexpressing this gene can be used as germplasm material for forage breeding. This invention provides a new solution for reducing the lignin content in alfalfa and improving forage quality. Attached Figure Description

[0014] Figure 1 Electrophoresis image of PCR products; Figure 1 In the diagram: M represents DNA Maker, and the DNA Maker band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; 1, 2 and 3 are all PCR products.

[0015] Figure 2 Electrophoresis diagram of PCR products from positive plants; Figure 2 In Chinese: WT represents wild-type alfalfa, while OE3, OE4, OE6, OE7, OE13, OE14, and OE16 are alfalfa varieties that have been modified with the target gene.

[0016] Figure 3 The results of lignin qualitative analysis staining of positive plants; Figure 3 In Chinese: WT represents wild-type alfalfa, while OE3, OE7, OE14, and OE16 are alfalfa varieties that have been modified with the target gene.

[0017] Figure 4 The statistical results of quantitative analysis of lignin in positive plants; Figure 4 In Chinese: WT represents wild-type alfalfa, OE3, OE7, OE14, and OE16 are alfalfa varieties that have been transformed with the target gene, and "*" indicates the degree of significant difference.

[0018] The specific content of the present invention will be further explained in detail below with reference to the embodiments. Detailed Implementation

[0019] It should be noted that, unless otherwise specified, all carriers, reagents, and kits used in this invention are those known in the art, such as:

[0020] The pCAMBIA1300 vector is a conventional vector known in the prior art, used for overexpression of alfalfa genes related to lignin synthesis inhibition.

[0021] RNA extraction kit The Super Total RNA Extraction Kit (manufactured by Promega (Beijing) Biotechnology Co., Ltd.) is used to extract total RNA from alfalfa stem tissue.

[0022] The reverse transcription PCR kit is from PrimeScript. TM II 1st Strand cDNA Synthesis Kit, purchased from Baori Biotechnology (Beijing) Co., Ltd., for synthesizing the first strand of cDNA.

[0023] The PCR kit was Phanta Max Super-Fidelity DNA Polymerase, purchased from Nanjing Novizan Biotechnology Co., Ltd., and was used to perform PCR to obtain the CDS region of the target gene.

[0024] The following are specific embodiments of the present invention. It should be noted that the present invention is not limited to the following specific embodiments. All equivalent modifications made based on the technical solutions of this application fall within the protection scope of the present invention.

[0025] Example 1:

[0026] In this embodiment, a gene related to inhibiting lignin synthesis in alfalfa is given, named MsMYB17. The full-length sequence of the CDS region of this gene is: 5’-atgggaagatcaccttgttgtgaaaaagctcatacaaacaaaggagcatggacaaaagaagaagatgatagacttatatcatatattagggcacatggtgaaggttgttggagatctctccctaaagcagctggcttactccgatgtggtaaaagttgccgtctccggtggattaactatctcaggccagaccttaaacgtggtaacttcacagaagaagaagatgaactcatcatcaaactccatagtcttcttggtaacaaatggtctttgatagctggaagattacctggaagaacagataatgagataaagaattattggaacactcatataagaagaaagcttttgaatagaggaattgaccctgctactcatagacctttaaacgaagtttctcattctcattctcaatctcaatcacaatctcaaactcttcatcttcaaaatcaagaagctgttacaatagctgtagcagcatctacatcagctcctactgctacaaaaaccctaccaacgactatatcttttgcatcatccattaaacaagaacaatatcatcatcatcatcatcaagaaatgaacgcaaacatggttaaagggttggttttagaacgttgtcctgatttgaatcttgagctaacaattagtccaccacgtgttcaagaacatgatgaacaattcagaaacagagagaggaacaatctctgttttgtttgtagtttgggtttgcagaatagtaaggattgtacctgtgatgaaattgttggaaattctagcagtggaaatggttctactgcacctgcttatgatttcttgggtttgaaaggtggtgtttgggattacaaaggcttagaaatgaaatga-3’。

[0027] The full-length amino acid sequence encoded by the cds region of this gene is: 5'-MGRSPCCEKAHTNKGAWTKEEDDRLISYIRAHGEGCWRSLPKAAGLLRCGKSCRLRWINYLRPDLKRGNFTEEEDELIIKLHSLLGNKWSLIAGRLPGRTDNEIKNYWNTHIRRKLLNRGIDPATHRPLNEVSHSHSQSQSQSQTLHLQNQEAVTIAVAASTSAPTATKTLPTTISFASSIKQEQYHHHHHQEMNANMVKGLVLERCPDLNLELTISPPRVQEHDEQFRNRERNNLCFVCSLGLQNSKDCTCDEIVGNSSSGNGSTAPAYDFLGLKGGVWDYKGLEMK*-3'.

[0028] In this embodiment, the process of obtaining the gene related to lignin synthesis inhibition in alfalfa was as follows: cDNA from stem tissue was used as a template and obtained by PCR. This alfalfa belongs to the genus *Medicago* of the legume family, and its Latin name is *Medicago sativa* L.

[0029] Example 2:

[0030] This embodiment describes the application of the alfalfa lignin synthesis inhibition gene from Example 1 in alfalfa breeding. The specific method for this application includes the following steps:

[0031] Step 1, Obtain the target gene:

[0032] Total RNA was extracted from alfalfa stem tissue, and reverse transcription PCR was performed using the total RNA as a template to obtain cDNA. Using the cDNA as a template, PCR was performed with 2×35S-1300-F and 2×35S-1300-R as upstream and downstream primers, respectively. After PCR, agarose gel electrophoresis was performed, and the results are shown below. Figure 1 As shown, by Figure 1 The presence of a band between 750bp and 1000bp indicates that the target gene has been obtained. Sequencing of the PCR product confirmed that it contained the full-length CDS region of the target gene.

[0033] In this embodiment, the nucleotide sequence of the upstream PCR primer 2×35S-1300-F is as follows: 5'-gatctcgagctcaagcttcgaattcatgggaagatcaccttgttgt-3'; the nucleotide sequence of the downstream PCR primer 2×35S-1300-R is as follows: 5'-ggtaccgtcgactgcagaattctttcatttctaagcctttgtaatccc-3'.

[0034] In this embodiment, the PCR system and reaction conditions are shown in Tables 1 and 2.

[0035] Table 1. PCR Reaction System

[0036] reagents volume Phanta Max Super-Fidelity DNA Polymerase 1μl 2×Phanta Max Buffer 25μl dNTP Mix (10mM) 1μl primer F (10μM) 2μl primer R (10μM) 2μl cDNA 1μl <![CDATA[ddH2O]]> up to 50μl

[0037] Table 2. PCR reaction procedure

[0038]

[0039] Step 2, constructing the expression carrier:

[0040] The PCR product obtained in step one was subjected to agarose gel electrophoresis. After electrophoresis, the PCR product was recovered from the gel to obtain purified PCR product. Using homologous recombination, the purified PCR product was ligated with the linearized vector pCAMBIA1300 to obtain ligation product. The ligation product was then transformed into E. coli DH5α and cultured. After single-clone colonies grew, single-clone colonies were picked for colony PCR identification. Single-clone colonies that were positive for colony PCR were sent for sequencing. The single-clone strains that were correctly sequenced were the strains containing the expression vector.

[0041] Step 3, constructing transgenic plants:

[0042] The strain containing the expression vector obtained in step two was expanded and cultured, and then plasmids were extracted to obtain the expression vector. The expression vector was transformed into Agrobacterium EHA105 and cultured. After single colonies grew, single colonies were picked for colony PCR identification. The correctly identified Agrobacterium strains were expanded and cultured. Alfalfa leaves were infected using the leaf disc method, and the expression vector containing the target gene was transformed into alfalfa using Agrobacterium-mediated genetic transformation and tissue culture was performed. Then, genomic DNA was extracted from successfully cultured tissue culture seedlings and wild-type alfalfa leaves using the CTAB method. Using the genomic DNA as a template, PCR amplification was performed using the glufosinate resistance gene primers BlpR-F and BlpR-R as upstream and downstream primers, respectively. After PCR, agarose gel electrophoresis was performed, and the results are shown below. Figure 2 As shown.

[0043] Depend on Figure 2 It was found that wild-type alfalfa (WT) did not show any bands between 100 and 250 bp, while alfalfa transformed with expression vectors (OE3, OE4, OE6, OE7, OE13, OE14, OE16) all showed bands between 100 and 250 bp. These bands were fragments of the glufosinate resistance gene from the vector pCAMBIA1300. The above results indicate that the gene has been transferred into the alfalfa genome.

[0044] In this embodiment, the nucleotide sequence of the upstream primer BlpR-F used for identification is as follows: 5'-agtcgaccgtgtacgtctcc-3'; the nucleotide sequence of the downstream primer BlpR-R used for identification is as follows: 5'-gaagtccagctgccagaaac-3'.

[0045] Effect verification:

[0046] (A) To verify the effect of gene overexpression on lignin synthesis in alfalfa, this invention performed phloroglucinol staining on cross-sections of stem tissues from overexpression-positive plants OE3, OE7, OE14, OE16, and wild-type alfalfa. The results are as follows: Figure 3 As shown. By Figure 3 It can be seen that, compared with wild-type alfalfa, the xylem staining range of the overexpression positive plants OE3, OE7, OE14 and OE16 was significantly reduced, indicating that there was less lignin in the overexpression positive plants.

[0047] (B) This invention further determined the lignin content of overexpression-positive plants and wild-type alfalfa, and the results are as follows: Figure 4 As shown. By Figure 4 It was found that, compared with wild-type alfalfa, the lignin content of the overexpressing positive plants OE3, OE7, OE14 and OE16 was reduced by 40.8%, 34.1%, 63.0% and 30.4%, respectively.

[0048] (C) Based on the results of (A) and (B) above, it can be seen that overexpression of the alfalfa lignin synthesis inhibition gene of the present invention in wild-type alfalfa will lead to a significant reduction in lignin content in the plant, which helps to provide new breeding materials for low-lignin forage breeding.

Claims

1. Genes related to the inhibition of lignin synthesis in alfalfa MsMYB17 Application for inhibiting lignin content in alfalfa MsMYB17 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The method used in this application involves: inhibiting genes related to lignin synthesis in alfalfa. MsMYB17 It has been expressed in wild-type alfalfa.

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