CN117535342B - Method for increasing alfalfa yield and/or branch number, protein used by method and related biological material - Google Patents
Method for increasing alfalfa yield and/or branch number, protein used by method and related biological material Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Abstract
The present invention discloses a method for increasing alfalfa yield and/or branch number, and its protein and related biological material. The invention solves the technical problem of how to improve the yield and drought resistance of plants. The method for improving the yield and/or the branch number of the alfalfa comprises the steps of up-regulating or enhancing or improving the expression of a coding gene of a protein in the alfalfa and/or the activity and/or the content of the protein so as to improve the yield and/or the branch number of the alfalfa, wherein the protein is any one of the following components: b1 Amino acid sequence is a protein shown in sequence 2; b2 A protein having 80% or more identity and the same function as the protein of B1) obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein of B1); b3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag. The invention can be used for improving the yield and drought resistance of plants.
Description
Technical Field
The present invention relates to a method for increasing alfalfa yield and/or branch number in the field of genetic engineering, and its protein and related biological material.
Background
In natural environments, plants often suffer from adverse environmental effects in their growth due to immobility. With the increase of global warming, drought stress becomes the most serious abiotic stress affecting plant growth. Alfalfa is mainly planted in some arid and semiarid regions in China. In the long-term domestication of plants, a plurality of molecules, physiology and biochemistry mechanisms for coping with drought stress are formed, wherein the molecular mechanisms are key determinants for controlling the physiology and biochemistry mechanisms, key drought stress response genes are identified and cloned from the plants, and the expression of the key genes, especially the expression of transcription factor genes, is controlled by biotechnology means, so that a plurality of physiological response processes can be comprehensively regulated, and the method has important significance for enhancing drought resistance of the plants. Studies have shown that plants often respond to drought stress by actively limiting growth, reducing water loss. Therefore, the key genes which can promote plant growth and obviously enhance drought resistance are excavated from plants, gene resources can be provided for improving crop drought resistance by utilizing biological breeding technology, and the key genes are important bases for solving the problems of serious crop yield reduction and the like in arid areas.
Disclosure of Invention
The invention solves the technical problem of how to improve the yield of alfalfa.
In order to solve the above problems, the present invention provides a method for increasing yield and/or branch number of alfalfa.
The method comprises up-regulating or enhancing or increasing expression of a gene encoding a protein in alfalfa and/or activity and/or content of the protein to increase yield and/or branch number of alfalfa, wherein the protein is any one of the following proteins:
b1 Amino acid sequence is a protein shown in sequence 2;
b2 A protein which has 80% or more identity with the protein represented by B1) and has the same function as the protein represented by B1) and is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein described by B1);
B3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag.
In the above method, said up-regulating or enhancing or increasing expression of a gene encoding a protein in alfalfa comprises introducing a gene encoding said protein into alfalfa.
In order to solve the above problems, the present invention also provides a method for cultivating alfalfa with high yield and/or high branch number.
The method comprises up-regulating or enhancing or increasing expression of a gene encoding a protein in a alfalfa of interest and/or activity and/or content of the protein, resulting in a high yield and/or high number of branches of alfalfa having a higher yield and/or high number of branches than the alfalfa of interest;
The protein is any one of the following proteins:
b1 Amino acid sequence is a protein shown in sequence 2;
b2 A protein which has 80% or more identity with the protein represented by B1) and has the same function as the protein represented by B1) and is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein described by B1);
B3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag.
In order to solve the problems, the invention also provides a method for improving drought resistance of alfalfa
The method comprises up-regulating or enhancing or increasing expression of a gene encoding a protein in alfalfa and/or activity and/or content of the protein to increase drought resistance of alfalfa;
The protein is any one of the following proteins:
b1 Amino acid sequence is a protein shown in sequence 2;
b2 A protein which has 80% or more identity with the protein represented by B1) and has the same function as the protein represented by B1) and is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein described by B1);
B3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag.
In the above method, said up-regulating or enhancing or increasing expression of a gene encoding a protein in alfalfa comprises introducing a gene encoding said protein into alfalfa.
In order to solve the problems, the invention also provides a method for cultivating alfalfa with strong drought resistance.
The method comprises the steps of up-regulating or enhancing or improving the expression of a coding gene of a protein in a target alfalfa and/or the activity and/or the content of the protein to obtain the alfalfa with strong drought resistance, wherein the drought resistance of the alfalfa with strong drought resistance is higher than that of the target alfalfa;
The protein is any one of the following proteins:
b1 Amino acid sequence is a protein shown in sequence 2;
b2 A protein which has 80% or more identity with the protein represented by B1) and has the same function as the protein represented by B1) and is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein described by B1);
B3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag.
The method of any one of the above, wherein said up-regulating or enhancing or increasing expression of a gene encoding a protein in alfalfa comprises introducing a gene encoding said protein into alfalfa.
In the above method, the gene encoding the protein may be introduced into alfalfa by any one of the following:
c1 An expression cassette containing the coding gene;
C3 A recombinant vector containing the coding gene;
b4 A recombinant microorganism containing the coding gene.
The coding gene may be a DNA molecule whose coding sequence (CDS) is SEQ ID No. 1.
The alfalfa may be alfalfa. The alfalfa may be alfalfa No. 1.
In any of the above methods, the substance that up-regulates or enhances or increases expression of a gene encoding a protein and/or activity and/or content of the protein in alfalfa is any of the following:
b1 A nucleic acid molecule encoding the above protein;
B2 An expression cassette comprising the nucleic acid molecule of B1);
B3 A recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2);
B4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);
B5 A transgenic plant cell line comprising B1) said nucleic acid molecule, or a transgenic plant cell line comprising B2) said expression cassette, or a transgenic plant cell line comprising B3) said recombinant vector;
B6 A transgenic plant tissue comprising B1) said nucleic acid molecule, or a transgenic plant tissue comprising B2) said expression cassette, or a transgenic plant tissue comprising B3) said recombinant vector;
B7 A transgenic plant organ comprising the nucleic acid molecule of B1), or a transgenic plant organ comprising the expression cassette of B2), or a transgenic plant organ comprising the recombinant vector of B3).
B1 In the nucleic acid molecules, the person skilled in the art can easily mutate the nucleotide sequence encoding the protein MsMYBH according to the invention by known methods, for example directed evolution or point mutation. Those artificially modified nucleotides having 80% or more identity to the nucleotide sequence of the protein MsMYBH isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein MsMYBH and have the function of the protein MsMYBH.
The 80% or more identity may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.
Herein, such vectors are well known to those skilled in the art, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, or viral vectors. The vector can be pROKII vector;
in the above biological material, the expression cassette of B2) or B10) means a DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for promoting transcription of the gene but also a terminator for terminating transcription of the gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S, a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with a jasmonates); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I985) Nature 313:810; rosenberg et al (1987) Gene, 56:125; guerineau et al (1991) mol. Gen. Genet. 262:141; proudfoot (1991) Cell, 64:671; sanfacon et al Genes Dev., 5:141; mogen et al (1990) PLANT CELL, 2:1261; munroe et al (1990) Gene, 91:151; ballad et al (1989) Nucleic Acids Res. 17:7891; jo et al (1987) Nucleic Acid Res, 15:9627).
In the above B3), the recombinant vector may be a recombinant expression vector comprising the gene expression cassette constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector, etc., such as pGWB411、pGWB412、pGWB405、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa、pMDC85 or pCAMBIA1391-Xb. When MsMYBH is used to construct recombinant expression vectors, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like may be added before the transcription initiation nucleotide thereof, and they may be used alone or in combination with other plant promoters; in addition, when the gene of the present application is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. As a specific example, the present application uses pBI121 vector as expression vector. As a specific example, the present application uses pK7WIWG2I vector as RNAi vector. As a specific example, the microorganism strain in the recombinant microorganism may be agrobacterium EHA105.
In the above method, the nucleic acid molecule of B1) is a DNA molecule whose nucleotide sequence is shown in sequence 1.
The method of any one of the above, wherein said up-regulating or enhancing or increasing the expression of a gene encoding a protein in alfalfa and/or the activity and/or content of said protein comprises introducing into said alfalfa of interest a nucleic acid molecule according to B1), an expression cassette according to B2) or a recombinant vector according to B3) of claim 6 or 7.
In the above, the nucleic acid molecule may be a nucleic acid molecule as described in sequence 1.
In order to solve the above problems, the present invention also provides the following applications.
Use of a protein, a substance that modulates the expression of a gene encoding the protein, or a substance that modulates the activity or content of the protein in any of the following;
a1 Application in regulating drought resistance of plants and/or application in preparing products for regulating drought resistance of plants;
a2 Application in regulating plant height and/or application in preparing products for regulating plant height;
A3 Application in regulating and controlling plant branch number and/or application in preparing a product for regulating and controlling plant branch number;
a4 Application in regulating and controlling the content of the malondialdehyde of the plants and/or application in preparing products for regulating and controlling the content of the malondialdehyde of the plants;
a5 Application in regulating and controlling the proline content of plants and/or application in preparing products for regulating and controlling the proline content of plants;
a6 Application in regulating and controlling the relative water content of the plant leaves and/or application in preparing products for regulating and controlling the relative water content of the plant leaves;
a7 Application in regulating and controlling the antioxidant capacity of plants and/or application in preparing products for regulating and controlling the antioxidant capacity;
a8 Use in regulating dry weight of plants and/or use in the preparation of products regulating dry weight of plants;
a9 Application in regulating and controlling the fresh weight of plants and/or application in preparing products for regulating and controlling the fresh weight of plants;
A10 Application in regulating and controlling the relative water content of the plant leaves and/or application in preparing products for regulating and controlling the relative water content of the plant leaves;
The protein is any one of the following proteins:
b1 Amino acid sequence is a protein shown in sequence 2;
b2 A protein which has 80% or more identity with the protein represented by B1) and has the same function as the protein represented by B1) and is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein described by B1);
b3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag;
the substance is any one of the following:
d1 A nucleic acid molecule encoding said protein;
D2 An expression cassette comprising D1) said nucleic acid molecule;
D3 A recombinant vector comprising D1) said nucleic acid molecule, or a recombinant vector comprising D2) said expression cassette;
d4 A recombinant microorganism comprising D1) said nucleic acid molecule, or a recombinant microorganism comprising D2) said expression cassette, or a recombinant microorganism comprising D3) said recombinant vector;
d5 A transgenic plant cell line comprising D1) said nucleic acid molecule, or a transgenic plant cell line comprising D2) said expression cassette, or a transgenic plant cell line comprising D3) said recombinant vector;
d6 A transgenic plant tissue comprising D1) said nucleic acid molecule, or a transgenic plant tissue comprising D2) said expression cassette, or a transgenic plant tissue comprising D3) said recombinant vector;
d7 A transgenic plant organ comprising D1) said nucleic acid molecule, or a transgenic plant organ comprising D2) said expression cassette, or a transgenic plant organ comprising D3) said recombinant vector.
In order to solve the above problems, the present invention also provides the following biological materials.
The biological material is a protein or substance as described above.
Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained.
In the above protein, the 80% or more identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
In the above protein, sequence 2 (SEQ ID No. 2) consists of 274 amino acid residues. This was designated MsMYBH protein. The coding gene is MsMYBH gene.
In the present application, the modulation may be upregulation or enhancement or increase, and/or knockout or decrease.
In the application, the substance which is used for up-regulating or enhancing or improving the expression of the coding gene of the protein or the activity or the content of the protein can improve the drought resistance of plants.
Knocking out or reducing the substance expressed by the coding gene of the protein or the activity or the content of the protein can reduce the drought resistance of plants.
In the present application, the substance that upregulates or enhances or increases expression of the gene encoding the protein or the activity or content of the protein may increase the branch number of a plant.
Knocking out or reducing the expression of a gene encoding said protein or the activity or content of said protein may reduce the number of plant branches.
In the present application, the substance that up-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein may reduce the malondialdehyde content of plants.
Knocking out or reducing the substance encoding the gene expression of the protein or the activity or content of the protein can increase the malondialdehyde content of plants.
In the present application, the substance that up-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein may decrease to increase the proline content of plants.
Knocking out or reducing the expression of a gene encoding said protein or the activity or content of said protein may reduce the proline content of a plant.
In the present application, the substance that over-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein may decrease to increase the fresh weight of the plant.
Knocking out or reducing the expression of a gene encoding said protein or the activity or content of said protein may reduce the fresh weight of the plant.
In the present application, the substance that up-regulates or enhances or increases expression of a gene encoding the protein or the activity or content of the protein may decrease to increase dry weight of a plant.
Knocking out or reducing the expression of a gene encoding said protein or the activity or content of said protein may reduce plant dry weight.
In the present application, the substance that up-regulates or enhances or increases expression of a gene encoding the protein or the activity or content of the protein may decrease to increase the relative water content of plant leaves under drought stress conditions.
Knocking out or reducing the expression of a gene encoding said protein or the activity or content of said protein under drought stress conditions can reduce the relative water content of the plant leaves.
In the present application, the substance that up-regulates or enhances or increases the expression of a gene encoding the protein or the activity or content of the protein may decrease to increase the antioxidant enzyme activity of a plant under drought stress conditions.
Knocking out or reducing the expression of a gene encoding said protein or the activity or content of said protein under drought stress conditions may reduce the plant antioxidant enzyme activity. The antioxidant may be POD activity (peroxidase activity), CAT activity (catalase activity), or SOD activity (superoxide dismutase activity).
In the present application, the substance that up-regulates or enhances or increases expression of a gene encoding the protein or the activity or content of the protein may decrease to increase plant Pn under drought stress conditions.
Knocking out or reducing the expression of a gene encoding said protein or the activity or content of said protein under drought stress conditions may reduce the plant Pn.
In the present application, under drought stress conditions, the substance that up-regulates or enhances or increases expression of a gene encoding the protein or the activity or content of the protein may decrease to increase plant Fm.
Knocking out or reducing the expression of a gene encoding said protein or the activity or content of said protein under drought stress conditions may reduce plant Fm.
In the above application, the plant is any one of the following:
j1 Dicotyledonous plants;
j2 Leguminous plants;
J3 Alfalfa plant;
J4 Alfalfa.
In the application, the alfalfa can be alfalfa variety 1.
In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).
In the above application, the substance regulating the expression of the coding gene of the protein is any one of the following:
b1 A nucleic acid molecule encoding the above protein;
B2 An expression cassette comprising the nucleic acid molecule of B1);
B3 A recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2);
B4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);
B5 A transgenic plant cell line comprising B1) said nucleic acid molecule, or a transgenic plant cell line comprising B2) said expression cassette, or a transgenic plant cell line comprising B3) said recombinant vector;
B6 A transgenic plant tissue comprising B1) said nucleic acid molecule, or a transgenic plant tissue comprising B2) said expression cassette, or a transgenic plant tissue comprising B3) said recombinant vector;
B7 A transgenic plant organ comprising B1) said nucleic acid molecule, or a transgenic plant organ comprising B2) said expression cassette, or a transgenic plant organ comprising B3) said recombinant vector;
B8 A nucleic acid molecule which inhibits or reduces or down-regulates the expression of a gene encoding the protein according to claim 1 or 2 or which inhibits or reduces or down-regulates the activity or content of the protein;
b9 A gene encoding the nucleic acid molecule of B8);
b10 An expression cassette comprising the gene of B9);
B11 A recombinant vector comprising the gene of B9), or a recombinant vector comprising the expression cassette of B10);
B12 A recombinant microorganism comprising the gene of B9), a recombinant microorganism comprising the expression cassette of B10), or a recombinant microorganism comprising the recombinant vector of B11);
b13 A transgenic plant cell line comprising the gene of B9), or a transgenic plant cell line comprising the expression cassette of B10), or a transgenic plant cell line comprising the recombinant vector of B11);
B14 A transgenic plant tissue containing the gene of B9), or a transgenic plant tissue containing the expression cassette of B10), or a transgenic plant tissue containing the recombinant vector of B11);
b15 A transgenic plant organ containing the gene of B9), or a transgenic plant organ containing the expression cassette of B10), or a transgenic plant organ containing the recombinant vector of B11).
B1 Or B9) the nucleotide sequence encoding the protein MsMYBH of the invention can be easily mutated by a person skilled in the art using known methods, for example directed evolution or point mutation. Those artificially modified nucleotides having 80% or more identity to the nucleotide sequence of the protein MsMYBH isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein MsMYBH and have the function of the protein MsMYBH.
The 80% or more identity may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.
Herein, such vectors are well known to those skilled in the art, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, or viral vectors. The vector can be pROKII vector;
in the above biological material, the expression cassette of B2) or B10) means a DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for promoting transcription of the gene but also a terminator for terminating transcription of the gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S, a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with a jasmonates); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I985) Nature 313:810; rosenberg et al (1987) Gene, 56:125; guerineau et al (1991) mol. Gen. Genet. 262:141; proudfoot (1991) Cell, 64:671; sanfacon et al Genes Dev., 5:141; mogen et al (1990) PLANT CELL, 2:1261; munroe et al (1990) Gene, 91:151; ballad et al (1989) Nucleic Acids Res. 17:7891; jo et al (1987) Nucleic Acid Res, 15:9627).
In the above B3) or B11), the recombinant vector may be a recombinant expression vector comprising the gene expression cassette constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector, etc., such as pGWB411、pGWB412、pGWB405、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa、pMDC85 or pCAMBIA1391-Xb. When MsMYBH is used to construct recombinant expression vectors, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like may be added before the transcription initiation nucleotide thereof, and they may be used alone or in combination with other plant promoters; in addition, when the gene of the present application is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. As a specific example, the present application uses pBI121 vector as expression vector. As a specific example, the present application uses pK7WIWG2I vector as RNAi vector. As a specific example, the microorganism strain in the recombinant microorganism may be agrobacterium EHA105.
In the above application, the nucleic acid molecule of B1) is a DNA molecule whose nucleotide sequence is shown in sequence 1.
In the above application, the nucleic acid molecule of B9) is RNA transcribed from a DNA molecule of formula (I):
SEQ forward-X-SEQ reverse (I);
The SEQ forward direction is a partial fragment of sequence 1; the sequence of the SEQ reverse direction is reversely complementary to the sequence of the SEQ forward direction; and X is a spacer sequence between the forward direction of the SEQ and the reverse direction of the SEQ, so that an RNA molecule transcribed from the DNA molecule shown in the formula (I) forms a stem-loop structure.
A method for cultivating a plant with high drought resistance comprises up-regulating or enhancing or increasing the expression level of the coding gene of the protein in a target plant, and/or obtaining the plant with high drought resistance by the activity and/or content of the protein, wherein the drought resistance of the plant with high drought resistance is higher than that of the target plant.
A method for improving drought resistance in a plant comprising increasing drought resistance in a plant by up-regulating or enhancing or increasing expression of a gene encoding said protein in a plant and/or activity and/or content of said protein.
In the present application, the plant may be alfalfa. The alfalfa may be alfalfa number 1 of alfalfa varieties.
In the above method, the up-regulating or enhancing or increasing expression of a gene encoding the above protein in a plant comprises introducing the nucleic acid molecule of B1), the expression cassette of B2) or the recombinant vector of B3) of claim 3 into the plant of interest.
In the above, the nucleic acid molecule may be a nucleic acid molecule as described in sequence 1.
A method for cultivating low drought resistance plants comprises knocking out or reducing the expression level of the coding gene of the protein in target plants, and/or obtaining low drought resistance plants with activity and/or content of the protein, wherein the drought resistance of the low drought resistance plants is lower than that of the target plants.
In the above application or the above method, the plant is any one of the following:
j1 Dicotyledonous plants;
j2 Leguminous plants;
J3 Alfalfa plant;
J4 Alfalfa.
In the application, the alfalfa can be alfalfa variety 1.
A protein, said protein being any one of the following:
a1 Amino acid sequence is a protein shown in sequence 2;
A2 A protein which is obtained by substituting and/or deleting and/or adding the amino acid residues in the amino acid sequence of A1), has more than 80% of identity with the protein shown in A1) and has the same function;
a3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).
A biomaterial associated with the protein of claim, said biomaterial being any one of the following:
B1 A nucleic acid molecule encoding a protein as defined in any one of the above;
B2 An expression cassette comprising the nucleic acid molecule of B1);
B3 A recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2);
B4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);
B5 A transgenic plant cell line comprising B1) said nucleic acid molecule, or a transgenic plant cell line comprising B2) said expression cassette, or a transgenic plant cell line comprising B3) said recombinant vector;
B6 A transgenic plant tissue comprising B1) said nucleic acid molecule, or a transgenic plant tissue comprising B2) said expression cassette, or a transgenic plant tissue comprising B3) said recombinant vector;
B7 A transgenic plant organ comprising the nucleic acid molecule of B1), or a transgenic plant organ comprising the expression cassette of B2), or a transgenic plant organ comprising the recombinant vector of B3).
Advantageous effects
The present invention discloses a method for increasing alfalfa yield and/or branch number, and its protein and related biological material. The invention solves the technical problem of regulating and controlling drought resistance of plants. The protein is shown in a sequence 2, the over-expression MsMYBH protein coding gene in the plant can increase the drought resistance of the plant, and the silencing of MsMYBH protein coding gene expression in the plant can reduce the drought resistance defect of the plant, so that the protein can be used for plant breeding and drought resistance research.
The invention constructs MsMYBH gene over-expression vector: recombinant vectors MsMYBH-pBI121 and MsMYBH RNAi vector recombinant vector MsMYBH-pK7WIWG I, over-expressed MsMYBH positive transgenic plants (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6 and OE-7) and RNAi MsMYBH positive transgenic plants (RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7) were obtained by the transgenic method, and OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3 with obvious effect were selected for the subsequent experiments. Drought resistance tests were performed by selecting over-expressed MsMYBH positive transgenic lines (OE-1, OE-2, and OE-3) and RNi MsMYBH positive transgenic lines (RNAi-1, RNAi-2, and RNAi-3) with WT (alfalfa 1) as a control.
The results show that: overexpression of MsMYBH protein coding genes in plants can increase drought resistance of plants and silencing MsMYBH protein coding gene expression in plants can reduce drought resistance of plants.
In particular, under normal culture conditions: 1. the branch number of the over-expression MsMYBH positive transgenic plant is higher than that of the WT, and the branch number of the WT is higher than that of the positive transgenic line; 2. the fresh and dry weights of the overexpressing MsMYBH positive transgenic lines were significantly higher than WT, which was significantly higher than the RNAi MsMYBH positive transgenic line.
Under drought conditions: 1. the drought resistance of the over-expression MsMYBH positive transgenic plant is higher than that of the WT, and the drought resistance of the WT is higher than that of a RNAi MsMYBH positive transgenic line; 2. the branch number of the over-expression MsMYBH positive transgenic plant is higher than that of the WT, and the branch number of the WT is higher than that of the RNAi MsMYBH positive transgenic line; 3. leaf wilting degree of the over-expressed MsMYBH positive transgenic plant is lower than that of the WT, and leaf wilting degree of the WT is lower than that of the RNAi MsMYBH positive transgenic line; 4. the leaf photosynthesis and water utilization efficiency of the over-expression MsMYBH positive transgenic plant is higher than that of the WT, and the leaf photosynthesis and water utilization efficiency of the WT is higher than that of the RNAi MsMYBH positive transgenic plant; 5. the antioxidant enzyme activity of the transgenic plant with positive MsMYBH over-expression is higher than that of the WT, and the antioxidant enzyme activity of the WT is higher than that of the transgenic line with positive RNAi MsMYBH.
Drawings
FIG. 1 is a cloning map of MsMYBH.
FIG. 2 is a diagram of the construction of vectors over-expressing MsMYSBH (A) and RNAi MsMYBH (B).
FIG. 3 is a graph of gene level and transcript level identification of overexpressing (A, B) and RNAi (C, D) MsMYBH transgenic positive alfalfa plants.
FIG. 4 is a diagram of MsMYBH tissue-specific expression (A) and subcellular localization (B).
FIG. 5 is a graph showing the expression pattern of MsMYBH under drought stress.
FIG. 6 is a phenotypic map of wild type and transgenic plants under control and drought treatment.
FIG. 7 is a graph of the photo-biological changes (A, B) of wild-type and transgenic lines under drought stress and biomass (C, D) and quality index (E-H) under control conditions.
FIG. 8 is a graph of H 2O2 accumulation (a) and antioxidant enzyme activity (b-d) for wild-type and transgenic lines under drought stress and control conditions.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The following examples were run using SPSS11.5 statistical software and the experimental results were expressed as mean ± standard deviation using One-way ANOVA test, P <0.05 (x) indicated significant differences and P < 0.01 (x) indicated very significant differences, and the different lower case letters in the multiple comparisons indicated significant differences (P < 0.05).
EXAMPLE 1 cloning of Gene and construction of transgenic vector
Extracting RNA of alfalfa in the 'alfalfa 1', carrying out reverse transcription by taking the RNA of alfalfa in the 'alfalfa 1' as a template to obtain cDNA of alfalfa in the 'alfalfa 1', and carrying out amplification by taking the cDNA of alfalfa in the 'alfalfa 1' as a template by using a cloning primer (F: ATGGGGAGAAGAAAGTGTTCGCATTG, a sequence 4;R: TTAAGTGACACTAATTGGGCCAAGA in a sequence table and a sequence 5 in the sequence table), wherein the reaction system is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 1min,34 cycles; extending at 72℃for 7min. cDNA cloned into MsMYBH gene (marker in FIG. 1, marker in the first lane, msMYBH gene in the second lane) was sequenced by single cloning, and the CDS sequence of MsMYBH (shown as sequence 1) was successfully obtained. The encoded amino acid sequence is shown in sequence 2 and is named MsMYBH protein. The genomic sequence is shown in SEQ ID NO. 3. This was designated MsMYBH gene.
The sequence 1 is specifically as follows:
ATGGGGAGAAGAAAGTGTTCGCATTGTGGTAAGATAGGACATAATTGTAGGACATGCACATCCTTCACTACCCTTGGAGGACTTCGTCTCTTTGGGGTCCAACTATCATCATCCTCCTCGTCATCATCTAGTACCATGATCAAGAAAAGCTTTAGCATGGACACCTTTCCCTCACCATCCTCTCCATCTTCCTCATTCTCTTCATCAACATCATTAACCAATATTGATGAAAATTATTATCACAAACCCACTTCTAATATTTCATATCTATCAGATTGTTTTATAGGTCCACCCCAAGAGAGAAAGAAAGGAGTTCCATGGACAGAAGAAGAGCACAGAACATTCCTTGTTGGATTAGAGAAGCTAGGAAAAGGAGACTGGAGAGGCATCTCTAAAAACTTTGTCACTTCAAGAACCCCTACACAAGTCGCTAGCCATGCTCAAAAATACTTTCTTAGATTGGCTACTATCAACAAGAAGCGACGTTCAAGTCTCTTCGACTTGGTTGGGAGCAAGAACACCAACACAAAAGAACAAGGGTATGCTAATTCAGTAGTAAATTTGGGTCATAAATTTGAGGATAAGTGTAAATGTGAAGTTGAGATGAATGATGGCACCACTTTGTCCTACTTTAAACAAGAAGAAGCAGCCAAATCAGAAAAGCAAGAAAATAATTACTCTACAGATAATTGGCTACATGACTCTTCAAATTGTGCAGCAGTGCCTAATTTGGACCTCACACTTTCAGTTGCATCCCCTAAGCTTGAACAAAATCAACCCTCCTCTGCTGGCTCATTCCTTCTTGGCCCAATTAGTGTCACTTAA.
The sequence 2 is specifically as follows:
MGRRKCSHCGKIGHNCRTCTSFTTLGGLRLFGVQLSSSSSSSSSTMIKKSFSMDTFPSPSSPSSSFSSSTSLTNIDENYYHKPTSNISYLSDCFIGPPQERKKGVPWTEEEHRTFLVGLEKLGKGDWRGISKNFVTSRTPTQVASHAQKYFLRLATINKKRRSSLFDLVGSKNTNTKEQGYANSVVNLGHKFEDKCKCEVEMNDGTTLSYFKQEEAAKSEKQENNYSTDNWLHDSSNCAAVPNLDLTLSVASPKLEQNQPSSAGSFLLGPISVT*.
the sequence 3 is specifically as follows:
ATGGGGAGAAGAAAGTGTTCGCATTGTGGTAAGATAGGACATAATTGTAGGACATGCACATCCTTCACTACCCTTGGAGGACTTCGTCTCTTCGGGGTCCAACTATCATCTTCCTCCTCATCATCATCTAGTACCATGATCAAGAAAAGCTTTAGCATGGACACCTTTCCCTCACCATCCTCTCCATCTTCCTCATTCTCTTCATCAACATCATTAACCAATATTGATGAAAATTATTATCACAAACCCACTTCTAATATTTCATATCTATCAGATTGTTTTATAGGTCCACCCCAAGAGAGAAAGAAAGGTATATTTATACATATGTTAGCGGAAAGATGGTCACTAATTAATGTTGTTTACGTTTTCTATTAATTTAATGTGTATTTTTTTATAGTATTTGTTGTATAGTTCATCAATTCAAAATTAATGAAAAATAAAATATATTTTTTTTTACAAATTTTATTGTTTGTAGTTGAAATGAAACTTCTTTCAAAACTAATTTATCATGTTCAAATTTCCAACAGCTTCCTTCATTTGTTTTTTTATTTATAATTTGTAATATACTAATATGTAACTACCAATAAGAATAGAATTGAAAGAAAAATGCTACACCAAAATCTTATATTCCTTTATATATTGGTATGGATGTATAGTCATGATCAATGTTAAGTAGTTATTCATACATGTATACTTCTAATTTGAGCTAAGGGAGTTAATGGATAAGTGTTTGAATCATGATGAATGAGAAAATACTTGGGATCTCTAGTCTAACATATATTATATACTGATTGATTGATCAGGAGTTCCATGGACAGAAGAAGAGCACAGAACATTCCTTGTTGGATTAGAGAAGCTAGGAAAAGGAGACTGGAGAGGCATCTCTAAAAACTTTGTCACTTCAAGAACCCTACACAAGTCGCTAGCCATGCTCAAAAATACTTTCTTAGATTGGCTACTATCAACAAGAAGCGACGTTCAAGTCTCTTTGACTTGGTATATACACTATACTATGTTAGACAAATTCAATTCATTCATGATCATCAATTGCTTAATTATTTAGCTATCTCTTGTCGTTCACCACTGAATATAAAGTAAGTCTTACATGGCCACAAGCCCATAACCACAAATAAAAATTATTTAATTAGTTACATTTCTTAATCTTTTCCTCTTTTAACTTTTTTTATTTGTGTGTTCAAGTAAGTATTTTTCCTAAATAAATATTTTATTATTTTTATTTTTATGGATCCTTGTCATTGAATTAATAATATGATATATTTGGCTGCAGGTTGGGAGCAAGAACACCAACACAAAAGAACAAGGGTATGCTAATTCAGTAGTAAATTTGGGTCATAAATTTGAGGATAAGTGTAAATGTGAAGTTGAGATGAATGATGGCACCACTTTGTCCTACTTTAAACAAGAAGAAGCAGCCAAATCAGAAAAGCAAGAAAATAATTACTCTACAGATAGTTGGCTACATGACTCTTCAAATTGTGCAGCAGTGCCTAATTTGGACCTCACACTTTCAGTTGCATCCCCTAAGCTTGAACAAAATCAACCCTCCCCTGCTGGCTCATTCCTTCTTGGCCCAATTAGCGTCACTTAA.
Further, over-expression MsMYBH primers (OEMsMYBH-F and OEMsMYBH-R, specifically shown in Table 1) and RNAiMsMYBH primers (RNAi MsMYBH-F and RNAi MsMYBH-R, specifically shown in Table 1) were designed by cloning the CDS sequence of MsMYBH, respectively. Recombinant plasmid MsMYBH-pBI121 was successfully constructed by homologous recombination (FIG. 2A, the first lane is recombinant plasmid MsMYBH-pBI121, the second lane is the result of double restriction enzyme digestion of recombinant plasmid MsMYBH-pBI121 with restriction enzymes Xbal and Xmal, and the third lane is Marker).
Recombinant plasmid MsMYBH-pBI121 is a recombinant vector obtained by replacing a small fragment between restriction enzymes Xbal and Xmal of a pBI121 plasmid (GenBank No. AF 485783.1) with a DNA fragment of sequence 1 and keeping other sequences of the pBI121 plasmid unchanged, and the recombinant vector is named as recombinant plasmid MsMYBH-pBI121.
Recombinant vector MsMYBH-pK7WIWG2I was obtained by replacing the DNA fragment of pK7WIWG2I plasmid (available from Soy Corp., cat. No. VT 001143) with the DNA fragment of nucleotide sequence 5'-GCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAACGCGTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCAGGTCGAC-3'（ sequence 17) and replacing the DNA fragment of nucleotide sequence 5'-GTCGACCTGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGAGACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGTCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCACACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCACGCGTTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGCCGGATCCTAACTCAAAATCCACACATTATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGCGGCCGC-3'（ sequence 19) with the DNA fragment of nucleotide sequence 5'-TGAAATATTAGAAGTGGGTTTGTGATAATAATTTTCATCAATATTGGTTAATGATGTTGATGAAGAGAATGAGGAAGATGGAGAGGATGGTGAGGGAAAGGTGTCCATGCTAAAGCTTTTCTTGATCATGGTACTAGATGATGACGAGGAGGATGATGATAGTTGGACCCCAAAGAGACGAAGTCCTCCAAGGGTAGTGAAGGATGTGCATGTCCTACAATTATGTCCTATCTTACCACAATGCGAACACTTTCTTCTCCCCAT-3'（ sequence 20) by gateway technology, and the other sequences of pK7WIWG I plasmid were kept unchanged, and the recombinant vector was designated as recombinant vector MsMYBH-pK7WIWG I. The result of electrophoresis of the recombinant vector MsMYBH-pK7WIWG I is shown in FIG. 2B (FIG. 2B, the first lane is the recombinant vector MsMYBH-pK7WIWG I, the second lane is the front of the identification of MsMYBH-pK7WIWG I (using the amplification results of primer 9 and primer 11 in the sequence Listing), the third lane is the back of the identification of MsMYBH-pK7WIWG I (using the amplification results of primer 10 and primer 11 in the sequence Listing), and the fourth lane is Marker).
Example 2: cultivation of transgenic alfalfa plants overexpressing and RNAi MsMYBH
Recombinant vector MsMYBH-pBI121 and recombinant vector MsMYBH-pK7WIWG I in example 1 were transformed into Agrobacterium EHA105 strain, respectively, then alfalfa leaves "Zhongqing 1" were transformed by using Agrobacterium-mediated genetic transformation, and 7 lines of positive transgenic lines of over-expression MsMYBH and RNi MsMYBH were obtained through the processes of dedifferentiation, re-differentiation, rooting, etc., respectively designated OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, OE-7, RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7, respectively.
The over-expressed MsMYBH positive transgenic lines (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, and OE-7) were subjected to Edley DNA extraction kit, RNA extraction kit, and reverse transcription kit, respectively, to obtain a DNA template for the over-expressed MsMYBH positive transgenic line and a cDNA template for the over-expressed MsMYBH positive transgenic line. DNA templates of the over-expressed MsMYBH positive transgenic lines were identified at the gene level and transcription level using gene level identification primers (OEMsMYBH-F and OEMsMYBH-R) using Edley DNA extraction kit (DN 14) and RNA extraction kit (RN 09), and as a result, 825bp bands were amplified as shown in FIG. 3A (Maker for the first lane, ddH 2 O control for the second lane, recombinant vector MsMYBH-pBI121 for the third lane, WT (medium-sized, alfalfa 1) for the fourth and fifth lanes, and DNA templates of the over-expressed MsMYBH positive transgenic lines (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, and OE-7) for the sixth to twelve lanes, as positive transgenic plants.
The transcript levels of the primers (MsMYBH-F and MsMYBH-R), the actin gene as an internal reference gene (detection primer: msActin-qPCR-F:5 '-CAAAAGATGGCAGATGCTGAGGAT-3',: sequence 15 in the sequence listing; msActin-qPCR-R:5 '-CATGACACCAGTATGACGAGGTCG-3',: sequence 16 in the sequence listing) and Taq enzyme and Norwegian qPCRmix were identified, and the results are shown in FIG. 3B (relative expression of MsMYBH gene on the ordinate, and WT being medium-order 1, OE-2, OE-3, OE-4, OE-5, OE-6 and OE-7, respectively, as overexpressed MsMYBH positive transgenic lines (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6 and OE-7)).
RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7) were used with the Edley DNA extraction kit (DN 14), RNA extraction kit (RN 09) and reverse transcription kit (PC 5801), respectively, to obtain the DNA template of RNAi MsMYBH positive transgenic line and the cDNA template of RNAi MsMYBH positive transgenic line. The DNA templates of the RNAi MsMYBH positive transgenic lines were identified at the gene level using the gene level identifying primers (pK 7WIWG2I-F, pK7WIWG I-R and RNAi MsMYBH-R2), as shown in FIG. 3C (the first lane is Maker, the second and third lanes are recombinant vectors MsMYBH-pK7WIWG I, the fourth and fifth lanes are WT (middle-size 1), the sixth to nineteenth lanes are DNA templates of the RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7) in this order, wherein even lanes 2-19 amplify the latter band (444 bp) using primers 9 and 11 in the sequence listing, odd lanes 2-19 amplify the former band (564 bp) using primers 10 and 11 in the sequence listing), and the transgenic plants amplify the same band as the recombinant plants, as shown in FIG. 3C, thus being positive transgenic plants.
The transcript levels were used to identify primers (MsMYBH-F and MsMYBH-R), the actin genes as reference genes (detection primers: msActin-qPCR-F and MsActin-qPCR-R), and Taq enzyme (PC 0902) and Nuo Weizan qPCRmix (R323-01) as described in FIG. 3 for the relative expression levels of D (ordinate MsMYBH gene, WT being Zhongjin No. 1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7 as RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7), respectively.
Further, by utilizing a cutting propagation mode, transgenic lines such as WT (in the number 1), OE-1, OE-2, OE-3, RNAi-1, RNAi-2, RNAi-3 and the like are propagated, so that enough materials are provided for MsMYBH phenotype identification.
Example 3 MsMYBH tissue-specific expression and subcellular localization
Collecting alfalfa root, stem, leaf and flower, extracting RNA, and reverse transcribing to obtain CDNA. Using MsMYBH qPCR primers (MsMYBH-F and MsMYBH-R), the expression level of MsMYBH in different tissues was detected using the actin gene as the reference gene (detection primers: msActin-qPCR-F and MsActin-qPCR-R), and the results showed that the expression level was highest in leaves, and then root, stem and flower were the lowest (FIG. 4A). The subcellular localization of the onion epidermis shows that MsMYBH is localized in the nucleus (B in fig. 4).
Example 4 MsMYBH expression Pattern under drought stress
Alfalfa seedlings were treated with 300mM mannitol, sampled at different times of treatment, RNA extracted, reverse transcribed to CDNA, and then tested for MsMYBH expression patterns under drought stress. The results show that: under drought stress, msMYBH had similar expression patterns in leaf and root, but the expression level in leaf was significantly higher than in root, and the expression level of MsMYBH was significantly induced by drought stress 4h before stress treatment (fig. 5, abscissa is drought stress treatment time, ordinate is relative expression level; be root and leaf) indicating that MsMYBH plays an important role in early response to drought stress.
Example 5 MsMYBH promotes alfalfa growth and enhances drought resistance
Drought resistance tests were performed by selecting over-expressed MsMYBH positive transgenic lines (OE-1, OE-2, and OE-3) and RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi-2, and RNAi-3) with WT (alfalfa 1) as a control, as follows:
WT, OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3 were propagated by means of cutting propagation (nutrient soil as cultivation substrate: vermiculite body=1:1), culture medium before sowing (culture medium is nutrient soil: vermiculite body=1:1), watering (excessive water at the bottom of the planting pot is discharged along with the holes), watering 100ml/100g of culture medium every 4 days to obtain cuttage offspring of WT, OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3, cutting the cuttage offspring after cutting for one month (uniformly cutting the cuttage offspring, leaving stubble height is about 15 cm), cutting for one month to obtain cuttage offspring seedlings of WT, OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3, randomly dividing the above cutting offspring seedlings (WT, OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3 cutting offspring seedlings) into two groups, namely a control group and a drought treatment group, wherein each group of WT cutting offspring seedlings, OE-1 cutting offspring seedlings, OE-2 cutting offspring seedlings, OE-3 cutting offspring seedlings, RNAi-1 cutting offspring seedlings, RNAi-2 cutting offspring seedlings and RNAi-3 cutting offspring seedlings has 1 repetition, repeating for 3 times, photographing, recording growth phenotype, recording, and processing (specifically, 3 in 1, OE MYBH in FIG. 6A, 3 in OE MYBH represent OE-1 cutting offspring, respectively), the OE-2 cutting offspring and the OE-3 cutting offspring, 3 in 2 and Ri MYBH in 1 and Ri MYBH in Ri MYBH are RNAi-1 cutting offspring, RNAi-2 cutting offspring and RNAi-3 cutting offspring respectively; WT is the cutting filial generation of alfalfa No. 1; control is control, drought is drought treatment, pre-treatment is pre-drought phenotype, post-treatment is post-drought phenotype) the following operations are continued:
Drought treatment group: the cutting offspring seedlings (WT, OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3) are subjected to natural drought treatment (stopping watering) for 20 days in a sunlight greenhouse (temperature 25+/-2 ℃ C., light for 16 hours/dark for 8 hours), and after the drought treatment, shooting is carried out to record the growth phenotype, and the growth phenotype is recorded after the treatment (specifically, 3 in 1, OE MYBH and OE MYBH in A, OE MYBH in FIG. 6 respectively represent OE-1 cutting offspring, OE-2 cutting offspring and OE-3 cutting offspring, and 3 in 1, ri MYBH and Ri MYBH in Ri MYBH are RNAi-1 cutting offspring, RNAi-2 cutting offspring and RNAi-3 cutting offspring; WT is a Zhongqing No. 1 cutting filial, a control group, drought is an drought treatment group, the drought is a pre-drought treatment phenotype, the drought is a post-drought treatment phenotype), alfalfa plant height, branch number, leaf relative water content, malondialdehyde content and proline content are detected and recorded after the drought treatment (the results are shown as B-F in figure 6, 3 in 2 and OE MYBH in 1 and OE MYBH in OE MYBH respectively represent OE-1 cutting filial, OE-2 cutting filial and OE-3 cutting filial, 3 in 2 and Ri MYBH in Ri MYBH respectively RNAi-1 cutting filial, RNAi-2 cutting filial and RNAi-3 cutting filial, WT is Zhongqing No. 1 cutting filial, the control group and drought is a drought treatment group), POD activity, CAT activity and SOD activity (the results are shown as B-D in figure 8, 3 in 1 in OE MYBH, 2 in OE MYBH and OE MYBH represent OE-1, OE-2 and OE-3 cuttings, respectively, and 3 in 1, ri MYBH and Ri MYBH in Ri MYBH are RNAi-1, RNAi-2 and RNAi-3 cuttings, respectively; WT is the cutting filial generation of alfalfa No. 1; control is control, drought is drought treatment), and net photosynthetic rate Pn and maximum fluorescence yield Fm (results shown as a and B in fig. 7, OE MYBH1, OE MYBH2, and OE MYBH3 represent OE-1, OE-2, and OE-3 cuttings, ri MYBH, ri MYBH, and Ri MYBH3 are RNAi-1, RNAi-2, and RNAi-3 cuttings, respectively; WT is alfalfa No. 1 cutting filial generation), H 2O2 content detection is carried out on alfalfa leaves after drought treatment (the result is shown as A in FIG. 8, OE MYBH is respectively an OE-1 cutting filial generation, an OE-2 cutting filial generation and an OE-3 cutting filial generation from left to right, and Ri MYBH is respectively an RNAi-1 cutting filial generation, an RNAi-2 cutting filial generation and an RNAi-3 cutting filial generation from left to right; WT is the cutting filial generation of alfalfa No. 1; control, drought treatment), data were treated using SPSS11.5 statistical software, experimental results expressed as mean ± standard deviation, with One-way ANOVA test, different letters indicated significant differences (P < 0.05).
Control group: seedlings were grown normally (100 ml/100G watered every 4 days, nutrient soil and vermiculite 1:1) in sunlight greenhouse (temperature 25.+ -. 2 ℃ C., light 16 h/dark 8 h, 4200 LUX) for 20 days, after normal cultivation, photographed to record growth phenotype, recorded as treated (3 in 1, OE MYBH and OE MYBH in FIG. 6A, OE MYBH represent OE-1, OE-2 and OE-3 cuttings, respectively, 3 in 1, ri MYBH and Ri MYBH in Ri MYBH are RNAi-1, RNAi-2 and RNAi-3 cuttings, respectively; WT is a well 1 cutting progeny, control is a drought treatment group, drought is a drought treatment group, the pre-treatment is a pre-drought phenotype, the post-treatment is a post-drought phenotype, normal culture is followed by recording and detecting alfalfa plant height, branch number, leaf relative moisture content, malondialdehyde content, proline content (results are shown as B-F in FIG. 6, 3 in 2 and OE MYBH in 1, OE MYBH in OE MYBH represent OE-1 cutting progeny, OE-2 cutting progeny and OE-3 cutting progeny, 3 in 2 and Ri MYBH in 1, ri MYBH in Ri MYBH are RNAi-1 cutting progeny, RNAi-2 cutting progeny and RNAi-3 cutting progeny, respectively; WT is a well 1 cutting progeny, control is a drought treatment group), fresh weight, dry weight, stem leaf ratio, crude protein, NDF and ADF (results are shown as C-G in FIG. 7, 3 in 2 and OE MYBH in 1, OE MYBH in OE MYBH represent-1, respectively), the OE-2 cutting offspring and the OE-3 cutting offspring, 3 in 2 and Ri MYBH in 1 and Ri MYBH in Ri MYBH are RNAi-1 cutting offspring, RNAi-2 cutting offspring and RNAi-3 cutting offspring respectively; WT is alfalfa No. 1 cutting filial generation), POD activity, CAT activity, SOD activity (as shown in B-D in FIG. 8, 3 in 2 and OE MYBH in 1, OE MYBH in OE MYBH respectively represent OE-1 cutting filial generation, OE-2 cutting filial generation and OE-3 cutting filial generation, and 3 in 2 and Ri MYBH in 1, ri MYBH in Ri MYBH respectively are RNAi-1 cutting filial generation, RNAi-2 cutting filial generation and RNAi-3 cutting filial generation; WT is the cutting filial generation of alfalfa No. 1; control is control, drought is drought treatment), and net photosynthetic rate Pn and maximum fluorescence yield Fm (results shown as a and B in fig. 7, OE MYBH1, OE MYBH2, and OE MYBH3 represent OE-1, OE-2, and OE-3 cuttings, ri MYBH, ri MYBH, and Ri MYBH3 are RNAi-1, RNAi-2, and RNAi-3 cuttings, respectively; WT is alfalfa No. 1 cutting filial generation), after normal culture, alfalfa leaves are taken for H 2O2 content detection (the result is shown as A in FIG. 8, OE MYBH is respectively an OE-1 cutting filial generation, an OE-2 cutting filial generation and an OE-3 cutting filial generation from left to right, and Ri MYBH is respectively an RNAi-1 cutting filial generation, an RNAi-2 cutting filial generation and an RNAi-3 cutting filial generation from left to right; WT is the cutting filial generation of alfalfa No. 1; controls are control groups and drought is drought treatment), phenotype observation is carried out in the whole course, and One-way ANOVA test is adopted, and different letters are adopted to represent significant differences.
Plant height: root neck to top height measurements were made on drought treated and control alfalfa, where top refers to the top of the main stem.
Branch number: the number of branches formed in the root parts of the ground was measured for the drought control and the control alfalfa.
Blade relative water content: the alfalfa of the drought treatment group and the alfalfa of the control group are respectively weighed 0.2g of the leaf, the leaf is marked as Wf, then the leaf is immersed in water for 8 hours, the saturated fresh weight Wt of the leaf is weighed, the leaf is deactivated in a baking oven at 105 ℃ for 15 minutes, the leaf is dried to constant weight at 75 ℃, the dry weight is weighed, the dry weight is marked as Wd, and the relative water content is calculated according to the formula: relative moisture content= (Wf-Wd)/(Wt-Wd) ×100%.
Malondialdehyde content: alfalfa leaves from drought and control groups were tested using the Soxhibao Malondialdehyde (MDA) content test kit (BC 0020), and each test was repeated 3 times.
Proline content: alfalfa leaves from drought treatment and control groups were tested using the test kit for proline (Pro) content from Soy Corp (BC 0290), and each test was repeated 3 times.
Fresh weight: and weighing alfalfa in the drought treatment group and the control group to obtain fresh weight.
Dry weight: and (3) sampling alfalfa in the drought treatment group and the control group, deactivating enzymes in an oven at 105 ℃ for 15 min, drying at 75 ℃ to constant weight, and weighing the dry weight.
Stem-leaf ratio: and (3) carrying out 105 ℃ de-enzyming on the whole alfalfa of the drought treatment group and the control group for 30 min, then drying at 60-65 ℃ to constant weight, and then separating and weighing stems and leaves, wherein the weight ratio of the stems to the leaves is the stem-leaf ratio.
Crude protein: the content of crude protein was determined for alfalfa in drought treatment and control groups according to GB/T6432-2018 determination of crude protein in feed.
NDF (neutral wash fiber.): neutral Detergent Fiber (NDF) was measured using a Van Soest detergent fiber assay. The specific determination method comprises the following steps:
(1) 0.5-1.0g (m) of 40 mesh sieve feed sample is accurately weighed, placed in a high-mouth-free beaker, and 100ml and 2ml of neutral detergent solution, decalin and 0.5g of anhydrous sodium sulfite are added. (2) Placing a condensing device, immediately placing on an electric furnace, boiling (5-10 min), slightly boiling for 1h (3), cooling for 10min, placing a glass crucible (m 1) with known mass on a suction filtration bottle, completely transferring residues into the suction filtration bottle, washing with boiling water, and suction-filtering. Then, the mixture was rinsed with 20ml of acetone and filtered with suction. And (4) taking off the crucible, drying at 105 ℃, and weighing (m 2). NDF: = (m 2-m 1)/m.
ADF (acid washed fiber): specific determination of acid washed fiber (ADF) using Van Soest's washed fiber assay:
(1) Accurately weighing the sample, 0.5-1.0g (m), placing in a beakers, adding 100ml of an acidic detergent solution and a plurality of drops of decalin. (2) the same NDF assay. (3) The residue was suction filtered on a suction flask with a glass crucible (m 3) of known mass, washed with boiling water, suction filtered repeatedly 3 times, then washed with a small amount of acetone, repeatedly rinsed until the filtrate was colorless, and the whole acetone was removed. (4) As measured by NDF, the mass of crucible+residue was (m 4). ADF= (m 4-m 3)/m.
POD activity (peroxidase activity): the alfalfa leaves of the drought treatment group and the control group were tested using the Soxhibao Peroxidase (POD) activity test kit (BC 0090), and each test was repeated 3 times.
CAT Activity (catalase Activity): alfalfa leaves from drought treatment and control groups were tested using the Soxhibao Catalase (CAT) activity test kit (BC 0200) and repeated 3 times per test.
SOD activity (superoxide dismutase activity): the alfalfa leaves of the drought treatment group and the control group were tested with the Soxhibao company superoxide dismutase (SOD) activity test kit (BC 0170) 3 times per test.
Pn (net photosynthetic rate): the net photosynthetic rate (Photosynthetic rate, pn) was determined for alfalfa leaves in drought control and control groups using a portable photosynthesis measurement system LI-6400XT instrument, 3 leaves each.
Fm (maximum fluorescence yield): the drought and control groups and alfalfa leaves were tested by a portable chlorophyll fluorometer MultispeQ (Photosynq, usa) as follows: after the instrument clamps tobacco leaves, a measuring key is clicked, and after about 15 seconds of measurement is completed, plants are kept upright during measurement, the angles of the leaves are not changed, 3 leaves are detected each time, and each leaf is repeated for 3 times.
H 2O2 content detection: the content of H 2O2 is detected by using a DAB method (3, 3-diaminobenzidine tetrahydrochloride), H 2O2 can rapidly react with DAB to generate a brownish red compound, so that hydrogen peroxide in tissues is positioned, and the darker the color is, the higher the H 2O2 content is. H 2O2 content of alfalfa leaves in drought treatment and control groups was measured using Seville DAB staining kit (servicebio, G1212-200T), 3 leaves per time.
The over-expression MsMYBH positive transgenic lines (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6 and OE-7) were identified at the gene level and the transcription level using the DNA extraction kit and the RNA extraction kit of Edley, respectively, the identification primer results are shown as A and B in FIG. 3, and the identification results of the RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7) at the gene level and the transcription level are shown as C and D in FIG. 3; WT is alfalfa No. 1, and the relative expression level is MsMYBH gene.
The results show that:
Under normal control conditions (control group), the over-expressed MsMYBH positive transgenic lines (OE MYBH: OE-1, OE-2 and OE-3), wild plants (WT) and RNAi MsMYBH positive transgenic plants (Ri MYBH: RNAi-1, RNAi-2 and RNAi-3) were not greatly different in height, but the OE MYBH transgenic lines had significantly more branches than WT, and the WT had significantly more branches than Ri MYBH transgenic plants (FIG. 6A-C; control group in FIG. 6), drought was drought treatment, after drought treatment, before drought treatment, OE MYBH was over-expressed MsMYBH positive transgenic lines, ri MYBH was RNAi MsMYBH positive transgenic plants, 1,2 and 3 in OE MYBH were respectively OE-1, OE-2 and OE-3 cuttings, 1,2 and 3 in Ri MYBH were respectively RNAi-1, RNAi-2 and RNAi-3 cuttings, and WT was 1 cuttings. Meanwhile, under the control condition, the biomass and related index data (fresh weight, dry weight, stem-leaf ratio, crude protein, DNF and ADF) of the transgenic plants and the WT were detected.
Whereas the fresh and dry weight of the over-expressed MsMYBH positive transgenic line was significantly higher than WT, whereas WT was significantly higher than RNAi MsMYBH positive transgenic line (C and D in fig. 7; OE MYBH in fig. 7C-D was the over-expressed MsMYBH positive transgenic line, ri MYBH was RNAi MsMYBH positive transgenic plant, 1,2 and 3 in OE MYBH were the OE-1, OE-2 and OE-3 cuttings, respectively, 1,2 and 3 in Ri MYBH were the RNAi-1, RNAi-2 and RNAi-3 cuttings, respectively, WT was the medium-sized and medium-sized cutting progeny, fig. 7 was a control test result map), and its pasture quality was not reduced (fig. 7F-H, OE MYBH was the over-expressed MsMYBH positive transgenic line, ri MYBH was RNAi MsMYBH positive transgenic plant, 1,2 and 3 in OE MYBH were the OE-1, OE-2 and OE-3 cuttings, respectively, 1,2 and 3 in Ri MYBH were the RNAi-1, RNAi-2 and RNAi-3 cuttings, respectively, WT was the medium-sized and RNAi-3 cuttings, respectively, and OE MYBH was the control test result map, and was the cutting progeny, and the control test result map was the cutting progeny, fig. 7, was the pasture was cut progeny. The results show that MsMYBH promotes the growth of alfalfa.
Following drought stress treatment (drought treatment group), OE MYBH transgenic lines exhibited stronger drought resistance, with more branches and minimal leaf wilting compared to WT (fig. 6A); and Ri MYBH transgenic plants had the weakest drought resistance, and a large number of leaves were wilted and shed (FIG. 6A). The physical index measurement also proves that OE MYBH transgenic lines have stronger drought resistance, and are mainly characterized by the highest relative water content of leaves, lowest malondialdehyde content and highest proline content (FIG. 6D-F, control group, drought is drought treatment group, after drought treatment, before drought treatment, OE MYBH is an over-expression MsMYBH positive transgenic line, ri MYBH is RNAi MsMYBH positive transgenic plant, 1,2 and 3 in OE MYBH are respectively OE-1 cutting progeny, OE-2 cutting progeny and OE-3 cutting progeny, 1,2 and 3 in Ri MYBH are respectively RNAi-1 cutting progeny, RNAi-2 cutting progeny and RNAi-3 cutting progeny, and WT is middle-alfalfa number 1 cutting progeny); before drought treatment for 12d, leaf photosynthesis and maximum fluorescence yields were also significantly higher than WT (FIGS. 7A and B, OE MYBH for OE-1, OE MYBH2 for OE-2, OE MYBH3 for OE-3, ri MYBH1 for RNAi-1, ri MYBH2 for RNAi-2, ri MYBH3 for RNAi-3); after drought stress, H 2O2 accumulated least (FIG. 8A), and antioxidant enzyme activity (POD activity, CAT activity and SOD activity) highest (B-D in FIG. 8, control group, drought treatment group, post-treatment, pre-treatment, OE MYBH, over-expressed MsMYBH positive transgenic line, ri MYBH, RNAi MsMYBH positive transgenic plant, 1,2 and 3 in OE MYBH were OE-1, OE-2 and OE-3 cuttings, respectively, 1,2 and 3 in Ri MYBH were RNAi-1, RNAi-2 and RNAi-3 cuttings, respectively, and WT was Zhongjin 1 cuttings). Ri MYBH transgenic plants performed in opposite fashion (D-F in FIG. 6, A-B in FIG. 7, and FIG. 8). The results show that MsMYBH enhances the drought resistance of the alfalfa.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (5)
1. A method for increasing alfalfa fresh weight, dry weight and/or branch number, characterized by: the method comprises up-regulating or enhancing or increasing expression of a gene encoding a protein in alfalfa and/or activity and/or content of the protein to increase fresh weight, dry weight and/or branch number of alfalfa, wherein the protein is any one of the following proteins:
b1 Amino acid sequence is a protein shown in sequence 2;
B2 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) with a protein tag.
2. The method of claim 1, wherein said up-regulating or enhancing or increasing expression of a gene encoding a protein in alfalfa comprises introducing the gene encoding the protein into alfalfa.
3. A method for cultivating alfalfa having a high fresh weight, a high dry weight and/or a high number of branches, comprising: the method comprises up-regulating or enhancing or increasing expression of a gene encoding a protein in a target alfalfa and/or activity and/or content of the protein to obtain an alfalfa with high fresh weight, high dry weight and/or number of branches, wherein the fresh weight, dry weight and/or number of branches of the alfalfa with high fresh weight, high dry weight and/or number of branches is higher than that of the target alfalfa;
The protein is any one of the following proteins:
b1 Amino acid sequence is a protein shown in sequence 2;
B2 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) with a protein tag.
4. The method of claim 3, wherein said up-regulating or enhancing or increasing expression of a gene encoding a protein in alfalfa comprises introducing a gene encoding said protein into alfalfa.
5. Protein, substance that up-regulates or enhances or increases expression of a gene encoding the protein or substance that up-regulates or enhances or increases activity or content of the protein for increasing fresh weight, dry weight and/or branch number of alfalfa and/or for preparing a product that increases fresh weight, dry weight and/or branch number of alfalfa;
The protein is any one of the following proteins:
b1 Amino acid sequence is a protein shown in sequence 2;
b2 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) with protein tags;
the substance is any one of the following:
d1 A nucleic acid molecule encoding said protein;
D2 An expression cassette comprising D1) said nucleic acid molecule;
D3 A recombinant vector comprising D1) said nucleic acid molecule, or a recombinant vector comprising D2) said expression cassette;
d4 A recombinant microorganism comprising D1) said nucleic acid molecule, or a recombinant microorganism comprising D2) said expression cassette, or a recombinant microorganism comprising D3) said recombinant vector;
d5 A transgenic plant cell line comprising D1) said nucleic acid molecule, or a transgenic plant cell line comprising D2) said expression cassette, or a transgenic plant cell line comprising D3) said recombinant vector;
d6 A transgenic plant tissue comprising D1) said nucleic acid molecule, or a transgenic plant tissue comprising D2) said expression cassette, or a transgenic plant tissue comprising D3) said recombinant vector;
d7 A transgenic plant organ comprising D1) said nucleic acid molecule, or a transgenic plant organ comprising D2) said expression cassette, or a transgenic plant organ comprising D3) said recombinant vector.
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