CN113481176B - Application of GA3ox1 protein in regulating alfalfa plant type - Google Patents

Application of GA3ox1 protein in regulating alfalfa plant type Download PDF

Info

Publication number
CN113481176B
CN113481176B CN202110885132.7A CN202110885132A CN113481176B CN 113481176 B CN113481176 B CN 113481176B CN 202110885132 A CN202110885132 A CN 202110885132A CN 113481176 B CN113481176 B CN 113481176B
Authority
CN
China
Prior art keywords
sequence
gene
msga3ox1
mtga3ox1
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110885132.7A
Other languages
Chinese (zh)
Other versions
CN113481176A (en
Inventor
王涛
董江丽
刘金玲
郑丽华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Agricultural University
Original Assignee
China Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by China Agricultural University filed Critical China Agricultural University
Priority to CN202110885132.7A priority Critical patent/CN113481176B/en
Publication of CN113481176A publication Critical patent/CN113481176A/en
Application granted granted Critical
Publication of CN113481176B publication Critical patent/CN113481176B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
    • C12Y114/11015—Gibberellin 3-beta-dioxygenase (1.14.11.15)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a method for cultivating dwarfed plant type and/or compact plant type and/or creeping plant type alfalfa by utilizing a gene editing technology. The invention provides application of GA3ox1 protein or its coding gene in regulating alfalfa plant type. The GA3ox1 protein coding gene is knocked out in alfalfa, the protein expression level and/or activity is reduced or inactivated, and the gibberellin content in alfalfa is reduced. Experiments prove that compared with wild plants, alfalfa GA3ox1 gene knockout plants have obviously reduced plant height and internode length and are dwarf and/or compact and/or creeping plant types. The gene editing technology is used for targeting GA3ox1, so that a new alfalfa plant type can be obtained quickly and efficiently, and the method has important significance for alfalfa interplanting, intercropping and the like.

Description

Application of GA3ox1 protein in regulating alfalfa plant type
Technical Field
The invention relates to the field of biotechnology, in particular to application of GA3ox1 protein in regulation of alfalfa plant types.
Background
Alfalfa is a perennial legume, has the characteristics of high yield, good quality, rich nutritional value, strong adaptability and the like, and is known as the king of pasture. The alfalfa has important agricultural production value, can be used as a high-quality feeding crop and can be interplanted with silage corns to enhance the nitrogen-fixing microbial activity of soil, improve the utilization efficiency of nutrient elements of rhizosphere soil, improve the soil fertility and promote the growth and development of plants; alfalfa is interplanted in the orchard, so that a virtuous circle ecosystem can be formed in the orchard, the economic effect generated by fruit tree planting is improved, the feed production is promoted, and the development of animal husbandry is promoted; the alfalfa is intercropped with other main-cultivated crops, so that the ground surface coverage rate can be effectively improved, the water loss is reduced, and the growth of weeds is inhibited. However, most alfalfa cultivars exhibit an erect character and are high in plant height, and the effectiveness and convenience of the farming activities are limited to a great extent; the creeping character is more beneficial to interplanting and intercropping application on the basis of keeping other production advantages. At present, natural creeping alfalfa materials are few, the comprehensive agronomic performance is poor, and genetic improvement is difficult to carry out through a traditional breeding mode; by utilizing biotechnology, particularly gene editing technology, the creeping alfalfa with excellent comprehensive performance can be efficiently constructed by taking the cultivated alfalfa as a genetic background.
Disclosure of Invention
The technical problem to be solved by the invention is how to quickly and efficiently obtain the dwarfed and/or compact and/or creeping plant type alfalfa.
In order to solve the above technical problems, the present invention provides the use of a protein or a substance regulating the expression of a gene encoding said protein, or a substance regulating the activity or content of said protein, said protein being MsGA3ox1 or MtGA3ox 1;
the MsGA3ox1 is any one of the following a1) -a 3):
a1) the amino acid sequence is protein shown as a sequence 1 in a sequence table;
a2) a protein which is obtained by substituting and/or deleting and/or adding one or more than one amino acid residues in the amino acid sequence shown in a1), has more than 60% of identity with the amino acid sequence shown in a1), and is related to the alfalfa plant type;
a3) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of a1) or a 2);
the MtGA3ox1 is any one of the following b1) -b 3):
b1) the amino acid sequence is protein shown as a sequence 2 in a sequence table;
b2) a protein which is obtained by substituting and/or deleting and/or adding one or more than one amino acid residues in the amino acid sequence shown in b1), has more than 60% of identity with the amino acid sequence shown in b1), and is related to the alfalfa plant type;
b3) a fusion protein obtained by connecting a label at the N end or/and the C end of b1) or b 2).
Further, in the application, the protein of a2) comprises MsGA3ox1/-8bp-L3, MsGA3ox1/-36bp, MsGA3ox1/-9bp, MsGA3ox1/+1bp, MsGA3ox1/-8bp-L15, MsGA3ox1/-7bp, MsGA3ox1/-72bp,
the MsGA3ox1/-8bp-L3 is a protein coded by an MsGA3ox1/-8bp-L3 gene, and the MsGA3ox1/-8bp-L3 gene is a DNA molecule obtained by deleting 8 bases in total from the nucleotides at the 350-position 357 in the sequence 3 in the sequence table and keeping other nucleotide sequences in the sequence 3 unchanged;
the MsGA3ox1/-36bp is a protein coded by an MsGA3ox1/-36bp gene, and the MsGA3ox1/-36bp gene is a DNA molecule obtained by deleting 36 bases in total from the 320-th and 355-th nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/-9bp is a protein coded by an MsGA3ox1/-9bp gene, and the MsGA3ox1/-9bp gene is a DNA molecule obtained by deleting 9 bases in total from the 351-359 th nucleotide of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/+1bp is a protein coded by an MsGA3ox1/+1bp gene, and the MsGA3ox1/+1bp gene is a DNA molecule obtained by inserting a base G into the nucleotide between the 350 th and 351 th nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/-8bp-L15 is a protein coded by an MsGA3ox1/-8bp-L15 gene, and the MsGA3ox1/-8bp-L15 gene is a DNA molecule obtained by deleting 8 bases in total of nucleotides at the 351-;
the MsGA3ox1/-7bp is a protein coded by an MsGA3ox1/-7bp gene, and the MsGA3ox1/-7bp gene is a DNA molecule obtained by deleting 7 bases in total from the 351-357 th nucleotide of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/-72bp is a protein coded by an MsGA3ox1/-72bp gene, and the MsGA3ox1/-72bp gene is a DNA molecule obtained by deleting 72 bases in total from the 351-422 th nucleotide of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
b2) the protein comprises MtGA3ox1/+1bp-L4, MtGA3ox1/-10bp, MtGA3ox1/-19bp, MtGA3ox1/+2bp, MtGA3ox1/-216bp, MtGA3ox1/+1bp-L11,
the MtGA3ox1/+1bp-L4 is protein coded by MtGA3ox1/+1bp-L4 gene, the MtGA3ox1/+1bp-L4 gene is DNA molecule obtained by adding a base C between the 255 th and 256 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/-10bp is protein coded by an MtGA3ox1/-10bp gene, and the MtGA3ox1/-10bp gene is a DNA molecule obtained by deleting 10 bases in total from the 330 th-339 th nucleotide of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/-19bp is a protein coded by an MtGA3ox1/-19bp gene, and the MtGA3ox1/-19bp gene is a DNA molecule obtained by deleting 19 bases in total from the 329 th 347 th nucleotide of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/+2bp is protein coded by MtGA3ox1/+2bp gene, the MtGA3ox1/+2bp gene is DNA molecule obtained by inserting two basic groups GA between the nucleotides of 331 st-332 th sequence in the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/-216bp is protein coded by an MtGA3ox1/-216bp gene, and the MtGA3ox1/-216bp gene is a DNA molecule obtained by deleting 216 bases in total from the 169 th 384 th nucleotide of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/+1bp-L11 is protein coded by MtGA3ox1/+1bp-L11 gene, and the MtGA3ox1/+1bp-L11 gene is DNA molecule obtained by adding one base G between the 331 st-332 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged.
Further, the protein may be derived from alfalfa.
Further, the alfalfa may be alfalfa or tribulus alfalfa.
The protein-tag refers to a polypeptide or protein which is expressed by fusion with a target protein by using a DNA in vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag, and/or a SUMO protein tag, etc.
The invention provides an application of a biological material related to the protein in regulating and controlling alfalfa plant types, wherein the biological material can be any one of the following materials:
A1) nucleic acid molecules encoding the above proteins;
A2) an expression cassette comprising the nucleic acid molecule of a 1);
A3) a recombinant vector comprising A1) said nucleic acid molecule or a recombinant vector comprising A2) said expression cassette;
A4) a recombinant microorganism containing A1) the nucleic acid molecule, or a recombinant microorganism containing A2) the expression cassette, or a recombinant microorganism containing A3) the recombinant vector;
A5) a transgenic plant cell line comprising A1) the nucleic acid molecule or a transgenic plant cell line comprising A2) the expression cassette;
A6) transgenic plant tissue comprising A1) the nucleic acid molecule or transgenic plant tissue comprising A2) the expression cassette;
A7) a transgenic plant organ containing A1) the nucleic acid molecule or a transgenic plant organ containing A2) the expression cassette;
A8) a nucleic acid molecule which inhibits or reduces the expression of a gene encoding the protein or the activity of the protein;
A9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to A8).
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
In the application, the alfalfa plant type can be regulated and controlled to reduce the alfalfa plant height and/or internode length and/or internode number, so that the alfalfa is dwarfed and/or compact and/or creeping form.
In the above application, the substance for regulating the activity or content of the protein may be a substance for knocking out a gene encoding the protein and/or a substance for regulating the expression of a gene encoding the protein.
In the above application, the substance for regulating gene expression may be a substance for regulating at least one of the following 6 kinds of 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 a 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 a protein translated from the gene).
In the above application, the regulation of gene expression may be the inhibition or reduction of gene expression, and the inhibition or reduction of gene expression may be achieved by gene knockout or by gene silencing.
The gene knockout (geneknockout) refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of a specific target gene by a change in the DNA sequence.
The gene silencing refers to the phenomenon that a gene is not expressed or is under expression under the condition of not damaging the original DNA. Gene silencing is premised on no change in DNA sequence, resulting in no or low expression of the gene. Gene silencing can occur at two levels, one at the transcriptional level due to DNA methylation, differential staining, and positional effects, and the other post-transcriptional gene silencing, i.e., inactivation of a gene at the post-transcriptional level by specific inhibition of a target RNA, including antisense RNA, co-suppression (co-suppression), gene suppression (quelling), RNA interference (RNAi), and micro-RNA (mirna) -mediated translational suppression, among others.
In the above application, the substance for regulating gene expression may be an agent for inhibiting or reducing the gene expression. The agent that inhibits or reduces the expression of the gene can be an agent that knocks out the gene, such as an agent that knocks out the gene by homologous recombination, or an agent that knocks out the gene by CRISPR-Cas 9. The agent that inhibits or reduces expression of the gene may comprise a polynucleotide that targets the gene, such as an siRNA, shRNA, sgRNA, miRNA, or antisense RNA.
Further, in the application, the nucleic acid molecule A1) can be a DNA molecule shown in any one of g1) to g4) as follows:
g1) the coding sequence of the coding chain is a DNA molecule of a sequence 3 in the sequence table;
g2) the DNA molecule has more than 60 percent of identity with the DNA molecule of g1) and codes and regulates and controls the related protein of alfalfa plant type;
g3) the coding sequence of the coding chain is a DNA molecule shown as a sequence 4 in the sequence table;
g4) the DNA molecule has more than 60 percent of identity with the DNA molecule of g3) and codes and regulates and controls the related protein of alfalfa plant type;
further, in the application, the DNA molecule of g2) comprises MsGA3ox1/-8bp-L3 gene, MsGA3ox1/-36bp gene, MsGA3ox1/-9bp gene, MsGA3ox1/+1bp gene, MsGA3ox1/-8bp-L15 gene, MsGA3ox1/-7bp gene, MsGA3ox1/-72bp gene,
the MsGA3ox1/-8bp-L3 gene is a DNA molecule obtained by deleting 8 bases in total from the 350 th-357 th nucleotide of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/-36bp gene is a DNA molecule obtained by deleting 36 bases of the 320-th and 355-th nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/-9bp gene is a DNA molecule obtained by deleting 9 bases in total from the 351-359 th nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/+1bp gene is a DNA molecule obtained by inserting a base G into the nucleotide between the 350 th and 351 rd nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/-8bp-L15 gene is a DNA molecule obtained by deleting 8 bases of the 351-358 th nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/-7bp gene is a DNA molecule obtained by deleting 7 bases in total from the 351-357 th nucleotide of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
the MsGA3ox1/-72bp gene is a DNA molecule obtained by deleting 72 bases in total from the 351-422 th nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
g4) the DNA molecule comprises MtGA3ox1/+1bp-L4 gene, MtGA3ox1/-10bp gene, MtGA3ox1/-19bp gene, MtGA3ox1/+2bp gene, MtGA3ox1/-216bp gene, MtGA3ox1/+1bp-L11 gene,
the MtGA3ox1/+1bp-L4 gene is a DNA molecule obtained by adding a base C between the 255 th and 256 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/-10bp gene is a DNA molecule obtained by deleting 10 bases of the nucleotides at the 330 nd-339 th site of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/-19bp gene is a DNA molecule obtained by deleting 19 bases in the 329 th 347 th nucleotide of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/+2bp gene is a DNA molecule obtained by inserting two bases GA between the 331 st-332 nd nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/-216bp gene is a DNA molecule obtained by deleting 216 bases of the 169 th 384 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
the MtGA3ox1/+1bp-L11 gene is a DNA molecule obtained by adding a base G between the 331 st-332 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged.
Further, in the application, A8) the nucleic acid molecule is: expressing a DNA molecule of a gRNA targeting the gene encoding the protein of a1) or b1) above or a gRNA targeting the gene encoding the protein of a1) or b1) above;
the grnas include sgRNA1 and sgRNA 2.
Further, B1), the reverse complement of the nucleotide indicated by the 347 nd and 365 th positions of the sequence 3 in the sequence table of the target sequence of the sgRNA1 (i.e., 5'-ACACCATCCGGGGAACGAA-3'), and the target sequence of the sgRNA2 is as the nucleotide sequence indicated by the 341 nd and 359 nd positions of the sequence 3 in the sequence table (i.e., 5'-AAGCCATTCGTTCCCCGGA-3');
B2) the reverse complement of the nucleotide sequence shown in the 329-position 347 of the sequence 4 in the sequence table of the target sequence of the sgRNA1 (namely 5'-ACACCATCTGGGGAACGAA-3'), and the nucleotide sequence shown in the 323-position 341 of the sequence 4 in the sequence table of the sgRNA2 (namely 5'-AAGCCATTCGTTCCCCAGA-3').
In the above applications, identity refers to the identity of a nucleotide sequence or an amino acid sequence. The identity of the nucleotide or amino acid sequence can be determined using homology search sites on the internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastn/blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and performing calculation by searching for the identity of a pair of nucleotides or amino acid sequences, a value (%) of identity can be obtained.
In the above applications, the 60% or greater identity may be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
The invention provides a method for producing alfalfa dwarf plant types and/or compact plant types and/or creeping plant types, which is characterized by comprising the following steps: comprises the step of generating the alfalfa dwarf plant type and/or compact plant type and/or creeping plant type by inhibiting or reducing the activity of the protein or the expression of the gene in the alfalfa genome.
Further, the alfalfa may be alfalfa or tribulus alfalfa.
Further, in the method, the inhibiting or reducing the activity of the protein or the expression of the gene in the alfalfa genome may be M1 or M2:
m1, carrying out at least one mutation of MsGA3ox1 gene shown in sequence 3 in the sequence table:
1) the MsGA3ox1 gene shown in the sequence 3 in the sequence table is mutated into an MsGA3ox1/-8bp-L3 gene, and the MsGA3ox1/-8bp-L3 gene is a DNA molecule obtained by deleting 8 bases in total at the 350-th 357 bit of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
2) the MsGA3ox1 gene shown in the sequence 3 in the sequence table is mutated into an MsGA3ox1/-36bp gene, and the MsGA3ox1/-36bp gene is a DNA molecule obtained by deleting 36 bases in the 320-th-355 bit of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
3) the MsGA3ox1 gene shown in the sequence 3 in the sequence table is mutated into an MsGA3ox1/-9bp gene, and the MsGA3ox1/-9bp gene is a DNA molecule obtained by deleting 9 bases in total from the 351-359 th nucleotide of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
4) the MsGA3ox1 gene shown in the sequence 3 in the sequence table is mutated into an MsGA3ox1/+1bp gene, and the MsGA3ox1/+1bp gene is a DNA molecule obtained by inserting a base G into the nucleotide between the 350 th and 351 th nucleotides in the sequence 3 in the sequence table and keeping other nucleotide sequences in the sequence 3 unchanged;
5) the MsGA3ox1 gene shown in the sequence 3 in the sequence table is mutated into an MsGA3ox1/-8bp-L15 gene, and the MsGA3ox1/-8bp-L15 gene is a DNA molecule obtained by deleting 8 bases in total at the 351-fold 358 th nucleotide of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
6) the MsGA3ox1 gene shown in the sequence 3 in the sequence table is mutated into an MsGA3ox1/-7bp gene, and the MsGA3ox1/-7bp gene is a DNA molecule obtained by deleting 7 bases in total from the 351-357 th nucleotide of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
7) the MsGA3ox1 gene shown in the sequence 3 in the sequence table is mutated into an MsGA3ox1/-72bp gene, and the MsGA3ox1/-72bp gene is a DNA molecule obtained by deleting 72 bases in total at the 351-422 th site of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged;
m2, carrying out at least one mutation on the MtGA3ox1 gene shown in a sequence 4 in a sequence table:
8) the MtGA3ox1 gene shown in a sequence 4 in a sequence table is mutated into an MtGA3ox1/+1bp-L4 gene, the MtGA3ox1/+1bp-L4 is protein coded by an MtGA3ox1/+1bp-L4 gene, and the MtGA3ox1/+1bp-L4 gene is a DNA molecule obtained by adding a base C between the 255 th and 256 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
9) the MtGA3ox1 gene shown in the sequence 4 in the sequence table is mutated into an MtGA3ox1/-10bp gene, the MtGA3ox1/-10bp gene is protein coded by the MtGA3ox1/-10bp gene, and the MtGA3ox1/-10bp gene is a DNA molecule obtained by deleting 10 bases of the nucleotides at the 330 th and the 339 th positions of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
10) the MtGA3ox1 gene shown in the sequence 4 in the sequence table is mutated into an MtGA3ox1/-19bp gene, the MtGA3ox1/-19bp gene is protein coded by the MtGA3ox1/-19bp gene, and the MtGA3ox1/-19bp gene is DNA molecule obtained by deleting 19 bases in total from the 329-th and 347-th nucleotides in the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
11) the MtGA3ox1 gene shown in the sequence 4 in the sequence table is mutated into MtGA3ox1/+2bp gene, the MtGA3ox1/+2bp is protein coded by the MtGA3ox1/+2bp gene, the MtGA3ox1/+2bp gene is DNA molecule obtained by inserting two basic groups GA between the nucleotides of the 331 st-332 th-sequence of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
12) the MtGA3ox1 gene shown in the sequence 4 in the sequence table is mutated into an MtGA3ox1/-216bp gene, the MtGA3ox1/-216bp gene is protein coded by the MtGA3ox1/-216bp gene, and the MtGA3ox1/-216bp gene is a DNA molecule obtained by deleting 216 bases of the nucleotides at the 169 th and 384 th positions of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged;
13) the MtGA3ox1 gene shown in the sequence 4 in the sequence table is mutated into an MtGA3ox1/+1bp-L11 gene, the MtGA3ox1/+1bp-L11 is protein coded by an MtGA3ox1/+1bp-L11 gene, and the MtGA3ox1/+1bp-L11 gene is DNA molecule obtained by adding a base G between the 331 st and 332 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged.
The invention provides a protein or a related biological material thereof, wherein the protein is the protein, and the biological material is the biological material.
The invention aims to provide a method for cultivating dwarfed plant type and/or compact plant type and/or creeping plant type alfalfa by utilizing a gene editing technology. Genetic transformation is carried out on a CRISPR/Cas9 gene editing vector carrying a targeted GA3ox1 gene in an agrobacterium-mediated manner, the genetic transformation is introduced into a receptor plant, a transgenic positive strain is obtained, materials with all alleles of GA3ox1 mutated are identified in the genetic transformation vector, and the phenotype is dwarfed and/or compact and/or creeping. Experiments prove that in medicago truncatula, compared with wild type, the plant type of the plant subjected to MtGA3ox1 gene knockout is creeping, the plant height, the length of the main root, the dry weight of the overground part and the root are reduced, the average root nodule number of each plant is reduced, but the azotobacter activity has no obvious difference; in alfalfa, the MsGA3ox1 knockout plant has a reduced plant height and reduced internode length compared to wild-type plants.
Drawings
FIG. 1 is a vector structure diagram of Medicago truncatula expression vector p6401-MtGA3ox 1.
FIG. 2 shows the results of identification of the Mtga3ox1 mutant; FIG. a shows the result of identifying a transgenic plant obtained by transferring a p6401-MtGA3ox1 vector into Medicago truncatula through mediation of Agrobacterium EHA105, wherein M represents DNA standard molecular weight, WT represents wild-type material, the positive control "+" is a plasmid p6401-MtGA3ox1, and the negative control "-" is ddH2O, serial numbers 1-4 are genetic transformation regeneration plants, and the results show that: 1-4 samples were regenerated plants transformed with p6401-MtGA3ox1 vector; FIG. b shows the genotype identification result of the transgenic plant target gene MtGA3ox1 obtained by transferring p6401-MtGA3ox1 vector into Medicago truncatula through mediation of Agrobacterium EHA105, wherein M represents DNA standard molecular weight, WT represents wild type material, and negative control "-" is ddH2O, serial numbers 1-4 are genetic transformation regeneration plants, and the results show that: 1-4 samples all have amplification bands, and are all positive transgenic plants obtained by transferring p6401-MtGA3ox1 vector into medicago truncatula through mediation of agrobacterium EHA 105.
FIG. 3 shows the result of an exemplary analysis of the mutation pattern of the positive transgenic plant Mtga3ox1 mutant obtained by Agrobacterium EHA105 mediated transfer of the p6401-Mtga3ox1 vector into Medicago truncatula.
FIG. 4 shows the growth of Medicago truncatula WT, Mtga3ox1 mutant material grown under 1/2MS culture conditions for five weeks; the overall morphology is shown in figure a, the plant height (figure b), the main root length (figure c), the dry weight of the overground part (figure d) and the dry weight of the root part (figure e) are detected, and the results show that: 1/2MS culture conditions, the plant height, the length of the main root, the dry weight of the overground part and the dry weight of the root of the Mtga3ox1 mutant are all obviously lower than the wild type.
FIG. 5 shows the growth of Medicago truncatula WT, Mtga3ox1 mutant material inoculated with Sinorhizobium japonicum 21d under nitrogen-free culture conditions, the plant morphology is shown as graph a, and the plant height (graph b), the length of main root (graph c), the dry weight of overground part (graph d), the dry weight of root (graph e), the average Rhizobium number per plant (graph f) and the azotobacter activity (graph g) are counted.
FIG. 6 shows the structure of the vector of alfalfa p6401-MsGA3ox 1.
FIG. 7 shows the result of electrophoresis detection of transgenic plants obtained by transferring p6401-MsGA3ox1 vector into alfalfa.
FIG. 8 shows the result of an exemplary analysis of the mutation pattern of the MsGA3ox1 mutant of a positive transgenic plant obtained by mediating the transfer of the p6401-MsGA3ox1 vector into alfalfa through Agrobacterium EHA 105.
FIG. 9 shows the alfalfa WT, Msga3ox1 mutant material phenotype. Growing for six weeks under 1/2MS culture conditions, wherein the plant morphology is shown in figure a and figure b, and the plant height (figure c) and internode length (figure d) are counted; exogenously applied 0.1mM GA4The Msga3ox1 mutant phenotype is shown in FIG. e.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The medicago truncatula R108 is a wild-type material preserved in the laboratory, and the medicago truncatula R108, a p6401 vector and a p5 CBC: described in "ZHU, F., YE, Q., CHEN, H., DONG, J. & WANG, T.2021.multigene recording results at MtCEP1/2/12 reduced regulated linear and non-number in medical guide project. J Exp Bot., the article is publicly available from the applicant and can be used only for the experiments of the instant invention.
The formula of the nitrogen-free culture medium is (1L): MgSO (MgSO)4·7H2O(0.25M)2mL，KH2PO4·2H2O(0.7M)1mL，NaH2PO4·2H2O(0.2M)4mL，Fe-EDTA·2Na(10mM)5mL，MnSO4(1mg/mL)100μL，CuSO4(1mg/mL)100μL，ZnSO4(1mg/mL)100μL，H3BO3(1mg/mL)100μL，NaMoO4(1mg/mL) 100. mu.L. Sterilizing at 121 deg.C for 22min, cooling to 50 deg.C, adding 1mL filter sterilized CaCl2Solution (1M).
1/2 the formula of MS culture medium is (1L): MURASHIGE&SKOOG(MS)BASAL MEDIUM(M519)(Phyto Technology LaboratoriesTM)2.2 g. 20g of sucrose and 8g of agar were added to the solid medium, and the pH was adjusted to 5.85.
Bulk mother liquor (1L) 10 xn 6: MgSO (MgSO)4·7H2O 1.85g，KNO3 28.3g，(NH4)2SO4 4.63g，CaCl2·2H2O 1.66g，KH2PO4 4g。
1000 × SH minimal mother liquor (100 mL): MnSO4·H2O 1g，H3BO3 500mg，ZnSO4·7H2O 100mg，KI 100mg，Na2MoO4·2H2O 10mg，CuSO4·5H2O 20mg，CoCl2·6H2O 10mg。
1000 × SH organic mother liquor (100 mL): 500mg of nicotinic acid, 500mg of pyridoxine hydrochloride and 500mg of thiamine hydrochloride.
50 XEDFS iron salt mother liquor (500 mL): 3.487g of NaFe EDTA.
The formula of SH3 alpha culture medium (1L) is as follows: 100mL of 10 XN 6 bulk mother liquor, 1mL of 1000 XSH trace mother liquor, 1mL of 1000 XSH organic mother liquor, 20mL of 50 XEDFS iron salt mother liquor, 0.4mL of 2, 4-D stock solution (10mg/mL), 0.5mL of 6-BAP stock solution (1mg/mL), 100mg of inositol, 30g of sucrose, and the pH is adjusted to 5.85. 3.2g of plant gel was added to the solid medium.
The formula of SH9 culture medium (1L) is as follows: 100mL of 10 XN 6 bulk mother liquor, 1mL of 1000 XSH micro mother liquor, 1mL of 1000 XSH organic mother liquor, 20mL of 50 XEDFS ferric salt mother liquor, 100mg of inositol and 20g of sucrose, and the pH is adjusted to 5.85. To the solid medium was added 8g of agar.
The formulation of SM4 medium (1L) was: MURASHIGE&SKOOG(MS)BASAL MEDIUM(M519)(Phyto Technology LaboratoriesTM)4.43g, 0.4mL of 2, 4-D stock solution (10mg/mL), 0.2mL of 6-BAP stock solution (1mg/mL), 30g of sucrose, and pH adjusted to 5.85. 3.2g of plant gel was added to the solid medium.
The formulation of MSBK medium (1L) was: MURASHIGE&SKOOG(MS)BASAL MEDIUM(M519)(Phyto Technology LaboratoriesTM)4.43g, 1mL of kinetin (1mg/mL), 0.5mL of 6-BAP stock solution (1mg/mL), 30g of sucrose, and pH adjusted to 5.85. 3.2g of plant gel was added to the solid medium.
The following examples use SPSS16.0 statistical software to process the data and the results are presented as mean ± standard deviation, with Kruskal-Wallis, nonimpatric test, P < 0.05 (x) for significant differences, P < 0.01 (x) for very significant differences and P < 0.001 (x) for very significant differences.
TABLE 1 primers
Figure BDA0003193752660000091
Example 1 acquisition and phenotypic testing of Medicago truncatula MtGA3ox1 knockout mutants
1. Vector p6401-MtGA3ox1 construction
Design of Gene editing target
The medicago truncatula R108 contains a coding sequence (CDS) which is a MtGA3ox1 coding gene shown in a sequence 4 in a sequence table, and the coding amino acid sequence is protein MtGA3ox1 shown in a sequence 2 in the sequence table.
Predicting websites through online targets (http://crispor.tefor.net/) Generate a list of targets, select target 1 to be 5'-ACACCATCTGGGGAACGAA-3' and target 2 to be 5'-AAGCCATTCGTTCCCCAGA-3', as shown in FIG. 1.
Construction of sgRNA Module
As shown in Table 1, primers MtGA3ox1-BsF, MtGA3ox1-F0, MtGA3ox1-R0, MtGA3ox1-BsF and MtGA3ox1-BsR were synthesized by Huada Gene Co.
Carrying out p5CBC-Target amplification reaction by bridging PCR, and amplifying to obtain a sgRNA module p5CBC-MtGA3ox1 with a Target sequence, wherein the reaction system is as follows:
Figure BDA0003193752660000092
the reaction procedure is as follows: a first round: denaturation at 94 deg.C for 2 min; and a second round: denaturation at 94 ℃ for 15sec, annealing at 60 ℃ for 30sec, extension at 68 ℃ for 1min, 30 cycles; and a third round: extension at 68 ℃ for 5 min. After the reaction is finished, detecting the product by 1% agarose gel electrophoresis, cutting a gel block where the target fragment is located, and recovering the fragment by using a gel recovery kit, wherein the size of the product fragment is 841 bp.
The gel recovered fragment is subjected to sample sequencing, and the sequence of the p5CBC-MtGA3ox1 is shown as the sequence 6 in the sequence table. The 18 th to 36 th positions of SEQ ID No.6 are the target 1 sequence of MtGA3ox1, and the 806 nd and 824 th positions are the target 2 sequence of MtGA3ox 1.
Construction of binary vector for gene editing
Golden Gate enzyme digestion ligation reaction: the p5CBC-MtGA3ox1 fragment with the target sequence and the p6401 vector were cut by Bsa I and ligated by T4 DNA Ligase to form p6401-MtGA3ox1 (see FIG. 1). The specific reaction system is as follows:
Figure BDA0003193752660000101
the reaction procedure is as follows: the first step is as follows: 5h at 37 ℃; the second step is that: 50 ℃ for 5 min; the third step: 80 ℃ for 10 min.
After the reaction is completed, the product is added into escherichia coli TOP10 competent cells, ice bath is carried out for 30min, heat shock is carried out for 90s at 42 ℃, LB liquid culture medium without adding antibiotics is added, shaking culture is carried out at 37 ℃ and 150rpm for 45min, then bacterial liquid is coated on LB solid culture medium containing Kanamycin (Kanamycin, 50mg/L), and dark inversion screening culture is carried out at 37 ℃ for 12 h. Single clones were picked to 400. mu.L of LB liquid medium containing Kanamycin (Kanamycin, 50mg/L) and shake-cultured at 37 ℃ and 230rpm for 6-8 h, followed by PCR identification of the bacterial liquid with primers MtGA3ox1-BsF and MtGA3ox1-BSR for amplification, and the positive clones had a specific band with a size of 841 bp. Positive cloning plasmids were extracted and verified by sequencing.
Sequencing results show that the p6401-MtGA3ox1 is a recombinant expression vector obtained by replacing the fragment between BsaI enzyme cutting sites of the vector p6401 with the base sequence shown in the 14 th-824 th sites of the fragment p5CBC-MtGA3ox1 and keeping other sequences of the vector p6401 unchanged. p6401-MtGA3ox1 expressed two sgrnas targeting sgRNA1 (target 1) and sgRNA2 (target 2) of the MtGA3ox1 gene.
2. Obtaining of MtGA3ox1 gene knockout mutant of medicago truncatula
2.1 transformation of Agrobacterium EHA105
A monoclonal colony of Agrobacterium EHA105 was picked, inoculated into 5mL of YEP medium, and cultured overnight at 28 ℃ with shaking in an incubator at 230 rpm. Transferring the bacterial liquid to 200mL YEP culture solution according to the proportion of 1:100, performing shaking culture at 28 ℃ and 230rpm until the bacterial liquid is OD600nm0.4-0.6. The suspension was transferred to 2 sterilized 250mL centrifuge bottles, centrifuged at 24 ℃ and 4000rpm for 10min, the supernatant was discarded in a clean bench, 150mL of pre-cooled sterilized MilliQ water was added to each suspension, and the procedure was repeated 2-3 times. After centrifugation at 5000rpm for 5min at 4 ℃ and supernatant discarded on a super clean bench, 2mL each of precooled MilliQ water containing 10% glycerol was added and the pellet was gently suspended. 200 mul of agrobacterium suspension per tube is subpackaged in a 1.5mL sterile eppendorf tube, frozen by liquid nitrogen and then frozen at-80 ℃.
To 200. mu.L of EHA105 competent cells thawed on ice, 0.5. mu.L (100 ng/. mu.L) of p6401-MtGA3ox1 plasmid was added, gently whipped and mixed well. Competence was transferred to a pre-cooled electric shock cup, 500. mu.L of pre-cooled blank YEP liquid medium was immediately added after 2100V electric shock, after mixing well by blowing, the solution in the electric shock cup was transferred to a 1.5mL centrifuge tube, cultured at 28 ℃ for 45min with shaking at 200 rpm. The pipette gun pipetted 50. mu.L of the inoculum spread on YEP solid medium containing 50. mu.g/mL Kan and 75. mu.g/mL rifampicin and incubated at 28 ℃ for about 48 h. A single colony on the YEP solid medium was picked, inoculated into a YEP medium containing the corresponding antibiotic, and cultured overnight at 28 ℃ with shaking at 230 rpm. Selecting clone to carry out PCR identification of bacterial liquid, wherein primers are MtGA3ox1-BsF and MtGA3ox1-BSR, positive clone has a specific band with the size of 841bp, and the detection result shows that the vector p6401-MtGA3ox1 has successfully transformed agrobacterium EHA105, and the recombinant agrobacterium tumefaciens is named as EHA105/p6401-MtGA3ox1 for the next infection.
2.2, EHA105/p6401-MtGA3ox1 Agrobacterium infection of Medicago truncatula
Placing the medicago truncatula R108 seeds in a 2mL centrifuge tube, treating the medicago truncatula R108 seeds for 8min by 1mL concentrated sulfuric acid, washing the medicago truncatula R108 seeds for 6 times by sterile water, adding 1mL of 0.5% sodium hypochlorite solution for disinfection for 12min, washing the medicago truncatula R108 seeds for 7 times by sterile deionized water in an ultra-clean bench, inoculating the medicago truncatula R108 seeds on a sterile 0.8% water agar culture medium, culturing the medicago truncatula R in the dark at 4 ℃ for 3d, culturing the medicago truncatula R108 seeds in the dark at 24 ℃ for 1d, and transferring the medicago truncatula R108 seeds to an 1/2MS culture medium for culturing for four weeks. The leaves of medicago truncatula are cut by a blade, cut into squares by sterile tweezers and a knife and placed in a glass vessel.
According to the following steps: transferring EHA105/p6401-MtGA3ox1 bacterial liquid into 20ml YEP liquid culture medium containing corresponding antibiotics at a ratio of 50, performing shaking culture at 28 ℃ and 230rpm until OD is reached600nmIs 0.6-0.8. Centrifuging at 24 deg.C and 3000rpm for 10min, discarding supernatant in ultra-clean bench, and suspending and precipitating with 100mL SH3 alpha (containing 100 μ M acetosyringone) to obtain staining solution.
Soaking the cut leaves in the infection solution, mixing well, vacuumizing, -0.1pka, and 30 min. The mixture was treated at 24 ℃ and 80rpm in the dark for 1.5 h. Taking out the leaf in a super clean bench, dipping redundant infection solution on sterilized filter paper, transferring to SH3 alpha solid culture medium containing 100 mu M acetosyringone and covered with filter paper on the surface, and culturing in dark for 3 d. The leaves were transferred to a new SH3 alpha solid medium (containing 200mg/L carbenicillin and 10mg/L hygromycin) with tweezers, and cultured in the dark for 6 weeks, and transferred to a new SH3 alpha solid medium containing the corresponding antibiotic every 2 revolutions. After 6 weeks of culture, the calli were transferred to SH9 solid medium (containing 200mg/L carbenicillin and 10mg/L hygromycin) and incubated under light for 9 weeks, during which time the calli were transferred to new SH9 medium containing the corresponding antibiotic every 3 weeks. Transferring the grown and shaped plants to 1/2MS culture medium, transferring to new 1/2MS culture medium every 3 weeks, transferring the plants to a greenhouse for culture after rooting, and collecting seeds after positive seedlings are identified.
2.3 detection of MtGA3ox1 Gene knockout mutant plants
And detecting positive plants of MtGA3ox1 gene knockout mutants by using DNA level. Lifting deviceTaking the genome DNA of the knockout mutant plant as a template, and taking MtGA3ox1-BsF and MtGA3ox1-BSR as primers to carry out amplification reaction. Wild type plant genomic DNA and ddH2O is a negative control. The reaction system is as follows:
Figure BDA0003193752660000111
the amplification reaction procedure was: a first round: denaturation at 95 deg.C for 5 min; and a second round: denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min, 34 cycles; and a third round: extension at 72 ℃ for 10 min. After the procedure was completed, the results were checked by electrophoresis on 1.0% agarose gel.
As shown in a in FIG. 2, the plants which can amplify specific p6401-MtGA3ox1 carrier segment (841bp) are transgenic positive plants.
T1 generation plants are taken as a template, MtGA3ox1-F and MtGA3ox1-R are taken as primers, amplification reaction is carried out, an amplification band (shown as b in figure 2) is cut, sequencing is carried out by Huada gene company, and the mutation mode of the MtGA3ox1 mutant is determined (shown as figure 3).
3. Phenotypic detection of MtGA3ox1 gene knockout mutant
Mtga3ox1-1 mutant T1The generation positive plants (FIG. 3) were subjected to phenotypic testing under total nutrient conditions. Wild type (R108) and T1Placing seeds of the generation positive plants in a 2mL centrifuge tube, treating the seeds with 1mL concentrated sulfuric acid for 8min, washing the seeds with sterile water for 6 times, adding 1mL of 0.5% sodium hypochlorite solution for disinfection for 12min, washing the seeds with sterile deionized water for 7 times in an ultra-clean bench, inoculating the seeds on a sterile 0.8% water agar culture medium, carrying out dark culture at 4 ℃ for 3d, carrying out dark culture at 24 ℃ for 1d, transferring the seeds to an 1/2MS culture medium for five weeks, and detecting the plant heights, the main root lengths, the overground parts and the dry roots of the wild type and mutant plants. The results are shown in fig. 4, where the mutant plants were significantly lower in plant height, main root length, aerial parts and dry root weight than the wild type plants.
T1And (5) carrying out phenotype detection on the generation positive plants under a nitrogen-free condition. Wild type (R108) and Mtga3ox1-1 mutant T1Placing the seeds of the plants with positive generation in a 2mL centrifuge tube, treating with 1mL concentrated sulfuric acid for 8min, and washing with sterile waterWashing for 6 times, adding 1mL of 0.5% sodium hypochlorite solution, sterilizing for 12min, washing for 7 times in a super clean bench with sterile deionized water, inoculating on sterile 0.8% water agar culture medium, culturing in dark at 4 deg.C for 3d, culturing in dark at 24 deg.C for 1d, and transferring to nitrogen-deficient culture medium for one week. Activating the Sinorhizobium strain Sm1021, picking the monoclonal colony in 5mL TY culture solution, shaking and culturing at 28 ℃, 230rpm for 2d, 1: transferring 50 percent of the culture solution to 100mL of TY culture solution, performing shaking culture at 28 ℃ and 230rpm until the culture solution reaches OD600nmUp to 1.0. Centrifuging at room temperature 4000rpm for 10min, discarding supernatant, and resuspending the thallus to OD with sterile deionized water600nmUp to 0.05. Inoculating 15mL of the heavy suspension liquid to each plant, and after 21d, detecting the plant height, the main root length, the dry weight of the overground part, the dry weight of the root, the average rhizobium number of each plant and the azotobacter activity of the wild type and mutant plants. The results are shown in FIG. 5, and the plant height, main root length, overground dry weight, root dry weight and average rhizobium number of the Mtga3ox1-1 mutant are all significantly lower than those of the wild type plant, but the azotobacter activity is not significantly different. Compared with wild plants, the Mtga3ox1-1 mutant plant has about 70% lower average plant height, about 20% lower overground biomass and about 40% lower main root length and underground biomass.
The mutant plants of the stolon phenotype correspond to the following gene mutants:
compared with the wild type medicago truncatula R108, the biallelic homozygous mutant plant with the MtGA3ox1/+1bp genotype has the advantages that for the MtGA3ox1 gene, the MtGA3ox1 gene in two chromosomes is mutated into the MtGA3ox1/+1bp gene, the MtGA3ox1/+1bp gene is a DNA molecule obtained by adding one base C between the 255 th and 256 th nucleotides of an MtGA3ox1 coding sequence (a sequence 4 in a sequence table) and keeping other nucleotide sequences of the sequence 4 unchanged, and the DNA molecule is named as MtGA3ox1/+1 bp-L4; the coding sequence of the MtGA3ox1/+1bp-L4 gene is a DNA molecule obtained by adding a base C between the 255 th and 256 th nucleotides of the sequence 4 in a sequence table and keeping other nucleotide sequences of the sequence 4 unchanged, and the coded protein is MtGA3ox1/+1bp-L4 [ Mt GA3ox1-4 ] in figure 3.
Compared with the wild-type medicago truncatula R108, the heterozygous mutant plant of the allele with the genotype of MtGA3ox1/-10bp/-19bp has the advantages that for the MtGA3ox1 gene, the MtGA3ox1 gene in one chromosome is mutated into the MtGA3ox1/-10bp gene, and the MtGA3ox1/-10bp gene is a DNA molecule obtained by deleting 10 bases in the 330 th and 339 bits of the MtGA3ox1 coding sequence (the sequence 4 in a sequence table) and keeping other nucleotide sequences of the sequence 4 unchanged; the coding sequence of the MtGA3ox1/-10bp gene is a DNA molecule obtained by deleting 10 bases of the nucleotides at the 330 nd-339 th site of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged, and the coded protein is MtGA3ox1/-10 bp; the MtGA3ox1 gene in one chromosome is mutated into the MtGA3ox1/-19bp gene, the MtGA3ox1/-19bp gene is a DNA molecule obtained by deleting 19 bases in the 329-; the coding sequence of the MtGA3ox1/-19bp gene is a DNA molecule obtained by deleting 19 bases of the 329 th and 347 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged, and the coded protein is MtGA3ox1/-19bp [ MtGA3ox1-1 ] in figure 3.
Compared with the wild-type medicago truncatula R108, the MtGA3ox1 gene of the heterozygote mutant plant of the allele with the genotype of MtGA3ox1/+2bp/-216bp has the advantages that the MtGA3ox1 gene in one chromosome is mutated into the MtGA3ox1/+2bp gene, and the MtGA3ox1/+2bp gene is a DNA molecule obtained by inserting two basic groups GA between the 331 st and 332 rd nucleotides of the MtGA3ox1 coding sequence (sequence 4 in a sequence table) and keeping other nucleotide sequences of the sequence 4 unchanged; the coding sequence of the MtGA3ox1/+2bp gene is a DNA molecule obtained by inserting two bases GA between the 331 st-332 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged, and the coded protein is MtGA3ox1/+2 bp; the MtGA3ox1 gene in one chromosome is mutated into MtGA3ox1/-216bp gene, the MtGA3ox1/-216bp gene is DNA molecule obtained by deleting 216 bases of the 169 th 384 th nucleotide of the MtGA3ox1 coding sequence (sequence 4 in the sequence table) and keeping other nucleotide sequences of the sequence 4 unchanged; the coding sequence of the MtGA3ox1/-216bp gene is a DNA molecule obtained by deleting 216 bases of the 169 th and 384 th nucleotides of the sequence 4 in the sequence table and keeping other nucleotide sequences of the sequence 4 unchanged, and the coded protein is MtGA3ox1/-216bp [ Mt GA3ox1-10 ] in figure 3.
Compared with the wild type medicago truncatula R108, the biallelic homozygous mutant plant with the MtGA3ox1/+1bp genotype has the advantages that for the MtGA3ox1 gene, the MtGA3ox1 gene in two chromosomes is mutated into the MtGA3ox1/+1bp gene, the MtGA3ox1/+1bp gene is a DNA molecule obtained by adding one base G between the 331 rd and 332 th nucleotides of the MtGA3ox1 coding sequence (sequence 4 in a sequence table) and keeping other nucleotide sequences of the sequence 4 unchanged, and the DNA molecule is named as MtGA3ox1/+1 bp-L11; the coding sequence of the MtGA3ox1/+1bp-L11 gene is a DNA molecule obtained by adding a base G between the 331 st and 332 th nucleotides of the sequence 4 in the sequence table and keeping the other nucleotide sequences of the sequence 4 unchanged, and the coded protein is MtGA3ox1/+1bp-L11 [ Mt GA3ox1-11 in figure 3 ].
Example 2 obtaining and phenotypic testing of MsgA3ox1 knockout mutants of alfalfa
1. Alfalfa MsGA3ox1 gene CRISPR/Cas9 system vector construction
Design of Gene editing target
The alfalfa contains a coding sequence (CDS) which is an MsGA3ox1 coding gene shown in a sequence 3 in a sequence table, and a protein MsGA3ox1 shown in a sequence 1 in the sequence table.
Predicting websites through online targets (http://crispor.tefor.net/) And generating a target point list, and selecting target point 1 as 5 ' -ACACCATCCGGGGAACGAA-3 and target point 2 as 5'-AAGCCATTCGTTCCCCGGA-3'.
Construction of sgRNA Module
Primers MsGA3ox1-BsF, MsGA3ox1-F0, MsGA3ox1-R0 and MsGA3ox1-BsR were synthesized by Huada Gene Co.
Carrying out p5CBC-Target amplification reaction by bridging PCR, and amplifying to obtain a sgRNA module p5CBC-MsGA3ox1 with a Target sequence, wherein the reaction system is as follows:
Figure BDA0003193752660000131
the reaction procedure is as follows: a first round: denaturation at 94 deg.C for 2 min; and a second round: denaturation at 94 ℃ for 15sec, annealing at 60 ℃ for 30sec, extension at 68 ℃ for 1min, 30 cycles; and a third round: extension at 68 ℃ for 5 min. After the reaction is finished, detecting the product by 1% agarose gel electrophoresis, cutting a gel block where the target fragment is located, and recovering the fragment by using a gel recovery kit, wherein the size of the product fragment is 841 bp.
The gel recovery fragment is sent to be sequenced, and the sequence of the p5CBC-MsGA3ox1 is shown as the sequence 5 in the sequence table. The 18 th to 36 th positions of SEQ ID No.5 are the target 1 sequence of MsGA3ox1, and the 806 th and 824 th positions are the target 2 sequence of MsGA3ox 1.
Construction of binary vector for gene editing
Golden Gate enzyme digestion ligation reaction: the p5CBC-MsGA3ox1 fragment with the target sequence and the p6401 vector were cleaved with Bsa I and ligated with T4 DNALigase to construct p6401-MsGA3ox1 (see FIG. 6). The specific reaction system is as follows:
Figure BDA0003193752660000141
the reaction procedure is as follows: the first step is as follows: 5h at 37 ℃; the second step is that: 50 ℃ for 5 min; the third step: 80 ℃ for 10 min.
After the reaction is completed, the product is added into escherichia coli TOP10 competent cells, ice bath is carried out for 30min, heat shock is carried out for 90s at 42 ℃, LB liquid culture medium without adding antibiotics is added, shaking culture is carried out at 37 ℃ and 150rpm for 45min, then bacterial liquid is coated on LB solid culture medium containing Kanamycin (Kanamycin, 50mg/L), and dark inversion screening culture is carried out at 37 ℃ for 12 h. Single clones are picked to 400 mu L LB liquid culture medium containing Kanamycin (Kanamycin, 50mg/L) and are subjected to shake-flask culture at 37 ℃ and 230rpm for 6-8 h, then bacterial liquid PCR identification is carried out, primers are MsGA3ox1-BsF and MsGA3ox1-BSR for amplification, and positive clones have specific bands with the size of 841 bp. Positive cloning plasmids were extracted and verified by sequencing.
Sequencing results show that the p6401-MsGA3ox1 is a recombinant expression vector obtained by replacing the fragment between BsaI enzyme cutting sites of the vector p6401 with the base sequence shown in the 14 th-824 th sites of the fragment p5CBC-MsGA3ox1 and keeping other sequences of the vector p6401 unchanged. p6401-MsGA3ox1 expressed two sgrnas targeting sgRNA1 (target 1) and sgRNA2 (target 2) of the MsGA3ox1 gene. The 18 th to 36 th positions of the SEQ ID No.5 are coding sequences of sgRNA1, and the 806 th and 824 th positions are coding sequences of sgRNA 2.
2. Obtaining of alfalfa MsGA3ox1 gene knockout mutant
2.1 transformation of Agrobacterium EHA105
A monoclonal colony of Agrobacterium EHA105 was picked, inoculated into 5mL of YEP medium, and cultured overnight at 28 ℃ with shaking in an incubator at 230 rpm. Transferring the bacterial liquid to 200mL YEP culture solution according to the proportion of 1:100, performing shaking culture at 28 ℃ and 230rpm until the bacterial liquid is OD6000.4-0.6. The suspension was transferred to 2 sterilized 250mL centrifuge bottles, centrifuged at 24 ℃ and 4000rpm for 10min, the supernatant was discarded in a clean bench, 150mL of pre-cooled sterilized MilliQ water was added to each suspension, and the procedure was repeated 2-3 times. After centrifugation at 5000rpm for 5min at 4 ℃ and supernatant discarded on a super clean bench, 2mL each of precooled MilliQ water containing 10% glycerol was added and the pellet was gently suspended. 200 mul of agrobacterium suspension per tube is subpackaged in a 1.5mL sterile eppendorf tube, frozen by liquid nitrogen and then frozen at-80 ℃.
To 200. mu.L of EHA105 competent cells thawed on ice, 0.5. mu.L (100 ng/. mu.L) of p6401-MsGA3ox1 plasmid was added, gently whipped and mixed well. Competence was transferred to a pre-cooled electric shock cup, 500. mu.L of pre-cooled blank YEP liquid medium was immediately added after 2100V electric shock, after mixing well by blowing, the solution in the electric shock cup was transferred to a 1.5mL centrifuge tube, cultured at 28 ℃ for 45min with shaking at 200 rpm. The liquid transfer gun aspirates 50. mu.L of the bacterial solution, spreads it on YEP solid medium containing Kan 50. mu.g/mL and rifampicin 75. mu.g/mL, and incubates it at 28 ℃ for about 48 hours. A single colony on the YEP solid medium was picked, inoculated into a YEP medium containing the corresponding antibiotic, and cultured overnight at 28 ℃ with shaking at 230 rpm. Selecting clones to carry out PCR identification on bacterial liquid, wherein primers are MsGA3ox1-BsF and MsGA3ox1-BSR, positive clones have specific bands with the size of 841bp, and detection results show that p6401-MsGA3ox1 vectors have successfully transformed agrobacterium EHA105, and the recombinant agrobacterium tumefaciens is named as EHA105/p6401-MsGA3ox1 to be used for the next infection.
2.2 infection of alfalfa by EHA105/p6401-MsGA3ox1 Agrobacterium
According to a ratio of 1:1000In the example, EHA105/p6401-MsGA3ox1 was inoculated into 200mL YEP culture medium (50. mu.g/mL Kan and 75. mu.g/mL rifampicin), cultured at 28 ℃ and 230rpm to OD600nmIs 0.4. Transferring EHA105/p6401-MsGA3ox1 bacterial solution to sterile 250mL centrifuge bottle, centrifuging at 24 deg.C and 4000g for 10min, discarding supernatant in ultra-clean bench, suspending the bacterial strain in SM4 (containing 100 μ M acetosyringone) liquid culture medium, adjusting OD600nmTo 0.2-0.4, as an invasion solution.
Soaking the leaves collected the previous day in 0.1% Tween 20 solution, treating for 5min, transferring to a sterile empty glass bottle, washing with deionized water in a super clean bench for 3 times, and transferring to a blue-cap bottle. Rinsing with 75% ethanol for 15s, discarding the excessive liquid, adding 30% bleaching water, treating for 10min, and rinsing with deionized water for 3 times.
The disinfected leaves are transferred to a sterile empty glass bottle, and the invasion dye solution is added. Vacuumizing for 5min, and performing ultrasonic treatment for 3 min; vacuumizing for 5 min. Removing the staining solution in a super clean bench, removing redundant liquid from the leaves by using filter paper, drying the leaves, cutting the leaves into small pieces by using a blade, flatly paving the leaves on an SM4 solid culture medium, and culturing for 3d in the dark at room temperature. Transferring to SM4 solid medium (containing 200mg/L of cephalosporin and 10mg/L of hygromycin), culturing at 24 ℃ in light, transferring to new SM4 medium every 2 turnovers, growing callus, transferring the callus to MSBK regeneration medium (containing 200mg/L of timentin and 10mg/L of hygromycin) for 2-3 weeks, and transferring to SH9 medium (containing 200mg/L of cephalosporin and 5mg/L of hygromycin). And transferring the grown plants to 1/2MS culture medium, rooting, transferring to a greenhouse for culture, and identifying positive seedlings.
2.3 detection of MsGA3ox1 Gene knockout mutant plants
Detecting the positive plants of the MsGA3ox1 gene knockout mutant at the DNA level. Extracting genome DNA of the knockout mutant plant, and performing amplification reaction by using the genome DNA as a template and using MsGA3ox1-BsF and MsGA3ox1-BSR as primers. Wild type plant genomic DNA and ddH2O is a negative control. The reaction system is as follows:
Figure BDA0003193752660000151
the amplification reaction procedure was: a first round: denaturation at 95 deg.C for 5 min; and a second round: denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min, 34 cycles; and a third round: extension at 72 ℃ for 10 min. After the end of the procedure, the results were detected by electrophoresis on a 1.0% agarose gel (a in FIG. 7). And (3) taking the regenerated plant genome DNA as a template and MsGA3ox1-F and MsGA3ox1-R as primers, carrying out amplification reaction, cutting an amplification band (shown as b in figure 7), and sending the amplification band to Huada gene company for sequencing to determine the mutation mode of the mutant (shown as figure 8).
The electrophoresis detection result is shown in FIG. 7, wherein a in FIG. 7 is the identification result of the transgenic plant obtained by transferring p6401-MsGA3ox1 vector into alfalfa through mediation of Agrobacterium EHA105, wherein M represents DNA standard molecular weight, WT represents wild type material, positive control "+" is plasmid p6401-MsGA3ox1, and negative control "-" is ddH2O, serial numbers 1-7 are genetic transformation regeneration plants; as shown in a in FIG. 7, the plants which can amplify specific p6401-MsGA3ox1 carrier segment (841bp) are transgenic positive plants, and the results show that: samples 3-7 were regenerated plants transfected with p6401-MsGA3ox1 vector, and samples 1 and 2 were regenerated plants not transfected with p6401-MsGA3ox1 vector.
In FIG. 7, b is the identification result of the gene type of the target gene MsGA3ox1 of the transgenic plant obtained by transferring the p6401-MsGA3ox1 vector into alfalfa through the mediation of Agrobacterium EHA 105; wherein M represents DNA standard molecular weight, WT represents wild-type material, and negative control "-" is ddH2O, serial numbers 1-7 are genetic transformation regeneration plants, 1-7 samples all have amplification bands, and results show that the 1-7 samples are positive transgenic plants obtained by transferring p6401-MsGA3ox1 vectors into alfalfa through mediation of agrobacterium EHA 105.
And (3) taking the regenerated plant genome DNA as a template and MsGA3ox1-F and MsGA3ox1-R as primers, carrying out amplification reaction, cutting an amplification band (shown as b in figure 7), and sending the amplification band to Huada gene company for sequencing to determine the mutation mode of the mutant (shown as figure 8).
3. Phenotypic detection of MsGA3ox1 knockout mutant
Wild type and Msga3ox1-3, Msga3ox1-12 and Msga3ox 1-15T0Performing phenotype detection on 15 mutant plants obtained after cutting generation positive plants under the total nutrient conditionAnd (fig. 9). Alfalfa WT, Msga3ox1 mutant material were grown under 1/2MS culture conditions for six weeks, and plant height and internode length were counted. The Msga3ox1 mutant plants (a in fig. 9 and b in fig. 9) had reduced plant height (c in fig. 9) and reduced internode length (d in fig. 9) compared to the control plants. The average plant height of the wild type is 28.9cm, the average plant height of the mutant strain is 3.0cm, and the average plant height is reduced by 89.6%; the average length of the first internodes of the wild type is 0.9cm, the average length of the first internodes of the mutant is 0.4cm, and the reduction is 55.5%; the average length of the second internodes of the wild type is 2.6cm, the average length of the second internodes of the mutant is 0.7cm, and the reduction is 73.1%; the average length of the wild type third internode is 4.6cm, the average length of the mutant third internode is 0.8cm, and the reduction is 82.6%; the average length of the fourth internode of the wild type is 5.6cm, the average length of the fourth internode of the mutant is 0.5cm, and the reduction is 91.1 percent; the average length of the wild type fifth internode is 6.1cm, the average length of the mutant fifth internode is 0.4cm, and the reduction is 93.4%; the average length of the wild type sixth internode is 5.6 cm; the average length of the wild type seventh internode is 3.6 cm; the average length of the wild type segment VIII is 1.7 cm. The mutant plant has no sixth, seventh and eighth internodes because the plant height and internode length are reduced compared with the wild type.
Exogenous application of 0.1mM plant gibberellin 4 (GA)4) The average plant height and average internode length of the Msga3ox1 mutant plants were restored to the level of wild type plants (e in FIG. 9).
The mutated gene sequence is as follows:
compared with wild alfalfa, the four-equipotential heterozygous mutant plant with the gene type of MsGA3ox1/-8bp/-8bp/-8bp/-36bp has the advantages that for the MsGA3ox1 coding gene, the MsGA3ox1 gene in three chromosomes is mutated into the MsGA3ox1/-8bp gene, the MsGA3ox1/-8bp gene is a DNA molecule obtained by deleting 8 bases in the 350-th and 357-th nucleotides of the MsGA3ox1 coding sequence (sequence 3 in a sequence table) and keeping other nucleotide sequences of the sequence 3 unchanged, and the DNA molecule is named as MsGA3ox1/-8 bp-L3; the coding sequence of the MsGA3ox1/-8bp-L3 gene is a DNA molecule obtained by deleting 8 bases of the 350 th and 357 th nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged, and the coded protein is MsGA3ox1/-8 bp-L3; the MsGA3ox1 gene in one chromosome is mutated into an MsGA3ox1/-36bp gene, and the MsGA3ox1/-36bp gene is a DNA molecule obtained by deleting 36 bases in the 320 th and 355 th nucleotides of an MsGA3ox1 coding sequence (sequence 3 in a sequence table) and keeping other nucleotide sequences of the sequence 3 unchanged; the coding sequence of the MsGA3ox1/-36bp gene is a DNA molecule obtained by deleting 36 bases of the nucleotides at the 320 th and 355 th positions of the sequence 3 in a sequence table and keeping other nucleotide sequences of the sequence 3 unchanged, and the coded protein is MsGA3ox1/-36bp [ MsGA3ox1-3 ] in a picture 8.
Compared with wild alfalfa, the four-equipotential heterozygous mutant plant with the genotype of MsGA3ox1/-9bp/+1bp/+1bp/+1bp, for the MsGA3ox1 coding gene, the MsGA3ox1 gene in one chromosome is mutated into the MsGA3ox1/-9bp gene, and the MsGA3ox1/-9bp gene is a DNA molecule obtained by deleting 9 bases in total from the 351-19 nucleotides of the MsGA3ox1 coding sequence (sequence 3 in a sequence table) and keeping other nucleotide sequences of the sequence 3 unchanged; the coding sequence of the MsGA3ox1/-9bp gene is a DNA molecule obtained by deleting 9 bases in total from the 351-359 th nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged, and the coded protein is MsGA3ox1/-9 bp; MsGA3ox1 gene in three chromosomes is mutated into MsGA3ox1/+1bp gene, the MsGA3ox1/+1bp gene is a DNA molecule obtained by inserting a base G into the nucleotide between the 350 th and the 351 th of the MsGA3ox1 coding sequence (sequence 3 in a sequence table) and keeping other nucleotide sequences of the sequence 3 unchanged; the coding sequence of the MsGA3ox1/+1bp gene is a DNA molecule obtained by inserting a base G into the nucleotide between the 350 th and 351 rd nucleotides of the sequence 3 in the sequence table and keeping the other nucleotide sequences of the sequence 3 unchanged, and the coded protein is MsGA3ox1/+1bp [ MsGA3ox1-12 ] in figure 8 ].
Compared with wild alfalfa, the four-equipotential heterozygous mutant plant with the gene type of MsGA3ox1/+1bp/-8bp/-7bp/+1bp has the advantages that for the MsGA3ox1 coding gene, the MsGA3ox1 gene in two chromosomes is mutated into the MsGA3ox1/+1bp gene, the MsGA3ox1/+1bp gene is a DNA molecule obtained by inserting a base G into the nucleotide between the 350 th and the 351 of the MsGA3ox1 coding sequence (sequence 3 in a sequence table) and keeping other nucleotide sequences of the sequence 3 unchanged; the coding sequence of the MsGA3ox1/+1bp gene is a DNA molecule obtained by inserting a base G into the nucleotide between the 350 th and 351 rd nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged, and the coded protein is MsGA3ox1/+1 bp; the MsGA3ox1 gene in one chromosome is mutated into an MsGA3ox1/-8bp gene, the MsGA3ox1/-8bp gene is a DNA molecule obtained by deleting 8 bases in the 351-th and 358-th nucleotides of an MsGA3ox1 coding sequence (sequence 3 in a sequence table) and keeping other nucleotide sequences of the sequence 3 unchanged, and the DNA molecule is named as MsGA3ox1/-8 bp-L15; the coding sequence of the MsGA3ox1/-8bp-L15 gene is a DNA molecule obtained by deleting 8 bases of the 351-th 358 th nucleotide of the sequence 3 in a sequence table and keeping other nucleotide sequences of the sequence 3 unchanged, and the coded protein is MsGA3ox1/-8 bp-L15; the MsGA3ox1 gene in one chromosome is mutated into an MsGA3ox1/-7bp gene, and the MsGA3ox1/-7bp gene is a DNA molecule obtained by deleting 7 bases in total from the 351-357 th nucleotide of the MsGA3ox1 coding sequence (sequence 3 in a sequence table) and keeping other nucleotide sequences of the sequence 3 unchanged; the coding sequence of the MsGA3ox1/-7bp gene is a DNA molecule obtained by deleting 7 bases in total from the 351-357 th nucleotide of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged, and the coded protein is MsGA3ox1/-7bp [ MsGA3ox1-15 ] in figure 8.
Compared with wild alfalfa, the MsGA3ox1/-72bp/-72bp/WT/WT hybrid plant has the advantages that for the MsGA3ox1 coding gene, the MsGA3ox1 gene in two chromosomes is mutated into the MsGA3ox1/-72bp gene, the MsGA3ox1/-72bp gene is a DNA molecule obtained by deleting 72 bases of the 351-422 bit nucleotide of the MsGA3ox1 coding sequence (sequence 3 in a sequence table) and keeping other nucleotide sequences of the sequence 3 unchanged; the coding sequence of the MsGA3ox1/-72bp gene is a DNA molecule obtained by deleting 72 bases of the 351-422 th nucleotides of the sequence 3 in the sequence table and keeping other nucleotide sequences of the sequence 3 unchanged, and the coded protein is MsGA3ox1/-72 bp; the nucleotide sequence of the MsGA3ox1 coding sequence (sequence 3 in the sequence table) in the alfalfa genome is not changed in the other two chromosomes, and the MsGA3ox1/WT gene is generated. It is noted that the phenotype of the heterozygous mutant is not significantly different from that of the wild type, indicating that the tetra-allelic mutation of MsGA3ox1 in alfalfa is essential for its dwarf phenotype.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Sequence listing
<110> university of agriculture in China
Application of <120> GA3ox1 protein in regulation of alfalfa plant type
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 377
<212> PRT
<213> alfalfa (Medicago sativa)
<400> 1
Met Pro Ser Leu Ser Glu Ala Tyr Arg Ala His Pro Val His Val Asn
1 5 10 15
His Lys His Pro Asp Phe Asn Ser Leu Gln Glu Leu Pro Glu Ser Tyr
20 25 30
Thr Trp Thr His Leu Asp Asp His Thr Leu Ile Asn Ser Asn Asn Thr
35 40 45
Met Lys Glu Ser Ala Asn Ser Ser Ser Val Pro Ile Ile Asp Leu Asn
50 55 60
Asp Pro Asn Ala Ser Lys Leu Ile Gly His Ala Cys Lys Thr Trp Gly
65 70 75 80
Val Tyr Gln Val Val Asn His Gly Ile Pro Ile Ser Leu Leu Asp Glu
85 90 95
Ile Gln Trp Leu Gly Gln Thr Leu Phe Thr Leu Pro Ser His Gln Lys
100 105 110
Leu Lys Ala Ile Arg Ser Pro Asp Gly Val Ser Gly Tyr Gly Leu Ala
115 120 125
Arg Ile Ser Ser Phe Phe Pro Lys Leu Met Trp Ser Glu Gly Phe Thr
130 135 140
Ile Val Gly Ser Pro Leu Asp His Phe Arg Gln Leu Trp Pro Gln Asp
145 150 155 160
Tyr Ala Lys His Cys Asp Thr Val Leu Gln Tyr Asp Glu Ala Met Lys
165 170 175
Lys Leu Ala Gly Lys Leu Met Trp Leu Met Leu Asp Ser Leu Gly Ile
180 185 190
Thr Met Glu Asp Ile Glu Trp Ala Gly Ser Lys Ala Gln Phe Asp Glu
195 200 205
Lys Ala Cys Ala Ala Met Gln Leu Asn Ser Tyr Pro Ser Cys Pro Asp
210 215 220
Pro Asp His Ala Met Gly Leu Ala Pro His Thr Asp Ser Thr Phe Leu
225 230 235 240
Thr Ile Leu Ser Gln Asn Asp Ile Ser Gly Leu Gln Val Gln Arg Glu
245 250 255
Gly Ser Gly Trp Val Asn Val Pro Pro Leu His Gly Gly Leu Val Val
260 265 270
Asn Val Gly Asp Leu Phe His Ile Leu Ser Asn Gly Leu Tyr Thr Ser
275 280 285
Val Leu His Arg Val Leu Val Asn Arg Thr Arg Gln Arg Phe Ser Val
290 295 300
Ala Tyr Leu Tyr Gly Pro Pro Ser Asn Val Glu Ile Cys Pro His Glu
305 310 315 320
Lys Leu Val Gly Pro Thr Gln Pro Pro Leu Tyr Arg Ser Val Thr Trp
325 330 335
Asn Glu Tyr Leu Gly Thr Lys Ala Lys His Phe Asn Lys Ala Leu Ser
340 345 350
Ser Val Ser Leu Cys Ala Pro Ile Asn Gly Leu Phe Asp Val Asn Asp
355 360 365
Ser Asn Lys Ser Ser Val Gln Val Gly
370 375
<210> 2
<211> 371
<212> PRT
<213> Medicago truncatula (Medicago truncatula)
<400> 2
Met Pro Ser Leu Ser Glu Ala Tyr Arg Ala His Pro Val His Val Asn
1 5 10 15
His Lys His Pro Asp Phe Asn Ser Leu Gln Glu Leu Pro Glu Ser Tyr
20 25 30
Thr Trp Asn His Leu Asp Asp His Thr Leu Ile Lys Glu Gly Thr Thr
35 40 45
Ser Ser Ile Val Pro Val Ile Asp Leu Asn Asp Pro Asn Ala Ser Lys
50 55 60
Leu Ile Gly His Ala Cys Lys Thr Trp Gly Val Tyr Gln Val Val Asn
65 70 75 80
His Gly Ile Pro Ile Ser Leu Leu Asp Glu Ile Gln Trp Leu Gly Gln
85 90 95
Thr Leu Phe Thr Leu Pro Ser His Gln Lys Leu Lys Ala Ile Arg Ser
100 105 110
Pro Asp Gly Val Ser Gly Tyr Gly Leu Ala Arg Ile Ser Ser Phe Phe
115 120 125
Pro Lys Leu Met Trp Ser Glu Gly Phe Thr Ile Val Gly Ser Pro Leu
130 135 140
Asp His Phe Gln Gln Leu Trp Pro Gln Asp Tyr Ala Lys His Cys Asp
145 150 155 160
Thr Val Leu Gln Tyr Asp Glu Ala Met Lys Lys Leu Ala Gly Lys Leu
165 170 175
Met Trp Leu Met Leu Asp Ser Leu Gly Ile Thr Met Glu Asp Ile Lys
180 185 190
Trp Ala Gly Ser Lys Ala Gln Phe Asp Glu Lys Ala Cys Ala Ala Met
195 200 205
Gln Leu Asn Ser Tyr Pro Ser Cys Pro Asp Pro Asp His Ala Met Gly
210 215 220
Leu Ala Pro His Thr Asp Ser Thr Phe Leu Thr Ile Leu Ser Gln Asn
225 230 235 240
Asp Ile Ser Gly Leu Gln Val Gln Arg Glu Gly Ser Gly Trp Val Thr
245 250 255
Val Pro Pro Leu His Gly Gly Leu Val Val Asn Val Gly Asp Leu Phe
260 265 270
His Ile Leu Ser Asn Gly Leu Tyr Thr Ser Val Leu His Arg Val Leu
275 280 285
Val Asn Arg Thr Arg Gln Arg Phe Ser Val Ala Tyr Leu Tyr Gly Pro
290 295 300
Pro Ser Asn Val Glu Ile Cys Pro His Glu Lys Leu Val Gly Pro Thr
305 310 315 320
Gln Pro Pro Leu Tyr Arg Ser Val Thr Trp Asn Glu Tyr Leu Gly Thr
325 330 335
Lys Ala Lys Tyr Phe Asn Lys Ala Leu Ser Ser Val Ser Leu Cys Ala
340 345 350
Pro Ile Asn Gly Leu Phe Asp Val Asn Asp Ser Asn Lys Ser Ser Val
355 360 365
Gln Val Gly
370
<210> 3
<211> 1134
<212> DNA
<213> alfalfa (Medicago sativa)
<400> 3
atgccttcac tctcagaagc ctatagagct catccggtgc atgttaacca caagcaccct 60
gatttcaact cactacaaga acttcctgaa tcatacactt ggacacacct tgatgatcac 120
acccttatta attccaataa tactatgaag gagagtgcta atagtagtag tgttcccata 180
attgatctca atgacccaaa tgcttcaaag ttaataggac atgcatgcaa aacatggggt 240
gtgtatcaag tggtgaacca tggcatccca ataagcctcc ttgatgaaat tcaatggctt 300
ggacaaactc tcttcaccct tccctctcac caaaaactca aagccattcg ttccccggat 360
ggtgtttcag gttatggcct cgctcgcatc tcctccttct tccccaaact catgtggtcc 420
gagggattta ccatcgttgg atcccctctt gatcattttc gacaactttg gcctcaagat 480
tatgccaaac actgtgatac tgtcttgcaa tatgatgaag ccatgaaaaa gttagcgggg 540
aaactaatgt ggctaatgtt ggactctctt ggtattacaa tggaagatat cgaatgggct 600
ggctcaaaag cccaatttga tgagaaagcc tgtgcagcca tgcaactcaa ctcctaccct 660
agttgtccgg atccggatca cgccatgggt ctcgccccgc acacggactc aacatttcta 720
acgatccttt cccaaaatga cataagcggg ttgcaagttc aacgagaagg ttccgggtgg 780
gtcaatgtac ccccactcca tggaggactc gtggtcaacg taggcgacct attccacatt 840
ttgtcgaacg gattgtacac aagtgtgctc catcgggttt tagtgaaccg aactcgtcag 900
aggttttcgg ttgcgtattt gtatgggccc ccatcaaatg tagagatttg tccacatgag 960
aaattagtag gcccaacaca acctcccctt tataggtcag tgacttggaa tgagtacctt 1020
ggcacaaaag caaagcattt caacaaagca ctctcatccg ttagtctttg tgcacctatt 1080
aacggtttgt ttgatgtaaa cgactctaac aaaagtagtg tgcaagtggg ttaa 1134
<210> 4
<211> 1116
<212> DNA
<213> Medicago truncatula (Medicago truncatula)
<400> 4
atgccttcac tctcagaagc ttatagagct caccctgtgc atgttaacca caagcaccct 60
gatttcaatt cactacaaga acttcctgaa tcttacactt ggaaccacct tgatgatcac 120
acccttatta aggagggtac tactagtagt attgttcccg ttattgatct caatgaccca 180
aatgcttcaa agttaatagg acatgcatgc aaaacatggg gtgtgtatca agtggtgaac 240
catggcatcc caataagcct ccttgatgaa attcaatggc ttggacaaac tctcttcacc 300
cttccctctc accaaaaact caaagccatt cgttccccag atggtgtttc gggctatggc 360
ctcgctcgca tatcctcctt cttccccaaa ctcatgtggt ccgagggatt taccatcgtt 420
ggatcccctc ttgatcattt tcaacaactt tggcctcaag attatgccaa acactgtgat 480
actgtcttgc aatatgatga agccatgaaa aagttagcag ggaaactaat gtggctaatg 540
ttggactctc ttggtattac aatggaagat atcaaatggg ctggctcaaa agcccaattt 600
gatgaaaaag cctgtgcagc catgcaactc aactcctacc caagttgtcc ggatccggat 660
cacgccatgg gtctcgcccc gcacacggac tcaacatttc taacgatcct ttctcaaaat 720
gacataagcg ggctgcaagt tcaacgagag ggttccgggt gggtcactgt gcccccgctc 780
cacggaggac tcgtggtcaa cgtaggcgac ctattccaca ttttgtcgaa cggattgtac 840
acaagtgtgc tccatcgggt tttagtgaac cgaacccgtc agaggttttc ggttgcttat 900
ttgtatgggc ccccttccaa tgtagagatt tgtccacatg agaaattagt aggcccaaca 960
caacctcccc tttataggtc agttacttgg aatgagtacc ttggcacaaa agcaaagtat 1020
ttcaacaaag cactctcatc cgttagtctt tgtgcaccta ttaacggttt gtttgatgta 1080
aacgactcta acaaaagtag tgtgcaagtg ggttaa 1116
<210> 5
<211> 841
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atatatggtc tcgcttgaca ccatccgggg aacgaagttt tagagctaga aatagcaagt 60
taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt 120
gcaaaatttt ccagatcgat ttcttcttcc tctgttcttc ggcgttcaat ttctggggtt 180
ttctcttcgt tttctgtaac tgaaacctaa aatttgacct aaaaaaaatc tcaaataata 240
tgattcagtg gttttgtact tttcagttag ttgagttttg cagttccgat gagataaacc 300
aatagagtgt cgttttagta aaaaaaatta ttttaaaatg aatatcatca cttttcaata 360
tagaattatt attttacttc caattatacc ctctaattaa tttccaaagc attataccaa 420
tagtaaataa agttagttta gtaaaattgt catatctttt aacattatta ttagatttct 480
taatttgtgt ttaaaagctt taaacgatga tcatttttaa acagagagta taaagtagta 540
aaatagtact attagaaatg aattgacgtg acatgctatg aaaagtctgg aagagtatcg 600
ataaaaggct acactagagg tagctactta tatgcgcagg aactgaaatc aaaaatgaaa 660
taaaggagaa ggaagatgca tgttgtgtta tataagtgaa ggagaaggac ttgcatgttg 720
tgttatattt gcttgtttta gtcccacatc gactgaaaca gaaagtatct cggcgtttat 780
atactacaag cgaaccatta aattgaagcc attcgttccc cggagtttcg agaccaataa 840
t 841
<210> 6
<211> 841
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atatatggtc tcgcttgaca ccatctgggg aacgaagttt tagagctaga aatagcaagt 60
taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt 120
gcaaaatttt ccagatcgat ttcttcttcc tctgttcttc ggcgttcaat ttctggggtt 180
ttctcttcgt tttctgtaac tgaaacctaa aatttgacct aaaaaaaatc tcaaataata 240
tgattcagtg gttttgtact tttcagttag ttgagttttg cagttccgat gagataaacc 300
aatagagtgt cgttttagta aaaaaaatta ttttaaaatg aatatcatca cttttcaata 360
tagaattatt attttacttc caattatacc ctctaattaa tttccaaagc attataccaa 420
tagtaaataa agttagttta gtaaaattgt catatctttt aacattatta ttagatttct 480
taatttgtgt ttaaaagctt taaacgatga tcatttttaa acagagagta taaagtagta 540
aaatagtact attagaaatg aattgacgtg acatgctatg aaaagtctgg aagagtatcg 600
ataaaaggct acactagagg tagctactta tatgcgcagg aactgaaatc aaaaatgaaa 660
taaaggagaa ggaagatgca tgttgtgtta tataagtgaa ggagaaggac ttgcatgttg 720
tgttatattt gcttgtttta gtcccacatc gactgaaaca gaaagtatct cggcgtttat 780
atactacaag cgaaccatta aattgaagcc attcgttccc cagagtttcg agaccaataa 840
t 841

Claims (8)

1. Use of a substance that regulates the activity or content of a protein, or a substance that regulates the expression of a gene, said gene encoding said protein, said protein being MsGA3ox1 or MtGA3ox 1;
the MsGA3ox1 is a protein with an amino acid sequence shown as a sequence 1 in a sequence table;
the MtGA3ox1 is a protein with an amino acid sequence shown as a sequence 2 in a sequence table;
the MsGA3ox1 coding gene is a DNA molecule with the coding sequence of a coding chain being a sequence 3 in a sequence table;
the encoding gene of the MtGA3ox1 is a DNA molecule of which the encoding sequence of an encoding chain is a sequence 4 in a sequence table;
the substance regulating the activity or content of the protein or the substance regulating the expression of the gene is a nucleic acid molecule which inhibits or reduces the expression of a gene encoding the protein or the activity of the protein;
the nucleic acid molecule is a DNA molecule expressing a gRNA targeting the gene or a gRNA targeting the gene;
the grnas include sgRNA1 and sgRNA 2;
the target sequence of the sgRNA1 is a reverse complementary sequence of the nucleotides indicated by the 347 nd and 365 th positions of the sequence 3 in the sequence table, and the target sequence of the sgRNA2 is a nucleotide sequence indicated by the 341 nd and 359 nd positions of the sequence 3 in the sequence table;
or the like, or, alternatively,
the target sequence of the sgRNA1 is the reverse complementary sequence of the nucleotide indicated by the 329-th and 341-th positions 347 of the sequence 4 in the sequence table, and the target sequence of the sgRNA2 is the nucleotide sequence indicated by the 323-th and 341-th positions 341 of the sequence 4 in the sequence table.
2. Use of an expression cassette expressing sgRNA1 and sgRNA2 according to claim 1 for regulating alfalfa plant types.
3. Use of a recombinant vector expressing sgRNA1 and sgRNA2 of claim 1 or a recombinant vector containing the expression cassette of claim 2 for regulating alfalfa plant type.
4. Use of a recombinant microorganism expressing sgRNA1 and sgRNA2 according to claim 1 or a recombinant microorganism comprising an expression cassette according to claim 2 or a recombinant microorganism comprising a recombinant vector according to claim 3 for regulating alfalfa plant types.
5. Use of a transgenic plant cell line expressing sgRNA1 and sgRNA2 according to claim 1 or comprising an expression cassette according to claim 2 or comprising a recombinant vector according to claim 3 for regulating alfalfa plant types.
6. An expression cassette as claimed in claim 2.
7. A recombinant vector as claimed in claim 3.
8. A recombinant microorganism as claimed in claim 4.
CN202110885132.7A 2021-08-03 2021-08-03 Application of GA3ox1 protein in regulating alfalfa plant type Active CN113481176B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110885132.7A CN113481176B (en) 2021-08-03 2021-08-03 Application of GA3ox1 protein in regulating alfalfa plant type

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110885132.7A CN113481176B (en) 2021-08-03 2021-08-03 Application of GA3ox1 protein in regulating alfalfa plant type

Publications (2)

Publication Number Publication Date
CN113481176A CN113481176A (en) 2021-10-08
CN113481176B true CN113481176B (en) 2022-04-19

Family

ID=77945209

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110885132.7A Active CN113481176B (en) 2021-08-03 2021-08-03 Application of GA3ox1 protein in regulating alfalfa plant type

Country Status (1)

Country Link
CN (1) CN113481176B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114277041B (en) * 2021-11-17 2023-03-31 南京农业大学 Application of soybean gibberellin 3 beta-hydroxylase encoding gene GmGA3ox1

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112980854A (en) * 2021-03-11 2021-06-18 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） Gene for encoding GA3 ox-oxidase and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3783777B2 (en) * 2002-09-20 2006-06-07 独立行政法人農業生物資源研究所 Gibberellin 2-oxidase gene having a new function and use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112980854A (en) * 2021-03-11 2021-06-18 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） Gene for encoding GA3 ox-oxidase and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A＇gnes Dalmadi等.Dwarf plants of diploid Medicago sativa carry a mutation in the gibberellin 3-b-hydroxylase gene.《Plant Cell Rep》.2008,第27卷第1271–1279页. *
Multigene editing reveals that MtCEP1/2/12 redundantly control lateral root and nodule number in Medicago truncatula;Fugui Zhu等;《Journal of Experimental Botany》;20210226;第72卷(第10期);第3661–3676页 *
植物中赤霉素代谢酶与株高的关系;乔枫等;《生物技术通报》;20110326(第03期);摘要 *
蒺藜苜蓿赤霉素合成基因MtGA3ox1生物学功能解析;孙浩;《中国博士学位论文全文数据库基础科学辑》;20210115;摘要 *

Also Published As

Publication number Publication date
CN113481176A (en) 2021-10-08

Similar Documents

Publication Publication Date Title
AU2019297209B2 (en) Method of obtaining multi-leaf alfalfa material by means of MsPALM1 artificial site-directed mutant
CN116179589B (en) SlPRMT5 gene and application of protein thereof in regulation and control of tomato fruit yield
CN111574605B (en) Application of rice gene OsLAT5 in regulation of absorption and accumulation of diquat
CN111087457B (en) Protein NGR5 for improving nitrogen utilization rate and crop yield, and coding gene and application thereof
CN111153976A (en) Rice defense mechanism regulation protein and application thereof in breeding
CN113481176B (en) Application of GA3ox1 protein in regulating alfalfa plant type
US11365423B2 (en) Method of obtaining multileaflet Medicago sativa materials by means of MsPALM1 artificial site-directed mutants
WO2021104542A1 (en) Use of rice gene osatl15 in adjusting pesticide absorption and transport
CN114213515B (en) Gene OsR498G0917707800.01 and application of encoded protein in regulation of rice chalkiness
CN113234720B (en) Wheat long-chain non-coding RNAlncR156 and application thereof in regulation and control of wheat response to drought stress
CN116064568A (en) Alfalfa MsASG166 gene and application thereof in improving drought tolerance of plants
CN111560055B (en) Application of rice gene OsLAT3 in regulation of absorption and accumulation of diquat
CN114657157A (en) ZmD13 protein in regulating corn plant height
CN113388015A (en) Pear protein fragment PyDwarf1-462 and coding sequence and application thereof
CN116515853B (en) Ryegrass salt-tolerant gene LpNAC022 and application thereof
CN114644701B (en) Use of proteins derived from corn and related biomaterials
CN116751791B (en) Application of PvPSK3 gene in improving genetic transformation efficiency of gramineous plants
CN116121298B (en) Application of inhibiting expression of HSRP1 gene in improving heat resistance of plants
CN114106120B (en) Heavy metal transport protein PhHMA5II-1 and related products and application thereof
CN116064572B (en) MdWOX11 gene and protein for promoting adventitious root development and application thereof
CN111607604B (en) Application of cotton GHPSAT2 gene in promoting flowering of plants
CN117987427A (en) Deep-sowing-resistant gene ZmSRO e and application thereof
CN118272423A (en) Application of transcription factor OsbZIP05 in negative regulation of rice seed vigor
CN115851767A (en) Salt-tolerant gene MsaH2A.W from miscanthus plant silvergrass and application thereof
CN116590331A (en) Application of SmD3-b in regulating drought and salt stress resistance of plants

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant