CN116987165A - Sorghum plant height SgSD1 protein, breeding material and application thereof - Google Patents
Sorghum plant height SgSD1 protein, breeding material and application thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The application discloses sorghum plant height SgSD1 protein, a breeding material and application thereof. The technical problem to be solved is to regulate and control the sorghum plant height, in particular to reduce the sorghum plant height. Specifically disclosed is the use of a protein as described in sequence 2, a substance that modulates the expression of a gene encoding the protein, or a substance that modulates the activity or content of the protein in any of the following: a1 Application in regulating plant height; a2 The application of the method in preparing products for regulating plant height can obviously reduce the sorghum plant height by down regulating the expression of the encoding genes of the proteins, and can be applied to sorghum breeding or low-plant-height variety breeding.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to sorghum plant height SgSD1 protein, a breeding material and application thereof.
Background
Sorghum is one of five crops in the world, has the characteristics of drought resistance, salt and alkali resistance and barren resistance, plays a remarkable planting advantage in arid, semiarid, salt and alkali and barren areas, has an irreplaceable effect in future agricultural development, and is considered as the most potential grain crop. Plant dwarfing is an important research focus in crop breeding, ye is a main characteristic of the first green revolution, and in the 40 th century, dwarf plants are utilized to cultivate new lodging-resistant crop varieties, so that the grain yield is greatly improved. The dwarf plant height can effectively promote the distribution of photosynthetic products in the seeds, improve the yield of the seeds, reduce lodging and is suitable for close planting.
At present, 60, 27 and 30 dwarf related regulatory genes, such as genes of OsGA20ox2, rht-B1B, zmCCT and the like, are respectively analyzed in main crops such as rice, wheat, corn and the like, and are involved in regulating the formation of plant height. In sorghum, only 4 genes such as Dw1, dw2, dw3 and Dw4 are found, only the first 3 genes are cloned, and the plant height of the reduced internode cell proliferation activity is regulated by encoding cell proliferation novel proteins, protein kinase and ABCB1 auxin efflux transport proteins respectively, so that the research on the plant height of the sorghum is far behind other crops. The sorghum genetic control network is unclear in analysis, the genetic foundation is relatively narrow, and the selection of new varieties of ideal strain types is hindered. Therefore, there is an urgent need to mine and identify superior genes that regulate the plant height of sorghum more, improving breeding efficiency.
Therefore, how to provide a gene related to sorghum plant height is a technical problem to be solved by the person skilled in the art.
Disclosure of Invention
The application solves the technical problem of regulating plant height, especially sorghum, especially inhibiting or reducing or down regulating sorghum plant height.
In order to solve the above-described problems, the present application provides the following applications;
use of a protein, a substance that modulates the expression of a gene encoding said protein, or a substance that modulates the activity or content of said protein in any of the following:
a1 Application in regulating plant height;
a2 The application of the plant height regulating product is prepared;
the protein is any one of the following:
b1 Amino acid sequence is a protein shown in sequence 2;
b2 A protein which is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein of B1), has 80% or more identity with the protein of B1), and has the same function as the protein;
b3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag.
Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.
In the above protein, the 80% or more identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
The above protein is called SgSD1 protein.
In the above protein, sequence 2 (SEQ ID No. 2) consists of 327 amino acid residues.
The sequence 2 is specifically as follows:
MVSQERQEPALPLPSNSSSAKRAAASMDASSPAPPLLLRAPTPSPSIDLPAAAGKAAAVFDLRREPKIPAPFLWPHEEARPTSAAELEVPVVDVGVLRNGDRAGLRRAAAQVASACATHGFFQVCGHGVDAALGRAALDGASDFFRLPLADKQRARRVPGTVSGYTSAHADRFASKLPWKETLSFGFHDGAASPVVVDYFTGTLGQDFEPMGRVYQRYCEKMKELSLTIMELLELSLGVERGYYREFFEDSRSIMRCNYYPPCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGEWRPVRPVPGAMVINIGDTFMSELNKAQM
the above protein is called SgSD1 protein.
In the present application, the modulation may be knockout or inhibition or reduction or downregulation. The regulation may also be enhancement, or increase or up-regulation
In the above, the knockout or inhibition or reduction or downregulation of the expression of the gene encoding the above protein or the activity or content of the above protein can achieve inhibition or reduction or downregulation of plant height.
In the above, the enhancement or upregulation of the expression of the gene encoding the above protein or the activity or content of the above protein may result in enhancement or upregulation of plant height.
In the above uses, the protein is derived from sorghum.
In the above, the sorghum may be sorghum P898012.
In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).
In the above application, the substance regulating the expression of the encoding gene of the protein is any one of the following:
d1 A nucleic acid molecule which inhibits or reduces or down-regulates the expression of a gene encoding the above protein;
d2 Expression of D1) the gene encoding the nucleic acid molecule;
d3 An expression cassette comprising D2) said gene;
d4 A recombinant vector comprising the gene of D2) or a recombinant vector comprising the expression cassette of D3);
d5 A recombinant microorganism comprising the gene of D2), or a recombinant microorganism comprising the expression cassette of D3), or a recombinant microorganism comprising the recombinant vector of D4);
d6 A transgenic plant cell line containing the gene of D2), or a transgenic plant cell line containing the expression cassette of D3), or a transgenic plant cell line containing the recombinant vector of D4);
d7 A transgenic plant tissue containing the gene of D2), or a transgenic plant tissue containing the expression cassette of D3), or a transgenic plant tissue containing the recombinant vector of D4);
d8 A transgenic plant organ containing the gene of D2), or a transgenic plant organ containing the expression cassette of D3), or a transgenic plant organ containing the recombinant vector of D4).
D1 In said nucleic acid molecules, the person skilled in the art can easily mutate the nucleotide sequence of the application which inhibits or reduces or down-regulates and/or enhances or increases or up-regulates the expression of the gene encoding the protein SgSD1 by means of known methods, for example directed evolution or point mutation. Those artificially modified nucleotides having 80% or more identity with the nucleotide sequence isolated according to the present application that inhibits or reduces or down-regulates expression of the gene encoding the protein SgSD1 and having a function of inhibiting or reducing or down-regulating expression of the gene encoding the protein SgSD1 are all derived from the nucleotide sequence according to the present application and are equivalent to the sequence according to the present application.
In the above biological material, the expression cassette of D3) means a DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for promoting transcription of the gene but also a terminator for terminating transcription of the gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present application include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta-cone (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I985) Nature 313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet. 262:141; proudfoot (1991) Cell,64:671; sanfacon et al Genes Dev.,5:141; mogen et al (1990) Plant Cell,2:1261; munroe et al (1990) Gene,91:151; ballad et al (1989) Nucleic Acids Res.17:7891; joid et al (1987) Nucleic Acid Res. 15:9627).
In D3) above, a recombinant expression vector containing the gene expression cassette may be constructed using a plant expression vector. The plant expression vector may be a Gateway system vector or a binary agrobacterium vector, etc., such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When the IbpPGM is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like can be added before the transcription initiation nucleotide thereof, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present application is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.
The 80% or more identity may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.
In the above D4), the backbone vector of the recombinant vector may be a Pcas9 vector.
The microorganism of the above D5) may be Agrobacterium. The agrobacterium is agrobacterium tumefaciens (Agrobacterium tumefaciens) strain EHA105.
In the above-mentioned use, the plant is any one of the following:
g1 Monocotyledonous plants;
g2 A gramineous plant;
g3 Sorghum plants;
g4 Sorghum).
The sorghum may be sorghum variety P898012.
In order to solve the problems, the application also provides a method for reducing the plant height.
The method comprises reducing plant height by knocking out or inhibiting or reducing or down regulating expression of genes encoding the above proteins in plants, and/or activity and/or content of the above proteins.
In order to solve the problems, the application also provides a method for cultivating low-plant-height plants.
The method comprises knocking out or inhibiting or reducing or down regulating the expression level of the coding gene of the protein in the target plant, and/or the activity and/or the content of the protein to obtain a low-plant-height plant, wherein the plant height of the low-plant-height plant is lower than that of the target plant.
In the present application, the plant may be sorghum. The sorghum may be sorghum variety P898012.
In the above method, the knocking out or inhibiting or reducing or down-regulating the expression of the gene encoding the above protein in the plant comprises introducing the above nucleic acid molecule, expression cassette or recombinant vector into the plant of interest.
In the above, the nucleic acid molecule may be a nucleic acid molecule as described in sequence 1.
The sequence 1 is specifically as follows:
CACTCGCACATCTCATGGTGTCCCAAGAACGGCAAGAGCCAGCACTGCCTCTGCCTAGCAACAGCAGCAGCGCCAAGCGAGCAGCCGCGTCCATGGACGCCAGCAGCCCGGCCCCGCCGCTCCTCCTCCGCGCCCCCACTCCCAGTCCCAGCATTGACCTCCCCGCTGCCGCTGGCAAGGCCGCGGCCGTGTTCGACCTGCGGCGGGAGCCCAAGATCCCGGCGCCATTCCTGTGGCCGCACGAGGAGGCGCGCCCGACCTCGGCCGCGGAGCTGGAGGTTCCGGTGGTGGACGTGGGCGTGCTGCGCAATGGCGACCGCGCGGGGCTGCGGCGCGCCGCGGCGCAGGTGGCCTCGGCGTGCGCGACGCACGGGTTCTTCCAGGTGTGCGGGCACGGCGTGGACGCGGCCCTGGGGCGCGCCGCGCTGGACGGCGCCAGCGACTTCTTCCGGCTGCCGCTGGCCGACAAGCAGCGCGCCCGGCGCGTCCCCGGCACCGTGTCCGGGTACACGAGCGCGCACGCCGACCGGTTCGCGTCCAAGCTCCCCTGGAAGGAGACCCTGTCCTTCGGCTTCCACGACGGCGCCGCGTCGCCCGTCGTCGTGGACTACTTCACCGGCACCCTCGGCCAAGATTTCGAGCCAATGGGGTAAGCGAAGCACCGATTTACATTTACCGCGCGTCGGCCCCTGAGGCCTGGGTCTTAGTCTTAGCACTGCATATACGGTCGGTAGCTCTGGATATGATACGTATATATGAAACCCCGTTCCAATCCCATGCACGGTGTACACAGGCGGGTGTACCAGAGGTACTGCGAGAAGATGAAGGAGCTGTCGCTGACGATCATGGAGCTGCTGGAGCTGAGCCTGGGCGTGGAGCGCGGCTACTACCGGGAGTTCTTCGAGGACAGCCGCTCCATCATGCGGTGCAACTACTACCCGCCGTGCCCGGAGCCGGAGCGCACGCTGGGCACGGGCCCGCACTGCGACCCTACGGCGCTGACCATCCTCCTGCAGGACGACGTCGGCGGGCTGGAGGTGCTGGTGGACGGCGAGTGGCGCCCCGTCCGGCCCGTCCCAGGCGCCATGGTCATCAACATCGGCGACACCTTCATGGTAACCCCTGCTCTGTTTTTTCTTGTCCTCCTCTTGTCCTGTGTGTGTGTATATTCACTTCTCTCTGTTTTTTTGCCCCGAATCCTAGTGGACCTAACTGGACGGATTACAGCACGCACACGTAGGCATGTCATGTAGCAGCAGTCTGCAGCACTGTAGTACTTAGCGATGCAATAGAGACATGCGTTCCAGTCGGTTCCATCTCGGTGGGCTACAGCTACAGTCCTACACGGACGCGGCTCGTAGTCGTAGGGACGGGCGCGTTCTCTGTATCCACACACGGCTGCGCCCAGGCCGAGGCTTCCGCCGCGGGAAAGTTGCGACAACAGAACGGGGTTTGTGCCGTTGGAGCGTTGCGGAGAGGCAGAGGCTTGGGGGGACGGGGGCGCGATACGCTGCGATGGGTGGGTGACCGAGGCGACGCTTTCGGCGGGGGCCCGGGCCTGCCCAGGTGCGCGCGGCCTCGTCGCCTTCCCCTGTTTTTTTGATGCCGCCGCTCGGTCCTCGGTGTTCTGGCTCCGCCCGCCCGCTCGCTGGGTGCCCATCCCATCTGATCCGATCCGCTCCGCTCCGCGGTGGCGGTCCTATGCGATGCCGCCGCACGAGCGCGGGGGGCCGCCCGTGGAGGAGTAGAAAGTGGTACAAGGTTGGTTGGAACTTGGAATTGTGGGGGGTTACTGCTGCTGGTGGCTGCTGCTTTGCAACTTGCCAGGCTGCTGCCTGTTGCCCCCCGCGTTTTCTAGCCGTTTCCGCTCGCGATCCGGCACGCGGCGCCCACACCGGGGCTCCAGCTCGGCCCCTTGGCCGTGTAGGTAGCAGGCACTTGCATCTGTCCGTTCGACACGATGATTCTTGTGCACTGTGTACGTATGTACTAACCCTTTCTGGTATGATGTACGCATGGCATGCAGGCGCTGTCGAACGGGCGGTACAAGAGCTGCCTGCACCGCGCGGTGGTGAACCAGCGGCAGGAGCGGCGGTCGCTGGCCTTCTTCCTGTGCCCGCGCGAGGACCGGGTGGTGCGGCCGCCGGCCAGCAGCGCCACGCCGCGGCAGTACCCGGACTTCACCTGGGCCGACCTCATGCGCTTCACGCAGCGCCACTACCGCGCCGACACCCGCACGCTGGACGCCTTCACCCGCTGGCTCTCCCACGGCCCAGTCCCAGCCCAGGAGGCGGCGGCTCCCTGCACCTAGCGAGCGAGCGAGCCGGGCCAAACAAACAAGGGGCAAAGGCCATCTCTTTCGCCGGGGCCCGCGCGCGGGGTTCGCCCACGTGCGCGCCCAGGTGGGCGCTGGCCGCGGGCAGGTGGCGGACATGTGGCCTGCGGGCCCCGCGCCGCCTTCCCATTTTTGGACGCTGCCGCGCATGCCGCATGCGTGCGTCGACGGCCCTACTACTTCTACTACTGCTACTGCGACTACTAGTGTACATACGCAAAAATACATATATACGTATTTTCTATATATATATATATAAGCAAGGCGGCCCCCCGGTGACCTTTTCTTTGTTTTTGTCGACAACTGTGTTTTGATCCCATTCTAGCTGTTCTATGGACCATGGATGGTTCGTTCAATGTTTGTACGTACTCCACGTAACCAAACTACTCTAGTGGACTAGTAGATCGGGCTCATGTGATGAAACTGGACCGACGCGGACGTCACGTGCGTCACCCGCGTCTGGTAGCGGTAGCGCACGAGCGCCGAATGTTTCCTGGGCCCGCAAGAGAATCGCTTCTCATCTCCTCTCACCATGAATGGGGAAAAATGCTGCGTCGAAAGTTCCAGACGTTTCCAAATTCCAAACGGTTTTGTGGCGTCCGATCCATGGGGCGCCCCAAACTTCCAAGACGTTTTCAGGTTCCAAATCTTCGTGCTCCACATCACCTTCTTCCCAGATTCATTTGCCTCGTCGCTTGCTCTCCTGTGTTATTCACGGGTCCCACTGTTGCCCCGTCTGCGAGAAAGAAATTTATTAGAGTTGAAGCATTCGACATTTCGACTGACTGATTGTTAGTATCACTAAATTTTGTGCACATGTTTCTTTGGTCATTCATCTCTGGATATTTTTTTTAGATAATGGATATAAATATCGGGCCTCTACATCTGAGGAAGTACACAGCCAATTATTTTCATCTCTGGACATGGGACGATGGAAGAGGCAGATAGATTTAGGAGACCCTTCAATTCAGAATTTCAGGTGCACAAGGCCTGCCTGGCTTGCCCGGATTCTTGTTTCGGACATGACCAACTAGGCCGCACTACTTGCACTGATAGCTGGAGAAAAAACAAAACTTTGCAAACAGCAGGATTATCTACAAGGGAAACTCCATCCACGTGAACCAGCATTTCAGGGAGAGATGCGACAAAAAAAAAGAGGCGGCAACAAAAAAATCTTACTGCAATTTTATCTCTGCATTGAACCTCTTCCAACCATGCCGCATCCTGTACTGTTTTGTATCTTTCCCGGTGGTCCGTTGCGTTCTCACGCAGTTGATAACATGCAGTCACGCACCACCGAATCCAGTGTACTAGGGGTAGTGACTTGTCACGCGGAACAACAGGTCGGTAGCACCAAGCAAGTCGCTGTAGACTTGGGCGTTTAACAACGACTTGCACAACAGTTCAAATATAGCATATGCAATTATGCACAAGATTGTTCGACTGCTATCCGACAAACTGAAGAAGCTGCCCAATTGAACAGAATGTACCAGTGATTTCCAGCACACTATCTTACAGCAGCGTTGAGAATGAAACAACAAATGGGGGAAAACAGATGTGTATTATTCTACAGTTACACCAAAGAGTTTGTCCTTTCAGCATCAACAAGAATCATATGCATATCTAGTGACAAAAATTCCTCTAATTTTACCCTACTTGGTAACAGTTCTCTTCAACACATATATTTCACGTGCTTGCATCGAGTTCCTTGGGCCGCCACATCGACTTCTCGACGCAAAGCAAGCCCTCGTTGCCCTTGGTGTAGGTCATTCGCACCTCCCACTGCAGGGACTTGGCCATGCTTTCCAGTTCGTTTATTGTGTCCGCAGTGTCCCTCACAATCAGTTTGCCTTGGGGCCTCAGTACACGATCAACCTCGGCAAAAACTGCCATCAATTTGCATCTGTAAACAAGCAACACAGATTTAGCATCTGTAAACACCACAGGTTTCATTGCAAGAAGCATAAAGCATGCAAACATGCTACTTGTACATGTCAAAGAAACATGTCAAACTCAAACACATGAAAATCATTATTATTGTTTTCTTGCTGAACTGATCACATTAGTTGGTTTCAATTTCTGAGTTCCACTAGTAATCTATACCAGAAGGATAGAATAATGTCAAGAACAAGAGATACAAACCTCTTTGTGAGCTTTGAGAATAGATGGTCCGCGTGCAGAAGGTCATAAGTTCTTGGGTAAGTGCTCAAAGACTCGCACCAGTCATGGTACATACCAAACAAACCACGCTCGTAAATGATGGGCAGTGTGTCTGGTGAATCAATCGGCACAATATTCATGACCCAGACCTTTTGGTCCCTCAGAGCTGCAGCAAAACTGCCATGCAACGATGTAAAGCATTAGTAAAAATATTGGGTTTTTTAAACCAAAACCAAGAAAGATAATTCCTCCAGCTTAACTGAAAGAAAGAAAGAAAAAAACTGCTTAATGACTTATGGTGGACAAGTTGCCTGTTATGTTTTATGATAGCTATGTGCCAGCTTGGCTAACTGGTAGTTATGTAGTGTGATCTGAATTACCAAAAAAGAGAAGAAAAAAAAATCATGCCCAAGAAACTGAGAAAGACACCCATTTACTTACCCTCCATACACAGCTCTCATGTCCATTACATTTCTCACTTTGGACCAGTCAATTCCCATGCCATTCACATACGATTTACTTACAACCCTTTTCCAGTGAGCATTATCTGCCTCGAAATCTTCATTTGCAGGCTTTCCATAGACCCCAACCTTGGAACCATCAATCCAGAAAGGAGTCTTCTCAAGCCTTTGTGGCCAAAACTCTGGCCATTTTGACCCTCGAACTTTCGAGCCAACAGGCAGTTTGTGCATGCATGCTTCCAAAGGTACATTCCTGCAAATCAAAAGATTGTGTAAGCAAAGCAGAGGAAGCACTTCGCCGCATTGAAAATACGTTCTTCTCAAAGAAACAAAACCATACCAAGCTGCATCTGCATCATCAGATTCCTTGCACAATGGTGGGTTGTTTTCGGATCTTTTCTCATAGCAAATGTTGTCCATTGGTTTCTGAAATATGACCATACCAACTTGATTTAACTTATCCTTAGTCTTGTTGACCATCTTCCAGCACATGGACTTTGTCAAAGTGGACATCGCTGAAAAGATTAAGGGGTCATATGTTATGATAGAAATAAAATTCAATTTTGCACTGTTGGTACATAGCATCTGTTTTGAACAAATGCAATCCTTCCTTATCCATGAAAGAAGTTAACCCCTGATACTTAGGATTATTCAGTACTTTCACTCATGAACTGCTGAATTTGTTCTGCCAGTAGTTGCTATACTAGAAATGTTCAGTGTACCAAACATAAATTTGGTACGGGTTCCTTATTAAAGATGGGAGGCTGTATGGTATTTCGACGTAACAAATCAAGTTAGCAGCTACCCTACTTATGGATATACACTTCTCAAAATGAATATACATAGTTTTGATAGGTGACATTAATTAATATAAGAACTTCATGCAGTTAGGGTGAAACTAAACTAAGCAGTTACGGAAATACCATTCCAAATCTCAACATCCTCTGGGAGCTTTTGGTAAACAGGAGTGGCAGACCAGACAAAGTAACCACCAGGGCGTAACAAGCGGTTCAATTCCAGCAAAAGCATGCCACCTAAAAGGAGTCAGTAATAAGATTCAGTTCTATAGCAAATCAATAAATGAAAGGAAGACATGTCACCAACAAGACAAACCTTCAATGTGCCAAGGGACCCTGCAGCGAGCGCAATGAATGACATCAAAGACTCTGCTGGGGTATGGAAGTCTCTTGGTGCCCATCACAGCTGATATTGCTGGAATTCCCCTTTCTAATGCAAATTGTACTTGAGCTTCATGCTCATCTTTCGGAGCAAAAGACATGGTAAGCACATCTCTATCAAACATGTAGCCTCCAAAGCTGGCAACTCCACAACCGACATCTAGAATGACGCGGCTTCGTTTGCCCCATGCAATATCAGGCAGTGCCTGTGAATGACAGTTTAATCAGCATATGATGAAAGCAAGTGTGATAATATCAAGTTCAAAGATGCAACATGAAACTTTCATAATCATGGACAGTACTAAGCTTGCTTGATAGATTAATGTATGGATGAGACTAAAAAAAAGGAAAGTTGTATCCATCAGAACGAGAGGCTGAAAACACATGGCTGGCTGTGAAAGCCTGATGTCGTTTAGTCTAGCATAAACAAACTGTCCTCAGCATGTAGATTTCCATAGGGTGGCATTTGACAAATTATGATTGTGGACTAGCGAATCAATCACTGATTCTCAAAAGTGTGAGACAGATGAGTTCAAGTCTAAGGGGTGACTAATATGGGATGCTGGGATGATGATGATGATATATACCTGCTGAATAGTATCAATATAGTGGAGGGCACCATTCTTGAACTGAGTCCCACCCCCAGGGAACAGGAGGTAGTCACCTGATACTTTAACCCAATTTTGATGTCCCTTGTACTCTGCGAGCCTAGTGTGAGGAACATTGCTGTACCATACCTGCAAAAAGCAGCACAAGATGGTAATAAGTAAACAGAGATCTTGGTCAGCTAAAGATGATTCAGTGTCGTACAATTTAGAATAGACAGAATCACCTTGTCCCTGCTCCTTGGCCACTCAATTGGGCGTTTATATCCTTCTGGGAGTGGAACAAGGCAGGTAGGAGGCTCCTCAGGGCAATGCCTCTCACGATGTTCATAATGTTTAGTAGTTCGAAGCTTCTTGATAGCCTTCTCGTTGTCAAGGCAAGGTATGTAATCTGTCGAGGCACTGCTATTACATAGTTTCCAGGAATAGCTAGTCGCATCACCTGAAGACTTTGATGACGCTTGGACTTCCTTTTCATTCTTGGACTCTGCAGCCTGTGTGGGGAATGAACCATTCTGGGTATTTGACTCCTTCAGAAGCTCTGATTGGGCCCCATCAGGAAATACCTCGTTGGAGTTTGAGCTCTGATCCTTCTCTCCATTTTCTTCCACCTTCTCTTCTATCTGAGGTTGCTCCTCCTGAGTGGCATCACCTTCAGGCTTCTCTTCTTGATCATCCTTGCTCTCTCCATCAGGTTTTTCATCACCACTCTCATTTGTGATTTCATCATCTTTCTTCTCCCCACTCTTCTCCCCGTCACCATCGTTCTTCATGTCATCTGACCGCCCTTCTGATTTTCCATTTGCATCATCAAACATATCCTTGGTCTCAGCTTTCTCTGTCGGCACTTCCGGCTCCTTCTCTTCAGGCTTCTCCTCTGGCTTCTCAGTGAACTTCTCTTCCATCGTGGCATCCTTGTTATTCGGCTCCTCCGGCATCGTGGCATCATTGTTGTCGGTGTCCTCAAATTTCTCAGAACCTTCACCAGCATTGTCCTGTGAGGCCCCAAAATTGACAGGCGCAGGCTGCTGCTTCACCACCGGCTTCTTATTCGAGGAGATCTCCAGCGGGAAGACAGTGGACGAGGTCATCATCCACGCGCCGACCAGGCAGAGCGCCACAAAGACGACGACCGTGGTGGTCGTGCAGAACGACGACGACGTCGAGGACGGCCGGCGGCCGTCCATCTTCCCACCTCGGCCAAATGCCATTAGTGCCTGGCGAACATGTACCAGAGCACCGACCTTCACGCGATTTATCTCCACCAACTACTGCTGGACCAAGAACCCCCAAAAAAATCGCACCTTTGTCTGCTTTGTGCTGCTACAGCCGCGCGGCACCTGAAGCAAACCACAAAAAAAACTTAAATCGCCGCGGACATAAATCAAGGTGCTGGATCTAAAGAACAAACGCTGGATCTACTCAAGCAACAACGGAAGGAAGATCCGCTATTGGTGCTAGTATTAGCTTCTTGTTTCCTAGTACTACAGCGGCTCTTTCCCAGTATAAGAACACGGGAAAACGCGGAGAAATCCCCCTTCGTGGCCAAACATGGAAAGAAAATTAGTAAAGCGTGTGCTTTAAAACCCCCTCGTTCTGTTCCTTCCGCGGAGAGCTACCGCATCTTCCAATTGAGCTGGTTCTCAGCTGGGCGCAAAACGCGCACTAATCAATGTCCGATTCCATCCACAAAGAAAAAAAAGACGGGAACAGCTAATCCAGCAGCTCGCTCGCTAGCTAGCTAGCTCATCGGCGGAAGGACGGAACCAGCTTTGCTGGATCCAGGACAGCAAGAGTGTGCAAGGAGAAAGAACGGAGCAGCAATGCGGATTGCGGAGGCGGTGGATTGGTACCTCGCCGGAACCGACCGGAGTGGTCGCGGTGGCCCTCCGCGCGGATCTCGAAGAGGAGCGAGGAAGGGGAAGGCGGATGCGCGTCCTTGGGTTCTCTGCCACCGCACTGGGCCTCGCCGCGTTATAAAGGCGGGCGGGCGGGCGGGCAGCGCAGTGTGAGTGGAGTGCAATCTGTTGTGTAGTGTGTGAAGAGGCGGAAGCGGAAGCGGAGGAGATGGGTTCGCATTAGACGACCGTACGTAATTATACGCTATACTAGTACTTGGGTTAGATTACTCGGGAGATCTTGGCCAAAATGTCCGGTCTGAGTGTTTGGTAGTTTTATGGATTTGCCCTTTTAAGATGTTGGTATTTCTCCGGGAGCTTAGAAAGAAGAAATGGCGATGCTTTAGGCCTTGTTTAGATGCGAAAAAAATTTGGATTTCGCTACTGTGGCATTTTTATTTGTTTGTAGCAAATATTGTCCAAACACGGACTAACTAAGATTCATCTCGCGATTTACAGTTAAACTGTACAATTAGTTTTTATTTTCATTTATATTTAATGTTTCATGGATGTGTCGAAAGATACGATATGATAGAAAATTTTGAAAACTTTTTAGTTATTGAGGTTAACTAAACAATGCCTTAATTGAGAATTTACTCGAGCAAAAAGAGTTAGGTCAGTCTCAGTGGAGAGTTTCATGGTGTTGTTTCCAAGACTGCCATATCATGTGAAATGAAATGAAACTTGGTTGAAACACTCACTCTCAATGGAGAGTTTCATTTTATAGTTTCATGGGCATTTAATTTCAATACTCATAGAGAGTTGATATCGTGCCAACTCATTTCTTCTCTCTCTTCTTAAATACACAGTCATATCATCAAAAAAAATCCTATGTAGCAACATATTTAATGCAAATAAAACTCATATGGTGGACTGTAGGAGTAGCATTAGGCCAAGGGCACACACACGGTCACGGTGTGAGTGCGACGGTGCGAGTGGGCCCGCGGCGGTAGTAAGTGCGTGCGCGCCCGGCGCCCCCCTCCGCGGCGACGACGCAGCGGCAGCGCGTCGTCCAGTGCACCGTCTGCTGTTCGGCGCTGCGGGTCCTCCGCGCCACGGCGCAGTGAACCGGGCGCGTGCATCCCGGGAGCGGCGGCTTGGCACTCCCCTGCTTGTCGGTGGCGGCCGTCGGCATCGCTCGGCCCCGGAGCGTCACGAGGCTGCTGATTGGGAGCGAGAGCGAGTAGTGGGGCTGGTTGGGGACAATCCCATTCCCACCCGGCCCACCAGGCTGGGACTGGCCCACTAGTCACTAGTGGGTGGCTCATGGGTGTGGGTGGGCTGGCTAATGCCGCCTGCCCAACAACCAACCCAACCCCTGTGGACGCTGGTACCGGTAGTTGCCGCGCCATGGTGGACTGCTGCCGCCTGATGCCTTTGCCTGCCACGCTCCACGAGTTGAGGCGCACCAAACTGTGCTGTGCTCCTGATTTGTGCTAATCGGCCGACGCGTACCATTCTTTCTTTCTTTCGTCTACGCGCAGAGAGGCCGGTTGACTGTTTCTTCGTTGGAGGGCCATGTTGACTCGTACTAATAATAAAAATAATAATACTAGGTTGACTTTTTCAATTCCAACGCAGCAGTGCAAAGCTGCCCACCTATGAGCACAGGTCCTTTTTTAACTCCGTTTTTGTACGTACACACGTACTGTCCAGCCTGTGTCTAATAATCTTACCAAAAACCTGTCATCTCACTATCAACCAATCAGGCTCTCCGCCTGTTCGTCGAGGAACAGCAGTTGTTTTCCCTACTCCAACATAGAGTACACTATGGACGCACATTACCATGCCAGCTTGAGCTTAGCATTGCCCACCGTTGGATAACTGCCATGCCATTCTCAGGCCCTGTTTAGTTCCCATCTAAAAATTTTTCATCCATTCCATCGAATCTTTGGACACATGCATGGAACATTAAATGTAGATAAAAAAATAAACTAATTACACAGTTTAGTTGAGAATCGCGAGACGAATCTTTTAAGTCTAGTTACTCCATAATTAGCCTTAAGTGCTACAGTAATCCACATATACTAATGACAGATTAATTATGCTTAATAAATTTGTCTTACAGTTTCCTGACGAGCTATGTAATTTGTTTTTTTATTAGTTTCTAAAAACCCCTCCCGACATCCTTCCGACATATCCGATGTGACAACCAAAAAATTTTCATCTTCAATCTAAACAGGCCCTCACTCTCATCATCTCATGCCGGGGCAGCAGGTCCGTCGTCAGGTCTGTCGTCCCGTCCCGTGCCGTCTGAAGCAACAGGCGAGAGAAACGCCGTTCCATCGGTTTGCCGAGCGTGCAGAGGATAGAGCTATACTCGATCCGGAGAGGATTGTGAAACGAAGCACGGTTAAGCAGTGCCGCGCACGTGCTGCTCTGCTCCTGGATCCGATCCAGATCGACTCGGGGCGTCTCGGCCTCAGCGGCGATGGCAATCATCGCGCGCGCTGCTGGAGCTGGACGTTTTCGTCTTGCATTGCAGGAGGCGGAACAGAACGGAGAAAGCCACGGCGCGCTTTGCCGACGCCACGCGCTGACACGAGGGACCCGTTCAGCGGCCAGCACGCAGCCTAATCATGCCTGTCGGGGGGAGCTCATCCGTTCCTGAATTTGGGTCATGCTCCAGTATCAGGTATTCAGGTACTAGTACTCCTGAGCCATGTGCTGCGACAAAAAAGCGAGGCTCCTGTAGTAGAGCCTTGTTTACTTACAAAATTTTTTACATTCTCAGTTATATTAAATCTTGTGACACATGCATAAAGCATTAAATATACATAAAAGAAATAATTATTTACACAGTTACTTATAATTTGCGAAACGAATCTTTTAAGACTGGTTAGTTTATGATTAGATAATATTTATTAAATACAAATGAAAGTAATATTATTTATATTTTGCAAAAAGTAAATAAGACCTAGGTAGCTAGGCCAACGTGAGCATGTCGGACCCGGACCGGTTCGTTCTACGGCGCGTCCCGCAAACCTGCAGCCAGGTAGTAGTAGTACACCGTGCACGGGAGAGGTGCGCCATGCATGCTCGGGCAAAAGATCATAGAGAAAGGTGCAGCGTTTCAGTTGCACACCTGACCGAGTGACGCCTCGCCTTGTTTGGCTTTGTTCCCAAAATTTTTTAAAATTCCTCATCACATTAAATCTTTGAACGAATATATGGAGCATTAAATATAAATAAAAGAAATAATTAATCATACAATTTGTCTGTAATTTGCGAGACGAATCTTTTGAGCCTAGTTAGTTTATAATTAAATAATATTTGTTAAATACAAACGAAAATGCTACGTTAGCCAAAACTAAAATTTTTCTCCAAACGTGACCCAGCACCTTCCGATCAATCATCACTCAGCGGGTCACGTCAGAAGATCAGATGGACCTTGCCGTCCGGGCCTGTCTCTCGGCCTCCTCCCCATCTGGAACGAACAGAGGTCCAGTCCTGTTTCGAGTCGAGCTGAGTCGATCAGATGGGCCTAAATAGGCCGAAGACGTAGGCAAAGGGCCCGCTGATTTATCTGATTCTTCTAGGACCGTGCATGCGCGGATGGGCCTAGGTGGAAACCCAACAGATGTGAGGCTTCAAAGAGGAAGAAGTCCGTTACACATGGAGAGTTAGTCTATAATGGGATAATATTTACCACAAACAAATAAAAATACTACAGTAGCGAAATCCAAAATTTTTCACATCTAAACAAGGCCCTAGATGTTTTGTCAGTGCCAGACCAGAGAAAATCTCGTCTTCTGCTGTCAATAGCTTTGATGATTCCTGGCGGCAGAGGTAAAGCTTGCCTGGGCCTTGTTTAGTTCCGAAAAGTGAAAAGTTTTCGGTACTGTAGCACTTTTGTTTGTTCGTGACAAATATCATCCAATTATGGACTAACTAGAATTAAAAGATTCGTCTCGTGATCTACAGCTAAACTGTGTAATTAGTTTTTGTTTTCGTCTATATTTAATGTTTCATGCATGTGCCACAAGATTCGATGTGACGGAGAATTTTGAAAATTTTTTGGTTTTCAGAGTGAACTAAACAAGGCCCAGATGTAATTGACCATGCCATCGAGCGCGAGTTGACTAGAGTGAGTCGGCCCTGATGGTTAAGTAGTGCAGACTGCCAAGTGGACAACCGTCTATCAACTTTGCAGAGTGGGGCGAATGCACTGAGGATGTTGGAGAGGGGCAAGCCAAGGTAAACTTGAGGAAAGATGCTTGTTGACACTGTAGTATGTGAACAATCCTGTTTAATTTTGTGTCCTCGACG。
the introduction may be achieved by genetic transformation.
In the above method, the target sequence of the nucleic acid molecule is nucleotides 248 to 267 of SEQ ID NO. 1.
In the above, the method is to insert/replace/delete the 248 th to 267 th positions of the sequence 1 region in sorghum genome to cause gene function deletion.
Above, causing a loss of gene function may result in a significant decrease in sorghum plant height.
In the above application and method, the plant is any one of the following:
g1 Monocotyledonous plants;
g2 A gramineous plant;
g3 Sorghum plants;
g4 Sorghum).
The protein or the substance described above.
Advantageous effects
According to the application, gene editing is carried out on the SgSD1 gene in the sorghum resource material plant, a gene knockout vector Pcas9-SgSD1 (also called recombinant plasmid Pcas9-SgSD 1) is constructed through a Pcas9 vector, and the gene knockout vector is introduced into a sorghum callus to obtain gene knockout plants DB03, DB06, DB08 and DB09. Homozygous plants of the knockout plants DB03, DB06, DB08 and DB09 were mutated in the SgSD1 gene in the genome compared to sorghum P898012, and the nucleotide sequences of DB03, DB06, DB08 and DB09 planted in the two homologous chromosomes were changed as follows: 14 deletions between positions 254-255 of SEQ ID No 1 and 6 bases (TGCGGA) insertions in the genome of DB 03; 1 base (C) is inserted between positions 264-265 of SEQ ID No 1 in the genome of DB 06; 1 base (C) deleted between position 266 of SEQ ID No 1 in the genome of DB 08; 1 base (G) is inserted between 264-265 of SEQ ID No.1 in the genome of DB09, the deletion and insertion mutation of the above 4 sites cause frame shift, leading to premature translation termination and the deletion of SgSD1 protein functions, so that the SgSD1 gene is knocked out. The plant heights of the gene editing plants were reduced by about 1/3 of the plant height found by field and laboratory planting of DB03, DB06, DB08 and DB09 with sorghum P898012.
Drawings
The plant heights of sorghum P898012 and gene editing DB03, DB06, DB08 and DB09 plants of fig. 1;
fig. 2 field photographs of sorghum P898012 and gene editing plants.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The following examples were run using SPSS11.5 statistical software and the experimental results were expressed as mean ± standard deviation, using One-way ANOVA test, P < 0.05 (x) indicated significant differences and P < 0.01 (x) indicated very significant differences.
EXAMPLE 1 construction of Gene knockout vector
The inventor of the application discovers a gene capable of regulating and controlling the plant height of sorghum, the genome sequence of the gene is shown as a sequence 1 in a sequence table, the gene is named as an SgSD1 gene, the gene comprises 3 exons, and the exons are respectively positioned at positions 1-649, 794-1115 and 12666-12906 of a sequence of SEQ ID NO. 1; the coding sequence is shown as a sequence 3 in a sequence table. The coding protein sequence is shown as sequence 2 in a sequence table, wherein the sequence 2 (SEQ ID No. 2) consists of 327 amino acid residues, and the protein is named as SgSD1 protein.
1. Vector construction
TABLE 1 primer list
In target-F and target-R in Table 1, the sequences with single underlines and wavy lines are sequences homologous to the linearization vectors described below, and the double underlined sequences are target sequences.
1. Primer design: the escherichia coli shuttle vector pCas9 (also called pCas9 vector and purchased by conventional commercial methods) is used as a skeleton vector, a website is used for predicting and selecting targets, and target primers are designed.
Cloning of the sgsd1 fragment: 1ul of each of the primers (target-F and target-R in Table 1) was added to a10 ul system, and the mixture was annealed at 94℃for 10min,0.1℃per second to 15℃and maintained at 15℃for 10min, to complete the annealing, to give an annealed product which was a SgSD1 fragment. The annealed product targets the SgSD1 gene in the sorghum genome, and targets 248-267 th sites of SEQ ID NO. 1.
Obtaining of the PCas9 Linear vector: the Pcas9 vector was digested with AarI, and a large vector fragment (about 15 kb) was recovered using a gel recovery kit (AxyPrep DNA), to obtain a Pcas9 linear vector.
Homologous recombination ligation of the SgSD1 fragment with the Pcas9 Linear vector: 1ul of the annealed product (SgSD 1 fragment) obtained above was taken together with the above Pcas9 linear vector (after cleavage of the Pcas9 vector), and was usedHomologous recombination enzyme [ ]UniSeamless Cloning and Assembly Kit) and carrying out homologous recombination connection to obtain a homologous recombinase reaction solution.
5. E.coli transformed with the recombinant vector: 10ul of the homologous recombination enzyme reaction solution is added into 100ul of competent cells of escherichia coli Trans-T1 (TransGen Biotech), kept stand on ice for 30min, subjected to heat shock at 42 ℃ for 30s, quickly taken out of the ice bath for 2min, coated on LB solid medium containing 50mg/ml of spectinomycin (spec) under the sterile environment of a super clean bench, and inversely cultured overnight at 37 ℃ until positive clones are obtained.
6. Identification of positive colonies: the positive plaques were picked up, transferred to LB liquid medium containing spectinomycin (spec), cultured overnight at 37℃at 200rpm, and the cells were collected by centrifugation, recombinant plasmids were extracted using a plasmid extraction kit (AxyPrep plasmid DNA small extraction kit), and the correct fragments were detected and confirmed to be inserted. The plasmid with correct sequence is named as Pcas9-SgSD1 homologous recombination vector, which is also named as recombinant plasmid Pcas9-SgSD1.
Recombinant plasmid Pcas9-SgSD1 is a 5' scheme in which the Pcas9 vector is replaced by DNA fragment 1 (nucleotide sequence is 248-267 of SEQ ID NO. 1)AGATGATCCGTGGCA-3' and 5 -The fragment between 3' keeps other nucleotide sequences of the Pcas9 vector unchanged, and a recombinant expression vector expressing the gRNA of the targeted sorghum SgSD1 gene and cas9 protein is obtained and is called as a Pcas9-SgSD1 homologous recombination vector (recombinant plasmid Pcas9-SgSD 1).
Genetic transformation of the Pcas9-SgSD1 expression vector: taking 5ul of recombinant plasmid Pcas9-SgSD1, adding into 100ul of competent cells of agrobacterium tumefaciens (EHA 105), ice-bathing for 30min, placing into liquid nitrogen for 5min, immediately transferring into a water bath kettle at 37 ℃ for water bath for 5min, taking out a centrifuge tube, adding 0.5ml of LB culture medium, and oscillating at 28 ℃ and 220rpm for 3-5h to complete the conversion of agrobacterium. The bacterial liquid is taken out and cultured for 2-3d in an inversion way under the condition of 28 ℃ in an LB culture medium containing 50mg/ml spec and 50mg/ml Rif antibiotics.
8. Identification of recombinant agrobacteria: single colonies were picked and inoculated in liquid medium containing the corresponding antibiotics and cultured overnight at 28℃with shaking. And re-extracting the agrobacterium plasmid by using a plasmid extraction kit, and then verifying the correctness of agrobacterium cloning by using the escherichia coli for standby, wherein the agrobacterium with the correct agrobacterium verification by using the escherichia coli is called recombinant agrobacterium EHA105/Pcas9-SgSD1. The recombinant agrobacterium EHA105/Pcas9-SgSD1 is the agrobacterium EHA105 (also called agrobacterium EHA105/Pcas9-SgSD 1) containing the recombinant plasmid Pcas9-SgSD1.
9. Obtaining and identifying knockout plants: transformation of Sorghum P898012 (Sorghum P898012) by Agrobacterium-mediated methods (ref: do P T, lee H, mookkan M, et al Rapid and efficient Agrobacterium-mediated transformation of Sorghum (Sorgum bicolor) employing standard binary vectors and bar gene as a selectable marker plant Cell Reports,2016,35 (10): 2065-2076.) is described in Rapid and efficient Agrobacterium-mediated transformation of Sorghum (Sorgum bicolor) employing standard binary vectors and bar gene as a selectable marker plant Cell Reports,2016,35 (10): 2065-2076., in which young embryos of material, named P898012) were obtained as T 0 The generation plants, total 27T plants are obtained 0 The generation plants were designated as DB 01-DB 27 (Gene editing plants DB 01-DB 27). Will T 0 Transplanting the plants in a seedling raising basin for cultivation, grinding sorghum leaves 2-3cm, extracting plant DNA by using a DNA extraction kit (DP 360), and performing PCR and sequencing by using a detection primer (Check-F1/Check-R1) to finish the identification of genotypes. The results show that the genes of SgSD1 in sorghum DB03, DB06, DB08 and DB09 are subjected to homozygous mutation for gene editing in various gene editing plants, and the genes are called DB 03T 0 generation gene deletion homozygous sorghum plants, DB 06T 0 generation gene deletion homozygous sorghum plants, DB 08T 0 generation gene deletion homozygous sorghum plants and DB 09T 0 generation gene deletion homozygous sorghum plants.
Sequencing results showed that:
the gene of SgSD1 in the high genome of the homozygous sorghum plant with the DB 03T 0 generation gene deletion is mutated compared with that of sorghum P898012: in both homologous chromosomes, the genomic genes of SgSD1 whose nucleotide sequences are sequence 1 (SEQ ID No 1) in the sequence listing were changed as follows: deletion of 14 and insertion of 6 bases between positions 254-255 of sequence 1 (TGCGGA);
the gene of SgSD1 in the high genome of the homozygous sorghum plant with the DB 06T 0 generation gene deletion is mutated compared with that of sorghum P898012: in both homologous chromosomes, the genome gene of SgSD1, the nucleotide sequence of which is sequence 1 in the sequence table, is changed as follows: 1 base (T) is inserted between positions 264-265 of sequence 1;
the homozygous sorghum plant with the DB 08T 0 generation gene deletion has mutation of the SgSD1 gene in the sorghum P898012: in both homologous chromosomes, the genome gene of SgSD1, the nucleotide sequence of which is sequence 3 in the sequence table, was changed as follows: a base (C) deletion at position 266 of sequence 1;
the gene of SgSD1 in the high genome of the homozygous sorghum plant with the DB 09T 0 generation gene deletion is mutated compared with that of sorghum P898012: in both homologous chromosomes, the genome gene of SgSD1, the nucleotide sequence of which is sequence 3 in the sequence table, was changed as follows: 1 base (G) was inserted between positions 264 to 265 of sequence 1.
The deletion and insertion mutation at the above 4 sites cause frame shift, so that translation is terminated in advance, and the function of the SgSD1 protein is deleted, thereby knocking out the SgSD1 gene.
The DB 03T 0 generation gene deletion homozygous sorghum plant, the DB 06T 0 generation gene deletion homozygous sorghum plant, the DB 08T 0 generation gene deletion homozygous sorghum plant and the DB 09T 0 generation gene deletion homozygous sorghum plant are respectively selfed to respectively obtain sorghum DB03, DB06, DB08 and DB 09T 1 Seeds of generation gene-deleted homozygous plants (from sorghum DB03, DB06, DB08 and DB 09T) 0 And the generation gene deletion homozygous plants are obtained by selfing and used for the next detection.
Example 2 phenotypic observations of knockout lines
Respectively subjecting the sorghum DB03, DB06, DB08 and DB 09T to the processes of 1 Seeds of generation gene-deleted homozygous plants (seeds grown into DB03, DB06, DB08 and DB 09T) 1 Generation gene-deleted homozygous plants) 10 plants each, at 2022, 6 and 5 days, sown in Beijing landThe spots were planted with sorghum P898012 as a control, and when the plants to be planted were mature, DB03, DB06, DB08 and DB 09T were measured, respectively 1 The plant height of the homozygous plant with the gene deletion and sorghum P898012.
Data were processed using SPSS11.5 statistical software, experimental results were expressed as mean ± standard deviation, with significant differences as indicated by P < 0.05 (x) and extremely significant differences as indicated by P < 0.01 (x) using One-way ANOVA.
The results show that: when the plants mature, DB03, DB06, DB08 and DB 09T were observed 1 The generation gene deletion homozygous plants and sorghum P898012 were cultivated in the field and laboratory to find that SgSD1 gene deletion homozygous plants (DB 03, DB06, DB08 and DB 09T) were compared with sorghum P898012 1 Plant height of the generation gene deletion homozygous plant is reduced by about 1/3 (see FIGS. 1 and 2, in FIG. 1, control is sorghum P898012, and DB03, DB06, DB08 and DB09 are DB03, DB06, DB08 and DB 09T, respectively) 1 The plant height unit of the generation gene deletion homozygous plant is centimeter (cm), in figure 2, control is sorghum P898012, and DB03 is DB 03T 1 Homozygous plants with deleted genes, DB06 of DB 06T 1 Homozygous plants with deleted gene, DB08 is DB 08T 1 Homozygous plants with deleted gene, DB09 is DB 09T 1 Homozygous plants with deleted genes).
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. Use of a protein, a substance that modulates the expression of a gene encoding said protein, or a substance that modulates the activity or content of said protein in any of the following:
a1 Application in regulating plant height;
a2 The application of the plant height regulating product is prepared;
the protein is any one of the following:
b1 Amino acid sequence is a protein shown in sequence 2;
b2 A protein which is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein of B1), has 80% or more identity with the protein of B1), and has the same function as the protein;
b3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag.
2. The use according to claim 1, wherein the protein is derived from sorghum.
3. The use according to claim 1 or 2, wherein the substance regulating the expression of the gene encoding the protein is any of the following:
d1 A nucleic acid molecule which inhibits or reduces or down-regulates the expression of a gene encoding the protein according to claim 1 or 2;
d2 Expression of D1) the gene encoding the nucleic acid molecule;
d3 An expression cassette comprising D2) said gene;
d4 A recombinant vector comprising the gene of D2) or a recombinant vector comprising the expression cassette of D3);
d5 A recombinant microorganism comprising the gene of D2), or a recombinant microorganism comprising the expression cassette of D3), or a recombinant microorganism comprising the recombinant vector of D4);
d6 A transgenic plant cell line containing the gene of D2), or a transgenic plant cell line containing the expression cassette of D3), or a transgenic plant cell line containing the recombinant vector of D4);
d7 A transgenic plant tissue containing the gene of D2), or a transgenic plant tissue containing the expression cassette of D3), or a transgenic plant tissue containing the recombinant vector of D4);
d8 A transgenic plant organ containing the gene of D2), or a transgenic plant organ containing the expression cassette of D3), or a transgenic plant organ containing the recombinant vector of D4).
4. A use according to any one of claims 1 to 3, wherein the plant is any one of the following:
g1 Monocotyledonous plants;
g2 A gramineous plant;
g3 Sorghum plants;
g4 Sorghum).
5. A method of reducing plant height, comprising reducing plant height by knocking out or inhibiting or reducing or down-regulating expression in a plant of a gene encoding a protein according to claim 1 or 2 and/or activity and/or content of a protein according to claim 1 or 2.
6. A method of growing a low plant height plant, said method comprising knocking out or inhibiting or reducing or down regulating the expression level of a gene encoding a protein according to claim 1 or 2 in a plant of interest and/or the activity and/or content of said protein to obtain a low plant height plant, said low plant height plant being lower than said plant of interest.
7. The method of claim 5 or 6, wherein said knocking out or inhibiting or reducing or down regulating expression of a gene encoding a protein according to claim 1 or 2 in a plant comprises introducing into said plant of interest a nucleic acid molecule, expression cassette or recombinant vector according to claim 3.
8. The method of any one of claims 5-7, wherein the target sequence of the nucleic acid molecule is nucleotides 248 to 267 of SEQ ID No. 1.
9. The use according to any one of claims 1 to 4, the method according to any one of claims 5 to 8, wherein the plant is any one of the following:
g1 Monocotyledonous plants;
g2 A gramineous plant;
g3 Sorghum plants;
g4 Sorghum).
10. The protein or the substance of any one of claims 1-4.
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