WO2020108620A1 - 提高植物再生能力的靶基因、调控分子及其应用 - Google Patents

提高植物再生能力的靶基因、调控分子及其应用 Download PDF

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WO2020108620A1
WO2020108620A1 PCT/CN2019/121984 CN2019121984W WO2020108620A1 WO 2020108620 A1 WO2020108620 A1 WO 2020108620A1 CN 2019121984 W CN2019121984 W CN 2019121984W WO 2020108620 A1 WO2020108620 A1 WO 2020108620A1
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ham
plant
regeneration
genes
mir171
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French (fr)
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王佳伟
吴连宇
王龙
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中国科学院分子植物科学卓越创新中心
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8259—Phytoremediation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
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    • 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)
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention belongs to the field of botany, and more specifically, the present invention relates to target genes, regulatory molecules and their applications for improving the regeneration ability of plants.
  • Regeneration refers to a process in which the whole body or organ of a living body is partially lost due to trauma, and on the basis of the remaining part, the same structure as the lost part in form and function is grown.
  • hydra, flatworms, echinoderms (starfish, sea lily) and other species can regenerate the complete shape of the wound after cutting their body parts.
  • Plant cells are also totipotent, and isolated plant organs (such as roots, hypocotyls, and leaves) can be regenerated by tissue culture. For example, take a branch from the willow tree, cut it up and down, and insert the lower end into damp sand or water. After a period of time, buds and adventitious roots will grow from the cut surface. Separating the leaves of Begonia or Crassulaceae from the mother body and placing them in a humid environment can easily produce adventitious roots and adventitious buds.
  • Plants establish shoot tip meristem (SAM) and root tip meristem (RAM) during embryonic development. These two meristems have the ability to continue to divide and differentiate, so that new lateral organs (such as leaves, roots, and flowers) can be continuously produced after the embryo, resulting in infinite development of the plant.
  • SAM shoot tip meristem
  • RAM root tip meristem
  • Tissue regeneration means that after injury or loss of tissue or organ, it can repair or re-grow a structure that can replace the function of the original tissue or organ.
  • Somatic embryo regeneration refers to the differentiation of differentiated cells in vitro, which has the ability to differentiate under certain conditions and can form a complete plant through a process similar to embryonic development.
  • Regeneration of plant organs refers to the process in which injured or isolated plant tissue grows adventitious roots or adventitious buds.
  • De novo organ regeneration is an important method of plant regeneration. Unlike the regeneration of somatic embryos, the process of de novo regeneration of plant organs only needs to induce explants (explants or organs) to form SAM and RAM, without the need for embryonic development. process.
  • auxin and cytokinin were the determinants of de novo regeneration of plant organs.
  • CIM exogenous auxin
  • RIM adventitious roots
  • SIM adventitious buds
  • WUSCHEL WUS
  • WUSCHEL is an important regulator of de novo bud regeneration.
  • WUS is the first WOX gene to be discovered. It is expressed in the organizing center (OC) of SAM stem cell niches and is essential for maintaining the activity of SAM stem cells.
  • CLAVATA3 CLV3 is expressed in stem cells and is the direct downstream target gene of WUS. CLV3 can inhibit the expression of WUS through the CLV1-CLV2 receptor kinase pathway, forming the WUS-CLV3 feedback inhibition pathway, which restricts the expression of WUS to the OC region.
  • CLV-WUS The feedback control mechanism of CLV-WUS has played an important role in promoting the cell differentiation process and maintaining the balance of SAM stem cell division and differentiation.
  • cytokinin is highly accumulated at the OC, suggesting that cytokinin may establish SAM by directly activating WUS expression.
  • non-coding RNA In addition to hormones, non-coding RNA is also involved in the regulation of plant regeneration. miRNA is a small molecule non-coding RNA commonly found in plants. It plays an important role in various life activities of plants and is an important regulator of gene expression. It has been found that some key genes that regulate the maintenance and establishment of shoot tip meristems are regulated by miRNA at the post-transcriptional level.
  • miR164 target NAC transcription factors CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 participate in the maintenance and establishment of the SAM border area
  • HD-ZIP III transcription factors PHB/PHV/REV are miR165/6 target genes, phb and phb
  • the rev three mutants exhibited a SAM-deleted phenotype
  • the F-box-like gene LCR is the target gene of miR394.
  • miR394 is expressed in the outermost cell of SAM, and participates in the maintenance of SAM through a short-distance movement to form a concentration gradient.
  • the purpose of the present invention is to provide target genes, regulatory molecules and their applications for improving plant regeneration ability
  • a method for improving the regeneration ability of plants comprising down-regulating HAM (HAIRYMERISTEM) in plants; the HAM includes homologues thereof.
  • the down-regulation of HAM in plants includes down-regulation of its expression or activity.
  • the down-regulation of HAM includes: knocking out or silencing the HAM gene in plants, or inhibiting the activity of the HAM protein; preferably, including but not limited to: interfering molecules that specifically interfere with the expression of the HAM gene
  • knock out the HAM gene by gene editing methods such as gene editing based on the CRISPR system
  • knock out the HAM gene by homologous recombination methods or inhibit the expression of HAM under ultraviolet stress.
  • the interfering molecule is a dsRNA, an antisense nucleic acid, a small interfering RNA, or a microRNA targeting the HAM coding gene or its transcript as an inhibitory or silencing target, or can express or form the dsRNA, Construction of antisense nucleic acid, small interfering RNA, micro RNA.
  • the interfering molecule is a microRNA that encodes HAM or its transcript as a target for inhibition or silencing, which is miR171; preferably, miR171 or its homologous gene in plants is upregulated, or They encode genes or precursor genes, thereby down-regulating HAM expression.
  • the up-regulation of miR171 or its homologous genes, or their coding genes or precursor genes in plants includes up-regulation of their expression or activity.
  • the miRNA precursor can be processed into miR171 or its homologous genes in plants.
  • the method for improving plant regeneration ability is a transgenic method.
  • the up-regulation by a miR171 up-regulator includes a miR171 up-regulator selected from the group consisting of: (a) polynucleotides, which can be transcribed or processed by plants into miR171 or its homologous genes , Or their coding genes or precursor genes, preferably, the polynucleotide has the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10; (b) Expression construct, said expression construct Containing miR171 or its homologous genes, or their coding genes or precursor genes, or the polynucleotides described in (a); (c) miR171 or their homologous genes, or their coding genes or precursor genes Agonist.
  • a polynucleotides, which can be transcribed or processed by plants into miR171 or its homologous genes , Or their coding genes or precursor genes, preferably, the polynucleotide has the sequence shown in SEQ ID NO: 9 or SEQ ID NO:
  • any of the upregulators described in (a) to (c) is introduced into plants to achieve the upregulation.
  • the upregulator is introduced into the plant using the Agrobacterium transformation method; preferably, the method includes: (1) providing Agrobacterium, which carries (a) to (c) Any of the upregulators; (2) contacting plant cells, tissues, or organs with the Agrobacterium in step (1), so that the upregulators of any of (a) to (c) are transferred into plants ; And (3) Selection of plants transformed with the upregulator.
  • the nucleotide sequence of the miR171 is shown in SEQ ID NO: 7 or SEQ ID NO: 8; or the homologous gene of the miR171 and the sequence homology of the miR171 are ⁇ 80% , Preferably ⁇ 90%, more preferably ⁇ 95%, and even more preferably ⁇ 99%.
  • the miR171 is miR171a, miR171b or miR171c.
  • the plant regeneration includes: bud regeneration, root regeneration, cell embryo regeneration; or the plant regeneration includes: plant explant-based regeneration, plant callus-based regeneration;
  • the plant explants or callus include (but are not limited to) explants or callus from the following group of plant tissues: hypocotyls, cotyledons, roots, leaves, embryos, floral organs.
  • the plant includes or the HAM or its homologue is from: dicotyledonous plant; preferably the plant includes a plant selected from the group consisting of Gramineae, Cruciferae , Solanaceae, Leguminosae, Chenopodiaceae, Salixaceae, Malvaceae, Tiliaceae, Rutaceae, Asteraceae, Cucurbitaceae, Papaya, Kapokaceae, Sycamore, Rhamnaceae, Euphorbiaceae, Morusaceae , Plants of the family Acerolaceae, Flaxaceae, Oleaceae, Actinidiaceae, Rosaceae; preferably, the plant includes or the HAM or its homologue is from a plant selected from the group consisting of: Arabidopsis thaliana , Ericium brevifolia, rice, tomato, tobacco, sugar beet, soybean, cabbage, corn, cotton, potato, wheat, Alpine Arabidopsis, mustard green, fla
  • the plant includes or the HAM or its homologue is derived from : Arabidopsis, sugar beet, soybean, cabbage, cotton, rape, tomato, tobacco.
  • the HAM includes: HAM1, HAM2, HAM3.
  • the HAM1 is selected from: (a) a protein such as SEQ ID NO: 1 amino acid sequence; (b) passing the SEQ ID NO: 1 amino acid sequence through one or more (such as 1-20 ; Preferably 1-15; more preferably 1-10, such as 5, 3) formed by substitution, deletion or addition of amino acid residues, and having (a) protein function derived from (a) Protein; or (c) and the protein sequence defined by (a) have more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology And a protein derived from (a) having the function of (a) protein; or (d) adding a tag sequence at the N or C terminus of the protein (a) or (b) or (c), or at its N terminus The protein formed after the signal peptide sequence.
  • a protein such as SEQ ID NO: 1 amino acid sequence
  • the SEQ ID NO: 1 amino acid sequence passes the SEQ ID NO: 1 amino acid sequence through one or
  • the HAM2 is selected from: (a) a protein such as SEQ ID NO: 3 amino acid sequence; (b) passing the SEQ ID NO: 3 amino acid sequence through one or more (such as 1-20 ; Preferably 1-15; more preferably 1-10, such as 5, 3) formed by substitution, deletion or addition of amino acid residues, and having (a) protein function derived from (a) Protein; or (c) and the protein sequence defined by (a) have more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology And a protein derived from (a) having the function of (a) protein; or (d) adding a tag sequence at the N or C terminus of the protein (a) or (b) or (c), or at its N terminus The protein formed after the signal peptide sequence.
  • a protein such as SEQ ID NO: 3 amino acid sequence
  • one or more such as 1-20 ; Preferably 1-15; more preferably 1-10, such as 5,
  • the HAM3 is selected from: (a) a protein such as SEQ ID NO: 5 amino acid sequence; (b) a SEQ ID NO: 5 amino acid sequence through one or more (such as 1-20 ; Preferably 1-15; more preferably 1-10, such as 5, 3) formed by substitution, deletion or addition of amino acid residues, and having (a) protein function derived from (a) Protein; or (c) and the protein sequence defined by (a) have more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology And a protein derived from (a) having the function of (a) protein; or (d) adding a tag sequence at the N or C terminus of the protein (a) or (b) or (c), or at its N terminus The protein formed after the signal peptide sequence.
  • a protein such as SEQ ID NO: 5 amino acid sequence
  • a SEQ ID NO: 5 amino acid sequence through one or more (such as 1-20 ; Prefer
  • the present invention also includes polynucleotides encoding the preceding HAM1, HAM2, HAM3.
  • HAM as a down-regulation target to improve plant regeneration ability; or for screening agents targeting HAM to increase plant regeneration rate.
  • a HAM down-regulating agent for increasing plant regeneration rate; preferably, the HAM down-regulating agent increases the expression of bud regeneration marker genes by down-regulating HAM; more preferably, The bud regeneration marker genes include: WUS, CLV3, CUC1 or CUC2.
  • the HAM down-regulator includes (but is not limited to): a down-regulator that knocks out or silences the HAM gene or inhibits the activity of the HAM protein; preferably, it includes: an interference molecule that specifically interferes with the expression of the HAM gene, Gene editing reagents for knocking out the HAM gene (such as gene editing based on the CRISPR system), reagents for knocking out the HAM gene based on homologous recombination.
  • the interfering molecule is a dsRNA, an antisense nucleic acid, a small interfering RNA, or a microRNA targeting the HAM coding gene or its transcript as an inhibitory or silencing target, or can express or form the dsRNA, Construction of antisense nucleic acid, small interfering RNA, micro RNA.
  • the interfering molecule is a miR171 or miR171 up-regulator that uses the HAM-encoding gene or its transcript as a target for inhibition or silencing.
  • the miR171 up-regulator includes: (a) a polynucleotide, the The polynucleotide can be transcribed or processed by plants into miR171 or its homologous genes, or their coding genes or precursor genes.
  • the polynucleotide has SEQ ID NO: 9 or SEQ ID NO: 10
  • B Expression constructs containing miR171 or its homologous genes, or their coding genes or precursor genes, or the polynucleotides described in (a);
  • the plant regeneration includes: plant explant-based regeneration, plant callus-based regeneration; preferably, the plant explants or callus includes (but is not limited to) from the following group of plants Tissue explants or callus: hypocotyls, cotyledons, roots, leaves, embryos, floral organs.
  • HAM as a molecular marker for identifying regenerative ability; the HAM includes homologues thereof.
  • miR171 or its homologous genes, or their coding genes or precursor genes as molecular markers for identifying regenerative ability.
  • a method for directional selection of plants with enhanced regenerative ability comprising: identifying the expression of HAM in the plant to be tested, if the HAM expression of the plant to be tested is significantly lower than that of the class This species) the average expression value of the HAM of the plant, then it is (potentially) a plant with enhanced regenerative ability; said HAM includes its homologues.
  • a method for directional selection of plants with enhanced regeneration capacity comprising: identifying the expression of miR171 or its homologous genes, or their coding genes or precursor genes in the plant to be tested, If the expression of miR171 or its homologous genes, or their coding genes or precursor genes of the plant to be tested is significantly higher than the average expression value of the plant of this type (or this kind), it is (potentially) the regeneration ability Enhanced plant.
  • a method for screening agents for improving plant regeneration ability comprising: (1) adding a candidate substance to a system containing HAM; (2) observing the system of (1) HAM expression or activity; if the candidate substance inhibits (preferably statistically inhibits; if it is reduced by more than 20%, preferably by more than 50%, more preferably by more than 80%), the expression or activity of HAM, This indicates that the candidate substance is an agent that enhances plant regeneration ability; the HAM includes its homologues.
  • a method for screening agents for improving plant regeneration ability comprising: (1) adding a candidate substance to a gene or precursor containing miR171 or a homologous gene thereof, or a coding gene or precursor thereof Gene system; (2) observe the expression or activity of miR171 or its homologous genes, or their coding genes or precursor genes in the system of (1); if the candidate substance increases (preferably statistically Increase; if it is increased by more than 20%, preferably by more than 50%, and more preferably by more than 80%) the expression or activity of miR171 or its homologous genes, or their coding genes or precursor genes, it indicates that the candidate substance It is a reagent to improve plant regeneration ability.
  • the method further includes setting a control group and a test group to observe the difference between the candidate substance in the test group and the control group.
  • the candidate substances include (but are not limited to): interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands) designed for HAM, or its upstream or downstream proteins or genes, small Molecular compounds (such as hormones) and so on.
  • the candidate substances include (but are not limited to): expression constructs, agonists, etc. designed for miR171 or its coding gene or precursor gene.
  • the system is selected from: a cell system (cell culture system), a subcellular system, a solution system, a plant tissue system, and a plant organ system.
  • the method further includes: performing further cell experiments and/or transgenic tests on the obtained potential substances, to further determine, from the candidate substances, substances that are excellent in improving plant regeneration ability.
  • an expression construct or a kit containing the expression construct containing a polynucleotide, which can be transcribed or processed by a plant into miR171 or a homologue thereof Genes, or their coding genes or precursor genes; or, they contain coding genes or precursor genes of miR171 or its homologous genes; preferably, the expression construct is an expression vector.
  • a plant cell which contains the expression construct, or contains miR171 or its homologous gene introduced from outside, or its coding gene or precursor gene.
  • HAM protein is highly homologous in terrestrial plants.
  • the black frame highlights the protein sequence corresponding to the nucleotide site recognized and cleaved by miR171 in plants of different species, which is highly conserved.
  • MiR171 regulates bud regeneration. Wild type (WT) and mir171a, mir171b and mir171ab double mutants were grown in SIM, and the shoot regeneration rate was counted. n 30.
  • FIG. 1 MiR171 and its target gene HAM regulate bud regeneration.
  • FIG. 5 Experiment of bud regeneration from root and leaf explants.
  • the left half of the petri dish is the budding of callus from the leaf on SIM.
  • the right part of the petri dish is the budding of callus from root on SIM.
  • miR171C-OX does not promote the bud regeneration ability of root-derived callus (right half)
  • miR171C-OX can significantly promote the bud of callus from explants derived from leaves Regeneration ability (left half).
  • FIG. 7 Inducing expression of miR171c can increase the regeneration rate of Arabidopsis buds.
  • FIG. 8 Induced expression of miR171c can increase the regeneration rate of Arabidopsis buds without undergoing callus formation.
  • FIG. 9 Inducing expression of miR171c promotes somatic embryogenesis.
  • FIG. 11 The expression trends of the bud regeneration marker genes WUS and CLV3 during the de novo bud regeneration of Arabidopsis wild-type Col-0 and MIR171C-OX materials; CIM3d, 5d, and 7d are respectively expressed on CIM medium Cultivation for 3, 5, and 7 days; SIM2d, 4d, and 8d indicate cultivation on SIM medium for 2, 4, and 8 days, respectively.
  • Targeting HAM can directly or indirectly amplify the CUC signaling pathway of the marker gene established by buds.
  • CIM3d, 5d, and 7d represent cultivation on CIM medium for 3, 5, and 7 days, respectively;
  • SIM1d, 2d, 4d, and 8d represent cultivation on SIM medium for 1, 2, 4, and 8 days, respectively.
  • miR171 targeting HAM plays a role in promoting plant regeneration by inhibiting HAM.
  • the invention provides a universal, effective and simple new technology for improving plant regeneration rate, provides a new way for improved breeding of plants, and has good application prospects.
  • the present invention discloses new genes involved in the regulation of plant regeneration ability, which are HAM and its homologues.
  • HAM exists highly conservatively in plants, and the present invention is not limited to HAM from Arabidopsis thaliana or corn.
  • the "plant” is a plant suitable for transgenic manipulation, and may be a dicotyledonous plant, a monocotyledonous plant or a gymnosperm plant; it may include crops, flower plants or forestry plants.
  • the "plant” includes dicotyledonous plants; more preferably (but not limited to): Gramineae, Cruciferae, Solanaceae, Leguminosae, Chenopodiaceae, Salixaceae, Malvaceae, Tilia Plants of the family, Rutaceae, Asteraceae, Cucurbitaceae, Papaya, Bombaceae, Sycamore, Rhamnaceae, Euphorbiaceae, Morusaceae, Cerberaceae, Flaxaceae, Osmanthaceae, Kiwiaceae, Rosaceae plants; For example, Arabidopsis spp.
  • the HAM genes/proteins targeted in the present invention their homologues are widely present in plants and exist in a highly conservative manner, which is shown in FIG. 2 of the present invention in.
  • miR171 or its homologous genes that target the HAM gene or its homologues are also widely present in plants and exist in a highly conservative manner. Therefore, it can be understood that the plants of the present invention are not limited to those listed in the examples.
  • the "homologue” includes homologous polypeptides or genes of HAM in multiple species, for example, HAM in Arabidopsis, SCL6, SCL22, SCL27 and SCL6 in Gossupium hirsutum. SCL15, etc.; SCL6, SCL15, SCL22, etc.
  • HAM1 has the amino acid sequence shown in SEQ ID NO:1, and the nucleotide sequence shown in SEQ ID NO:2, where 882-902 are the targeting sites of miR171;
  • the HAM2 has the amino acid shown in SEQ ID NO:3 Sequence, and the nucleotide sequence shown in SEQ ID NO: 4, where 801-821 are the miR171 targeting sites;
  • the HAM3 has the amino acid sequence shown in SEQ ID NO: 5, and the nucleus shown in SEQ ID NO: 6.
  • the sequence of nucleotides, of which 672-692 is the target site of miR171.
  • miR171a, miR171b, and miR171c can all target HAM1, HAM2, and HAM3 to play down the latter (HAM1, HAM2, HAM3).
  • the present invention also includes variant forms having the same functions as the indicated polypeptides.
  • variant forms include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , Insertion and/or substitution, and addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids Insertion and/or substitution, and addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • substitution of amino acids with similar or similar properties usually does not change the function of the protein.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the invention also provides analogues of the polypeptides.
  • the difference between these analogues and the natural polypeptide can be the difference in amino acid sequence, the difference in the modification form that does not affect the sequence, or both.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagen, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • the polynucleotide encoding HAM may be in the form of DNA or RNA.
  • Polynucleotides encoding HAM mature polypeptides include: coding sequences that only encode mature polypeptides; mature polypeptide coding sequences and various additional coding sequences; mature polypeptide coding sequences (and optional additional coding sequences) and non-coding sequences.
  • the invention provides a method for improving plant regeneration ability, which comprises: down-regulating HAM or its homologues in plants.
  • the plant regeneration includes bud regeneration, root regeneration, cell embryo regeneration and the like.
  • the plant regeneration includes plant explant-based regeneration, plant callus-based regeneration, and the like.
  • HAM Gene expression units such as expression vectors or viruses
  • it will carry antisense HAM Gene expression units (such as expression vectors or viruses) are delivered to the target site so that cells or plant tissues do not express or reduce the expression of HAM protein; or the HAM gene is knocked out.
  • the HAM gene may be knocked out, thereby down-regulating the expression of the HAM gene in the plant.
  • the CRISPR/Cas9 system can be used for gene editing, thereby knocking out the HAM gene and improving the plant regeneration ability.
  • Appropriate sgRNA target sites will bring about higher gene editing efficiency, so before embarking on gene editing, it is important to design and find suitable target sites. After designing specific target sites, in vitro cell activity screening is required to obtain effective target sites for subsequent experiments.
  • virus-induced gene silencing can be used to inhibit HAM, thereby improving plant regeneration ability.
  • HAM ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • microRNAs using HAM-encoding genes or their transcripts as targets for inhibition or silencing are used to down-regulate HAM, thereby improving plant regeneration ability.
  • the microRNA is miR171.
  • the present invention also includes miR171 homologous genes, as well as genes encoding or precursor genes of miRNA171 or its homologous genes.
  • the present invention also includes miR171 analogs or derivatives, and their coding genes or precursor genes.
  • homologous genes of miRNA171 include homologous genes of miRNA171 in multiple species, which have the same sequence, substantially the same sequence, or homology as miRNA171 in Arabidopsis, and they also have targeting HAM Performance. At the same time, the coding genes or precursor genes of these "homologous genes" are also included in the present invention.
  • a polynucleotide construct that can be processed to increase the expression of the corresponding miRNA171 or its homologous gene after being introduced can be designed, that is, the polynucleotide construct can be up-regulated in vivo The corresponding amount of miRNA171 or its homologous genes.
  • an isolated polynucleotide (construct) is prepared, which can be transcribed into a precursor miRNA171 by a plant cell, and the precursor miRNA171 can be sheared by a host (such as a plant cell) Cut and expressed into the miRNA171.
  • the polynucleotide construct is located on an expression vector. Therefore, the present invention also includes a vector, which contains the miRNA171, or the polynucleotide construct.
  • the expression vector usually also contains a promoter, an origin of replication, and/or marker genes. Methods well known to those skilled in the art can be used to construct the expression vector required by the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology and so on.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as karamycin, gentamicin, hygromycin, and ampicillin resistance.
  • the invention also relates to the use of HAM and/or miRNA171 as a tracking marker for the progeny of gene transformed plants.
  • the invention also relates to the use of HAM and/or miRNA171 as a molecular marker to determine the regenerative capacity of the plant by detecting the expression of HAM and/or miRNA171 in the plant.
  • HAM and/or miRNA171 of the present invention is preferably obtained from Arabidopsis thaliana, those obtained from other plants are highly homologous to Arabidopsis HAM and/or miRNA171 (such as having more than 80%, such as 85%, 90% , 95%, and even 98% sequence identity) are also within the scope of the present invention.
  • Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
  • the present invention provides a method for screening agents for improving plant regeneration ability, the method comprising: (1) adding a candidate substance to a system containing HAM; (2) observing the expression of HAM in the system of (1) Or activity; if the candidate substance inhibits the expression or activity of HAM, it indicates that the candidate substance is an agent that enhances plant regeneration ability; the HAM includes homologues thereof.
  • the present invention also provides a method for screening agents for improving plant regeneration ability, the method comprising: (1) adding a candidate substance to a system containing miR171 or its homologous genes, or their precursors; (2) Observe the expression or activity of miR171 or its homologous genes, or their precursors in the system of (1); if the candidate substance increases the expression or activity of miR171 or its homologous genes, or their precursors, it indicates that Candidate substances are agents that increase the plant's ability to regenerate.
  • the method of screening a substance acting on the target with the protein or a specific region on it as a target is well known to those skilled in the art, and these methods can be used in the present invention.
  • the candidate substance may be selected from: peptides, polymerized peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. Depending on the type of substance to be screened, those skilled in the art know how to select an appropriate screening method.
  • the Arabidopsis Col-0 ecotype is used as the wild-type Arabidopsis.
  • pHB-MIR171C vector construction PCR amplification of Arabidopsis MIR171C fragments was cloned into the pBSK vector between BamH I and Xba I sites. After sequencing to confirm the sequence was correct, the MIR171C fragment was cut out with BamH I and Xba I and cloned into the binary vector pHB vector. This vector carries a 2x35S promoter. The gene sequence of the fragment of MIR171C is shown in SEQ ID NO: 9 (of which the 497-517th position is the corresponding position of the mature sequence of miR171c).
  • p35S-MIR171A vector Amplify the fragment of Arabidopsis thaliana MIR171A by PCR, clone it into the binary vector JW807 expression vector Kpn I and Spe I using homologous recombinase, and use it in subsequent experiments after confirming the sequence is correct.
  • the gene sequence of the fragment of MIR171A is shown in SEQ ID NO: 10 (of which positions 591 to 611 are corresponding positions of the mature sequence of miR171a).
  • p35S-MIR171B vector construction PCR amplification of Arabidopsis MIR171B fragments, using homologous recombinase cloned into binary vector JW807 expression vector (Tian-Qi, Zhang et al, The Plant Plant Cell, Vol 27:349-360, Feb, 2015) Between Kpn I and Spe I sequenced to confirm the sequence was correct and used for subsequent experiments.
  • the gene sequence of the fragment of MIR171B is shown in SEQ ID NO: 11 (of which positions 522-542 are the corresponding positions of the mature sequence of miR171b).
  • pER8-MIR171C vector Amplify the fragment of Arabidopsis MIR171C by PCR and clone into the pBSK vector between Xho I and Spe I sites. After sequencing to confirm that the sequence is correct, the MIR171C fragment was cut out with Xho I and Spe I and cloned into the pER8 vector (Zuo et al., Plant J, 2000 (Volume 24, Issue 2)). The construction of pHB-MIR171A or pHB-MIR171B vector is similar.
  • pHB-GFP-rHAM1 and pHB-GFP-rHAM3 vectors PCR amplification was used to obtain the full-length sequence of GFP, cloned into the PBSK vector between Pst I and Spe I sites, and the intermediate vector was named pBS-GFP.
  • rHAM1 and rHAM3 are mutant forms of anti-miR171. They carry out site-directed mutation at the recognition site of miR171 in the coding region to keep the encoded amino acid sequence unchanged.
  • the construction method adopts fusion PCR.
  • Plants need to be planted in advance to bloom before transformation, and long-day sunshine conditions will grow for about 30 days.
  • the screening of plant transgene generally has three resistance markers of Basta, Kan and Hygro. After the T0 plants of Basta are suspended with 1 ⁇ Agrose, they are evenly planted in 0.05% Basta soil. Ton plants of Kan and Hygro, after aseptic treatment, were spread on Hygro-resistant 1/2MS medium containing 50 ⁇ g/mL Kan or 40 ⁇ g/mL Hygro. Gene expression analysis.
  • hypocotyl tissue was cut and placed in CIM medium and cultured at 22°C for 7 days.
  • 70s::miR171C expression vector construction replace the 35S promoter in the JW807 expression vector with the 70s promoter, and use the 70s promoter to drive the expression of miR171C (Arabidopsis origin, SEQ ID NO: 9).
  • Transgenic plants were regenerated by agrobacterium infecting sugar beet callus and de novo regeneration of callus buds.
  • the method of sugar beet transformation and determination can also be found in WO2019134884A1.
  • AtrHAM1 sequence is as SEQ ID NO:12.
  • 35s::AtrHAM1-3xFLAG was obtained.
  • Construction vector pRIBO::MIM171a (for silencing miR171):
  • AtIPS1 SEQ ID NO: 14; MIM171a sequence included:
  • RIBO::MIM171a was obtained.
  • the RIBO promoter sequence is as follows (SEQ ID NO: 15):
  • MIR171B SEQ ID NO: 11, Arabidopsis origin, AtMIR171B
  • 35s::AtMIR171B was obtained.
  • the tobacco leaf disc method was used to transgene, and T2 transgenic plants were obtained.
  • Arabidopsis bud regeneration rate characterized by the total number of regenerated buds in each treatment combination except the number of explants.
  • Arabidopsis adventitious root regeneration rate characterized by the total number of adventitious roots regenerated in each treatment combination except for the number of explants.
  • Arabidopsis somatic embryogenesis capacity characterized by the number of somatic embryos regenerated from each explant (embryonic callus).
  • the target gene of miR171 is a GRAS transcription factor, which includes three genes in the Arabidopsis genome, namely HAM1, HAM2 and HAM3.
  • HAM has homologous proteins in most terrestrial plants, and the sites recognized by miR171 are highly conserved. That is, the mechanism by which miR171 targets HAM is highly conserved in plants.
  • miR171 There are three coding genes for miR171 in the Arabidopsis genome, MIR171A, MIR171B, and MIR171C; their encoded products are the mature sequences of miR171a, miR171b, and miR171c, of which the miR171b and miR171c mature sequences are the same.
  • the inventors prepared mir171a, mir171b and mir171ab mutants, respectively, and examined the bud regeneration ability of the mutants.
  • HAM is the only target of miR171 in plants, and its or its homologous genes are conservatively present in various plants. According to the existing data, miR171 can cleave its target genes HAM1, HAM2 and HAM3. Therefore, in plants overexpressing miR171, the mRNA levels of HAM1, HAM2 and HAM3 will be significantly reduced (Wang et al., Mol Plant, 2010 vol 3. and Llave, C. et al., Science, 2002. vol 297). According to the foregoing Example 2, it can be seen that miR171 exerts a function of regulating regeneration by targeting its only target, HAM. The present inventors further studied the bud regeneration of Arabidopsis thaliana overexpressing miR171 and down-regulating HAM.
  • the inventors constructed Arabidopsis MIR171C overexpression plants (MIR171C-OX, obtained by transferring the pHB-MIR171C vector into the Arabidopsis Col-0 ecotype), ham1ham2ham3 triple mutants and plants overexpressing HAM1 and HAM3 (35S::rHAM1 and 35S::rHAM3).
  • the following hypocotyls are the test bud regeneration rate of explants.
  • Plant callus can be derived from different plant organs and tissues. Commonly used plant hypocotyls, cotyledons, roots and leaves. The present inventors further investigated whether miR171 overexpression can increase the regeneration rate of explants from other sources, and investigated the ability of callus derived from roots and leaves of wild type (Col-0 ecotype) and MIR171C-OX plant materials to regenerate buds. The results are shown in Figure 5. The callus regeneration ability of both plant materials is very strong, while the callus regeneration ability of wild type leaves is much lower than that of MIR171C-OX.
  • Example 4 The miR171-HAM pathway promotes the expression of bud regeneration marker genes
  • wild type and MIR171C-OX were selected as materials, and cultured on SIM medium for a period of 2 hours to 96 hours. The amount of expression.
  • the present inventors analyzed the changes in the expression levels of the bud regeneration marker genes CUC1 and CUC2 during the de novo regeneration of Arabidopsis wild-type Col-0 and MIR171C-OX materials. The results are shown in Figure 12, showing that overexpression of miR171 can directly or indirectly amplify the CUC signaling pathway established by buds through targeted down-regulation of HAM.
  • Example 5 Induced expression of miR171 significantly reduces the expression level of HAM
  • miR171c can be induced and expressed by estradiol (ES).
  • ES estradiol
  • PER8-MIR171C was transformed into Arabidopsis thaliana to obtain transgenic plants.
  • the obtained transgenic PER8-MIR171C plants were cultured on SIM medium, and after induction treatment of the plant material with 5 micromoles of estradiol/DMSO, quantitative PCR analysis of the changes in the expression levels of pri-mir171c and HAM2; the results are shown in FIG. 13 and shown The mRNA level of HAM2 in PER8-MIR171C plants was significantly reduced.
  • Example 6 Inducing expression of miR171 can also increase plant shoot regeneration rate
  • explants in Arabidopsis thaliana can greatly improve cell totipotency and bud regeneration rate through callus culture, and the bud regeneration rate of explants without going through the callus culture stage is extremely low. So can high miR171 expression bypass the callus culture stage and directly increase the bud regeneration rate of explants?
  • the inventors selected the roots of Arabidopsis seedlings cultured vertically on 1/2MS for 7 days as explants, and the roots of PER8-MIR171C and wild type (WT) were explants (1 cm long), directly placed
  • the bud regeneration ability was examined on SIM medium containing DMSO (20 ⁇ M, control group) or ES (20 ⁇ M, experimental group). As shown in Figure 8, inducing expression of MIR171C on SIM can directly significantly enhance the bud regeneration ability of explants, and explants do not need to go through the callus culture stage.
  • Example 7 Inducing expression of miR171 can increase plant somatic embryo regeneration rate
  • PER8-MIR171C material was used to further investigate the effect of MIR171C on Arabidopsis somatic embryogenesis.
  • Example 8 Excessive or induced expression of miR171 can increase plant root regeneration rate
  • Example 9 The miR171-HAM pathway promotes regeneration under hypercytokinin conditions
  • Higher plants generally regenerate in two ways: somatic cell generation and de novo organ regeneration.
  • the regeneration of dicotyledonous plants generally selects various tissues of seedlings, such as cotyledons, roots or hypocotyls. These explants are regenerated through a de novo organ regeneration pathway under high cytokinin conditions.
  • monocotyledonous plants lose their ability to regenerate after embryos, so monocotyledonous plants generally select immature embryos as explants to achieve regeneration through somatic embryonic pathways. In this process, high concentrations of auxin are essential of.
  • the above experimental results indicate that the miR171-HAM pathway plays a role in the de novo organ regeneration process mediated by hypercytokinin, which plays a role in improving the regeneration rate. It can be inferred that the miR171-HAM pathway can effectively promote the regeneration of dicotyledonous plants.
  • Example 10 overexpression of miR171 can improve the regeneration rate of sugar beet and tobacco
  • the inventors determined the effect of miR171C (from Arabidopsis thaliana) on the efficiency of Agrobacterium-mediated transgenesis of dicotyledonous sugar beet (Sugarbeet).
  • Agrobacterium-mediated callus transformation experiments were carried out using the ubiquitous expression of promoter-driven miR171C or tDT in sugar beet, or a mixture of two bacterial solutions for co-transformation, followed by plant resistance screening to obtain positive transgenic plants, statistical transformation Efficiency (Transformation). See WO2019134884A1 for the determination method.
  • the transformation operation of sugar beet is to infect the beet callus (that is, transgenic) by Agrobacterium and regenerate the transgenic plant by de novo regeneration of callus bud.
  • the efficiency of Agrobacterium infection is very high, and the bud regeneration rate is relatively low, which is the limiting factor of transformation. Therefore, the final transformation efficiency directly reflects the regeneration rate, that is, the regeneration ability of callus.
  • Transgenic plants using tobacco leaf disc method obtained T2 generation tobacco transgenic plants, cut the leaves into small pieces (0.5cm ⁇ 0.5cm), placed in MS0 culture for 2 days, and then transferred to MS1 culture, wait for the buds to grow, statistics The number of regenerated buds per explant was compared with wild-type tobacco. The results showed that overexpression of MIR171A/B can increase the number of bud regeneration of transgenic tobacco, while overexpression of rHAM1 and overexpression of MIM171a will reduce the number of bud regeneration of transgenic tobacco.
  • Test group Arabidopsis cell line (which endogenously expresses HAM), and the candidate substance is given;
  • Control group Arabidopsis cell line (with endogenously expressed HAM), no candidate substance was administered.
  • HAM in the test group and the control group were detected and compared. If the expression of HAM in the test group is statistically lower (eg, more than 30% lower) in the control group, it indicates that the candidate substance is an agent that improves plant regeneration ability.

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Abstract

提供了提高植物再生能力的靶基因、调控分子及其应用，所述调节植物再生能力的靶标为HAM，靶向HAM的miR171通过抑制HAM促进植物再生，可用于植物的改良育种。

Description

提高植物再生能力的靶基因、调控分子及其应用 技术领域
本发明属于植物学领域，更具体地，本发明涉及提高植物再生能力的靶基因、调控分子及其应用。
背景技术
再生(regeneration)是指生物体的整体或器官因创伤而发生部分丢失，在剩余部分的基础上又生长出与丢失部分在形态和功能上相同结构的过程。在动物中，九头蛇、涡虫、棘皮动物(海星、海百合)等物种将其身体部分切割后，都能在伤口处再生出完整的形态。植物细胞同样具有全能性(totipotent)，离体植物器官(比如根、下胚轴和叶)可以通过组织培养实现植株再生。例如从柳树上取下一段枝条，上下切断，下端插入潮湿的沙土或水中，经过一段时间，就会从上下切割表面分别长出芽和不定根。将秋海棠属或景天科植物的叶子从母体分离下来，放在潮湿环境下，容易产生不定根与不定芽。
植物在胚胎发育过程中建立起茎尖分生组织(shoot apical meristem，SAM)与根尖分生组织(root apical meristem，RAM)。这两个分生组织具有持续分裂分化的能力，从而可以在胚后不断产生新的侧生器官(lateral organ，例如叶、根和花)，导致植物的发育呈现无限性。
高等植物再生可分为三种：组织再生(tissue regeneration)，体细胞胚再生(somatic embryogenesis)和器官从头再生(de novo organogenesis)。组织再生是指组织或器官在损伤或缺失后，可以修复或重新生长出能够替代原来组织器官行使功能的结构。体细胞胚再生是指离体的已分化的细胞，在一定条件培养下，重新具备分化能力，并且可以经过类似胚胎发育的过程形成完整植株。植物器官从头再生是指受伤或离体的植物组织长出不定根或不定芽的过程。器官从头再生是植物再生的重要方式，与体细胞胚再生不同，植物器官从头再生的过程仅需诱导外植体(explant，即离体组织或器官)形成SAM和RAM，无需经过类似胚胎发育的过程。
1957年Skoog and Miller发现生长素和细胞分裂素是植物器官从头再生的决定因素。在组织培养过程中，首先利用高浓度外源生长素(CIM)诱导外植体形成愈伤组织(callus)；愈伤组织再经过进一步诱导形成不定根(高生长素/细胞分裂素配比，RIM)或不定芽(高细胞分裂素/生长素配比，SIM)。但是，通过不同激素配比诱导植物体细胞再生成为芽具有明显的物种特异性，一些重要农作物的栽培种和一些非模式植物的再生效率极低，大大延长了作物育种和分子生物学实验周期，是目前困扰植物基础科学研 究和创新农业发展的核心问题。
与根从头再生相比，芽从头再生过程的分子机制仍不清晰。WUSCHEL(WUS)是芽从头再生的重要调控因子。WUS是第一个被发现的WOX基因，它在SAM干细胞龛的组织中心(organizer center，OC)表达，对于维持SAM干细胞活性至关重要。CLAVATA3(CLV3)在茎尖干细胞(stem cell)中表达，是WUS的直接下游靶基因。CLV3可以通过CLV1-CLV2受体激酶通路抑制WUS的表达，形成WUS-CLV3反馈抑制通路，使得WUS的表达局限在OC区域。CLV-WUS这一反馈调控机制在促进细胞分化进程与维持SAM干细胞分裂及分化的平衡等方面发挥了重要作用。研究发现，wus突变体完全丧失芽再生能力，表明WUS也是调控芽再生的关键因子。有趣的是细胞分裂素在OC处高度积累，提示细胞分裂素可能通过直接激活WUS的表达建立SAM。
除了激素外，非编码RNA也参与了植物再生的调控。miRNA是植物中普遍存在的小分子非编码RNA，在植物的各项生命活动中发挥着重要作用，是基因表达的重要调控因子。目前已发现调控茎尖分生组织维持与建立的一些关键基因都在转录后水平受到miRNA的调控。例如，miR164靶标的NAC类转录因子CUP-SHAPED COTYLEDON1(CUC1)和CUC2参与SAM的边界区的维持和建立；HD-ZIP III类转录因子PHB/PHV/REV是miR165/6的靶标基因，phb phb rev三突变体呈现出SAM缺失的表型；F-box类基因LCR是miR394的靶基因，miR394在SAM的最外层细胞表达，通过短距离移动形成浓度梯度参与SAM的维持。但是，这些发现是不够的，还不能满足解释植物再生的机理所需，本领域仍需进一步的探索。
发明内容
本发明的目的在于提供提高植物再生能力的靶基因、调控分子及其应用
在本发明的第一方面，提供一种提高植物再生能力的方法，所述方法包括下调植物中HAM(HAIRY MERISTEM)；所述的HAM包括其同源物。
在一个优选例中，所述的下调植物中HAM包括下调其表达或活性。
在另一优选例中，所述的下调HAM包括：在植物中敲除或沉默HAM基因，或抑制HAM蛋白的活性；较佳地，包括但不限于：以特异性干扰HAM基因表达的干扰分子来沉默HAM，以基因编辑方法(如基于CRISPR系统的基因编辑)敲除HAM基因，以同源重组方法敲除HAM基因，或以紫外胁迫抑制HAM表达。
在另一优选例中，所述的干扰分子是以HAM的编码基因或其转录本为抑制或沉默靶标的dsRNA、反义核酸、小干扰RNA、微小RNA，或能表达或形成所述dsRNA、反义核酸、小干扰RNA、微小RNA的构建物。
在另一优选例中，所述的干扰分子是以HAM的编码基因或其转录本为抑制或沉 默靶标的微小RNA，其是miR171；较佳地，上调植物中miR171或其同源基因、或它们的编码基因或前体基因，从而下调HAM表达。
在另一优选例中，所述的上调植物中miR171或其同源基因、或它们的编码基因或前体基因包括上调它们的表达或活性。
在另一优选例中，所述的miRNA的前体能在植物中被加工成miR171或其同源基因。
在另一优选例中，所述的提高植物再生能力的方法为转基因方法。
在另一优选例中，通过miR171上调剂进行上调，包括选自下组的miR171上调剂：(a)多核苷酸，所述的多核苷酸能被植物转录或加工成miR171或其同源基因、或它们的编码基因或前体基因，较佳地，所述多核苷酸具有SEQ ID NO:9或SEQ ID NO:10所示的序列；(b)表达构建物，所述的表达构建物含有miR171或其同源基因、或它们的编码基因或前体基因、或(a)中所述的多核苷酸；(c)miR171或其同源基因、或它们的编码基因或前体基因的激动剂。
在另一优选例中，将(a)～(c)任一所述的上调剂引入到植物中，从而实现所述的上调。
在另一优选例中，利用农杆菌转化法将所述上调剂引入到植物中；较佳地，所述方法包括：(1)提供农杆菌，所述农杆菌携带(a)～(c)任一所述的上调剂；(2)将植物细胞、组织或器官与步骤(1)中的农杆菌接触，从而使所述(a)～(c)任一所述的上调剂转入植物；和(3)选择出转入了所述上调剂的植物。
在另一优选例中，所述的miR171的核苷酸序列如SEQ ID NO:7或SEQ ID NO:8所示；或所述的miR171的同源基因与miR171的序列同源性≥80％，较佳地≥90％，更佳地≥95％，进一步更佳地≥99％。
在另一优选例中，所述的miR171为miR171a，miR171b或miR171c。
在另一优选例中，所述的植物再生包括：芽再生，根再生，细胞胚再生；或所述的植物再生包括：植物外植体为基础的再生，植物愈伤组织为基础的再生；较佳地，所述的植物外植体或愈伤组织包括(但不限于)来自下组植物组织的外植体或愈伤组织：下胚轴，子叶，根，叶片，胚胎，花器官。
在另一优选例中，所述的植物包括或所述的HAM或其同源物来自于：双子叶植物；较佳地所述的植物包括选自下组的植物：禾本科，十字花科，茄科，豆科，藜科，杨柳科，锦葵科，椴树科，芸香科，菊科，葫芦科，番木瓜科，木棉科，梧桐科，鼠李科，大戟科，桑科，土瓶草科，胡麻科，木犀科，猕猴桃科，蔷薇科的植物；较佳地，所述的植物包括或所述的HAM或其同源物来自于选自下组的植物：拟南芥，二穗短柄草，稻，番茄，烟草，甜菜，大豆，白菜，玉米，棉花，马铃薯，麦，高山南芥， 芥菜，亚麻芥，油菜，Eutrema saisugineum，黄麻(包括糙叶山黄麻，异色山黄麻，长溯黄麻)，杨树，克里曼丁橘，莴苣，番南瓜，番木瓜，西葫芦，向日葵，榴莲，青蒿，南瓜，可可豆，枣树，三叶橡胶，川桑，胡杨，土瓶草，木豆，Cynara cardunculus var.scolymus，芝麻，甜橙，蜜柑，枸橘，油橄榄，猕猴桃，月季；更佳地，所述的植物包括或所述的HAM或其同源物来自于：拟南芥，甜菜，大豆，白菜，棉花，油菜，番茄，烟草。
在另一优选例中，所述的HAM包括：HAM1，HAM2，HAM3。
在另一优选例中，所述的HAM1选自：(a)如SEQ ID NO:1氨基酸序列的蛋白；(b)将SEQ ID NO:1氨基酸序列经过一个或多个(如1-20个；较佳地1-15个；更佳地1-10个，如5个，3个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)蛋白功能的由(a)衍生的蛋白；或(c)与(a)限定的蛋白序列有80％以上(较佳地85％以上；更佳地90％以上；更佳95％以上，如98％，99％)同源性且具有(a)蛋白功能的由(a)衍生的蛋白；或(d)在(a)或(b)或(c)所述蛋白的N或C末端添加标签序列，或在其N末端添加信号肽序列后形成的蛋白。
在另一优选例中，所述的HAM2选自：(a)如SEQ ID NO:3氨基酸序列的蛋白；(b)将SEQ ID NO:3氨基酸序列经过一个或多个(如1-20个；较佳地1-15个；更佳地1-10个，如5个，3个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)蛋白功能的由(a)衍生的蛋白；或(c)与(a)限定的蛋白序列有80％以上(较佳地85％以上；更佳地90％以上；更佳95％以上，如98％，99％)同源性且具有(a)蛋白功能的由(a)衍生的蛋白；或(d)在(a)或(b)或(c)所述蛋白的N或C末端添加标签序列，或在其N末端添加信号肽序列后形成的蛋白。
在另一优选例中，所述的HAM3选自：(a)如SEQ ID NO:5氨基酸序列的蛋白；(b)将SEQ ID NO:5氨基酸序列经过一个或多个(如1-20个；较佳地1-15个；更佳地1-10个，如5个，3个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)蛋白功能的由(a)衍生的蛋白；或(c)与(a)限定的蛋白序列有80％以上(较佳地85％以上；更佳地90％以上；更佳95％以上，如98％，99％)同源性且具有(a)蛋白功能的由(a)衍生的蛋白；或(d)在(a)或(b)或(c)所述蛋白的N或C末端添加标签序列，或在其N末端添加信号肽序列后形成的蛋白。
在另一优选例中，本发明还包括编码前面HAM1、HAM2、HAM3的多核苷酸。
在本发明的另一方面，提供一种HAM的用途，用于作为下调靶点，以提高植物再生能力；或用于筛选靶向于HAM从而提高植物再生率的试剂。
在本发明的另一方面，提供一种HAM下调剂的用途，用于提高植物再生率；较佳地，所述的HAM下调剂通过下调HAM，提高芽再生标志基因的表达；更佳地，所 述芽再生标志基因包括：WUS、CLV3、CUC1或CUC2。
在一个优选例中，所述的HAM下调剂包括(但不限于)：敲除或沉默HAM基因或抑制HAM蛋白活性的下调剂；较佳地，包括：特异性干扰HAM基因表达的干扰分子，敲除HAM基因的基因编辑(如基于CRISPR系统的基因编辑)试剂，基于同源重组的敲除HAM基因的试剂。
在另一优选例中，所述的干扰分子是以HAM的编码基因或其转录本为抑制或沉默靶标的dsRNA、反义核酸、小干扰RNA、微小RNA，或能表达或形成所述dsRNA、反义核酸、小干扰RNA、微小RNA的构建物。
在另一优选例中，所述的干扰分子是以HAM的编码基因或其转录本为抑制或沉默靶标的miR171或miR171上调剂，所述miR171上调剂包括：(a)多核苷酸，所述的多核苷酸能被植物转录或加工成miR171或其同源基因、或它们的编码基因或前体基因，较佳地，所述多核苷酸具有SEQ ID NO:9或SEQ ID NO:10所示的序列；(b)表达构建物，所述的表达构建物含有miR171或其同源基因、或它们的编码基因或前体基因、或(a)中所述的多核苷酸；(c)miR171或其同源基因、或它们的编码基因或前体基因的激动剂。
所述的植物再生包括：植物外植体为基础的再生，植物愈伤组织为基础的再生；较佳地，所述的植物外植体或愈伤组织包括(但不限于)来自下组植物组织的外植体或愈伤组织：下胚轴，子叶，根，叶片，胚胎，花器官。
在本发明的另一方面，提供一种HAM的用途，用于作为鉴定再生能力的分子标记；所述的HAM包括其同源物。
在本发明的另一方面，提供一种miR171或其同源基因、或它们的编码基因或前体基因的用途，用于作为鉴定再生能力的分子标记。
在本发明的另一方面，提供一种定向选择再生能力增强的植物的方法，所述方法包括：鉴定待测植物中HAM的表达，若是该待测植物的HAM表达显著低于该类(或该种)植物的HAM平均表达值，则其为(潜在地为)再生能力增强的植物；所述的HAM包括其同源物。
在本发明的另一方面，提供一种定向选择再生能力增强的植物的方法，所述方法包括：鉴定待测植物中miR171或其同源基因、或它们的编码基因或前体基因的表达，若是该待测植物的miR171或其同源基因、或它们的编码基因或前体基因的表达显著高于该类(或该种)植物的平均表达值，则其为(潜在地为)再生能力增强的植物。
在本发明的另一方面，提供一种筛选提高植物再生能力的试剂的方法，所述方法包括：(1)将候选物质加入到含有HAM的体系中；(2)观测(1)的体系中HAM的表达或活性；若所述候选物质抑制(较佳地是统计学上抑制；如降低20％以上，较佳地抑制50％ 以上，更佳地抑制80％以上)HAM的表达或活性，则表明该候选物质是提高植物再生能力的试剂；所述的HAM包括其同源物。
在本发明的另一方面，提供一种筛选提高植物再生能力的试剂的方法，所述方法包括：(1)将候选物质加入到含有miR171或其同源基因、或它们的编码基因或前体基因的体系中；(2)观测(1)的体系中miR171或其同源基因、或它们的编码基因或前体基因的表达或活性；若所述候选物质提高(较佳地是统计学上提高；如提高20％以上，较佳地提高50％以上，更佳地提高80％以上)miR171或其同源基因、或它们的编码基因或前体基因的表达或活性，则表明该候选物质是提高植物再生能力的试剂。
在一个优选例中，所述的方法还包括设置对照组与测试组，以观测候选物质在测试组与对照组中的区别。
在另一优选例中，所述的候选物质包括(但不限于)：针对HAM，或其上游或下游蛋白或基因设计的干扰分子、核酸抑制物、结合分子(如抗体或配体)、小分子化合物(如激素)等。
在另一优选例中，所述的候选物质包括(但不限于)：针对miR171或其编码基因或前体基因设计的表达构建物、激动剂等。
在另一优选例中，所述的体系选自：细胞体系(细胞培养物体系)、亚细胞体系、溶液体系、植物组织体系、植物器官体系。
在另一优选例中，所述方法还包括：对获得的潜在物质进行进一步的细胞实验和/或转基因试验，以从候选物质中进一步确定对于提高植物再生能力效果优异的物质。
在本发明的另一方面，提供表达构建物或含有该表达构建物的试剂盒，所述表达构建物含有多核苷酸，所述的多核苷酸能被植物转录或加工成miR171或其同源基因、或它们的编码基因或前体基因；或，其含有miR171或其同源基因的编码基因或前体基因；较佳地，所述的表达构建物为表达载体。
在本发明的另一方面，提供一种植物细胞，其中含有所述的表达构建物，或其中包含外源引入的miR171或其同源基因、或它们的编码基因或前体基因。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、miR171的进化分析。斜线标示区域代表在所有陆生植物中均能找到。
图2、HAM蛋白保守性分析。HAM蛋白在陆生植物中高度同源，黑框突出显示不同物种植物中被miR171所识别并剪切的核苷酸位点所对应的蛋白质序列，该位点高度保守。
图3、miR171调控芽再生。野生型(WT)和mir171a，mir171b和mir171ab双突变 体在SIM生长，统计芽再生率。n＝30。
图4、miR171与其靶基因HAM调控芽再生。野生型(WT)、MIR171C-OX(OVX)、ham1 ham2 ham3三突变体、35S::rHAM1和35S::rHAM3外植体在SIM生长，统计芽再生率。n＝30。
图5、来源于根和叶片外植体芽再生实验。培养皿左半部为来自于叶片的愈伤组织在SIM上的生芽情况。培养皿右半部为来自于根的愈伤组织在SIM上的生芽情况。相较于野生型(Col-0)，miR171C-OX并不会促进根来源愈伤组织的芽再生能力(右半部)，而miR171C-OX可以显著促进叶片来源外植体愈伤组织的芽再生能力(左半部)。
图6、WUS和CLV3的表达模式。野生型(WT)和miR171C-OX在SIM生长诱导芽再生。在不同时间点取材，通过定量PCR检测WUS和CLV3的表达量。内参选取UBQ10。n＝3。其中SIM2h、4h、8h、24h、48h、96h分别表示在SIM培养基上培养2、4、8、24、48、96小时。
图7、诱导表达miR171c可以提高拟南芥芽再生率。PER8-MIR171C外植体(下胚轴)先在含有DMSO(对照组)或ES(实验组)的CIM培养基上培养7天，然后转移至无ES或DMSO的SIM培养基上培养24天。统计再生芽数量。n＝32。
图8、诱导表达miR171c可以在不经历愈伤组织形成的条件下提高拟南芥芽再生率。野生型与PER8-MIR171C外植体(根)在含有DMSO(对照组)或ES(实验组)的SIM培养基上。统计再生芽数量。n＝32。
图9、诱导表达miR171c促进体细胞胚的发生。PER8-MIR171C幼胚在含有DMSO(对照组)或ES(实验组)的E5培养基上生长15天，然后转移到MS培养基上。n＝30。
图10、诱导表达miR171c促进根再生。不同植物在1/2MS培养基上生长12天，然后在B5，B5(20μM DMSO)或B5(含有20μM ES)的培养基上考察生根能力。统计每天的生根率(生根植物/总植物)，从处理后第6天统计到第16天。n＝60。
图11、拟南芥野生型Col-0及MIR171C-OX材料在芽从头再生过程中，芽再生标志基因WUS和CLV3的表达量变化趋势；其中，CIM3d、5d、7d分别表示在CIM培养基上培养3、5、7天；SIM2d、4d、8d分别表示在SIM培养基上培养2、4、8天。
图12、靶向HAM可以直接或间接放大芽建立的标志基因CUC信号途径。其中，CIM3d、5d、7d分别表示在CIM培养基上培养3、5、7天；SIM1d、2d、4d、8d分别表示在SIM培养基上培养1、2、4、8天。
图13、PER8-MIR171C植物中HAM2的mRNA水平被显著降低。
图14、转化过表达miR171C的质粒或者共转过表达miR171C的质粒，可以显著提高甜菜的转基因效率。
具体实施方式
本发明人经过深入的研究，揭示了调节植物再生能力的新的靶标HAM及其同源物。在植物体内，靶向于HAM的miR171通过抑制HAM，发挥促进植物再生的作用。本发明提供了具有普适性的、有效和简便的提高植物再生率的新技术，为植物的改良育种提供新的途径，具有良好的应用前景。
本发明揭示了新的参与植物再生能力调控的基因，为HAM及其同源物。HAM在植物中高度保守地存在，本发明中不仅限于来自拟南芥或玉米的HAM。
如本文所用，所述的“植物”是适用于进行转基因操作的植物，可以是双子叶植物、单子叶植物或裸子植物；可以包括农作物、花卉植物或林业植物等。较佳地，所述的“植物”包括双子叶植物；更佳地包括(但不仅限于)：禾本科，十字花科，茄科，豆科，藜科，杨柳科，锦葵科，椴树科，芸香科，菊科，葫芦科，番木瓜科，木棉科，梧桐科，鼠李科，大戟科，桑科，土瓶草科，胡麻科，木犀科，猕猴桃科，蔷薇科的植物；例如，十字花科鼠耳芥属如拟南芥；禾本科稻属植物如水稻，禾本科小麦属植物如小麦，禾本科玉米属植物如玉米等；十字花科芸薹属的大白菜、小白菜、油菜；锦葵科棉属作物如棉花；茄科番茄属植物如番茄，茄科烟草属植物如烟草，藜科甜菜属的甜菜，豆科大豆属的大豆等。根据本发明人对于植物的大规模分析，本发明中所靶向的HAM基因/蛋白，其同源物广泛地存在于植物中，且以高度保守的方式存在，这体现在本发明的图2中。同样地，靶向于HAM基因或其同源物的miR171或其同源基因也广泛地存在于植物中，且以高度保守的方式存在。因此，可以理解，本发明的植物并不限于实施例中所列举的。
如本文所用，所述的“同源物”包括在多个物种中HAM的同源多肽或基因，例如拟南芥中为HAM，在陆生棉(Gossupium hirsutum)中为SCL6，SCL22，SCL27和SCL15等；在烟草(Nicotiana tabacum)中SCL6，SCL15和SCL22等；在番茄中(Solanum lycopersicum)中为NP_001333839.1，NP_001333836.1(HAM)，XP_004232383.1(SCL15)和XP_004232402.1(SCL7)等；在油菜(Brassica napus)中为BnaAnng18540D和BnaAnng18550D；在玉米(Zea mays L.)中为GRMZM2G037792_T01(GRAS79)，GRMZM5G825321_T01(GRAS transcription factor)，GRMZM5G825321_T02(GRAS transcription factor)。在水稻中为OsHAM1，OsHAM2，OsHAM3，和OsHAM4。玉米ZmMIR171靶向于GRMZM2G037792(GRAS79)，GRMZM5G825321_T01(GRAS transcription factor)和GRMZM5G825321_T02(GRAS transcription factor)。
例如，在拟南芥中存在HAM1，HAM2，HAM3。该HAM1具有SEQ ID NO:1 所示氨基酸序列，以及SEQ ID NO:2所示核苷酸序列，其中第882～902为miR171的靶向位点；该HAM2具有SEQ ID NO:3所示氨基酸序列，以及SEQ ID NO:4所示核苷酸序列，其中第801～821为miR171的靶向位点；该HAM3具有SEQ ID NO:5所示氨基酸序列，以及SEQ ID NO:6所示核苷酸序列，其中第672～692为miR171的靶向位点。miR171的靶向位点是保守的，均包含“GGGATATTGGCGCGGCTCAA”的序列，因此，可以理解的是，miR171a、miR171b、miR171c均能够靶向于HAM1，HAM2，HAM3来发挥下调后者(HAM1，HAM2，HAM3)的作用。
本发明中，还包括具有与所示多肽具有相同功能的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。本发明还提供所述多肽的类似物。这些类似物与天然多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。编码HAM的多核苷酸可以是DNA形式或RNA形式。编码HAM成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
本发明提供了一种提高植物再生能力的方法，包括：下调植物中HAM或其同源物。
本发明中，所述的植物再生包括芽再生、根再生、细胞胚再生等。并且，所述的植物再生包括植物外植体为基础的再生、植物愈伤组织为基础的再生等。
根据本发明的解释，在得知了所述的HAM对于植物再生能力的调控机制后，可以采用本领域人员熟知的多种方法来降低HAM的表达或使之缺失表达，比如将携带反义HAM基因的表达单位(比如表达载体或病毒等)递送到靶点上，使得细胞或植物组织不表达或降低表达HAM蛋白；或将HAM基因进行敲除。
作为本发明的一种实施方式，可通过敲除HAM基因，从而下调植物中HAM基因的表达。
作为本发明的一种实施方式，可采用CRISPR/Cas9系统进行基因编辑，从而敲 除HAM基因，提高植物再生能力。合适的sgRNA靶位点，会带来更高的基因编辑效率，所以在着手进行基因编辑前，设计并找到合适的靶位点较为重要。在设计特异性靶位点后，还需要进行体外细胞活性筛选，以获得有效的靶位点用于后续实验。
作为本发明的一种实施方式，可采用病毒诱导的基因沉默(VIGS)抑制HAM，从而提高植物再生能力。
应理解，本领域技术人员在得知了HAM与植物性状的相关性以后，可以以各种途径制备出下调HAM的分子，从而用于调控植物性状。所述的干扰分子可通过转基因技术被输送到植物体内，或还可采用本领域已知的多种技术被输送到植物体内。
作为本发明的一种优选实施方式，利用以HAM的编码基因或其转录本为抑制或沉默靶标的微小RNA来下调HAM，从而提高植物再生能力。该微小RNA为miR171。本发明也包括miR171同源基因，也包括miRNA171或其同源基因的编码基因或前体基因。本发明也包括miR171的类似物或衍生物，以及它们的编码基因或前体基因。
如本文所用，miRNA171的“同源基因”包括在多个物种中miRNA171的同源基因，它们与拟南芥中的miRNA171的序列相同、基本相同或具有同源性，并且它们也具有靶向HAM的性能。同时，这些“同源基因”的编码基因或前体基因也涵盖在本发明中。
根据本发明所提供的信息，可设计出在被导入后可被加工成可提高相应的miRNA171或其同源基因表达的多核苷酸构建物，也即所述多核苷酸构建物能够在体内上调相应的miRNA171或其同源基因的量。例如，制备一种分离的多核苷酸(构建物)，所述的多核苷酸(构建物)可被植物细胞转录成前体miRNA171，所述的前体miRNA171可被宿主(如植物细胞)剪切且表达成所述的miRNA171。
通常，所述的多核苷酸构建位于表达载体上。因此，本发明还包括一种载体，它含有所述的miRNA171，或所述的多核苷酸构建物。所述的表达载体通常还含有启动子、复制起始点和/或标记基因等。本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如卡拉霉素、庆大霉素、潮霉素、氨苄青霉素抗性。
本发明还涉及利用HAM和/或miRNA171作为一种基因转化植株后代的追踪标记。本发明还涉及利用HAM和/或miRNA171作为一种分子标记，通过检测植物中HAM和/或miRNA171的表达情况，确定植物的再生能力。
应理解，虽然本发明的HAM和/或miRNA171优选获自拟南芥，但是获自其它植物的与拟南芥HAM和/或miRNA171高度同源(如具有80％以上，如85％、90％、95％、甚至98％序列相同性)的其它基因也在本发明的范围之内。比对序列相同性的方 法和工具也是本领域周知的，例如BLAST。
在得知了miR171靶向于HAM后，通过已知HAM促进植物再生能力及其分子机制以后，可基于该新发现来筛选通过调节HAM和/或miR171，从而定向调控植物再生能力的物质或潜在物质。
因此，本发明提供了一种筛选提高植物再生能力的试剂的方法，所述方法包括：(1)将候选物质加入到含有HAM的体系中；(2)观测(1)的体系中HAM的表达或活性；若所述候选物质抑制HAM的表达或活性，则表明该候选物质是提高植物再生能力的试剂；所述的HAM包括其同源物。
本发明还提供了一种筛选提高植物再生能力的试剂的方法，所述方法包括：(1)将候选物质加入到含有miR171或其同源基因、或它们的前体的体系中；(2)观测(1)的体系中miR171或其同源基因、或它们的前体的表达或活性；若所述候选物质提高miR171或其同源基因、或它们的前体的表达或活性，则表明该候选物质是提高植物再生能力的试剂。
以蛋白或其上特定的区域作为靶点，来筛选作用于该靶点的物质的方法是本领域人员所熟知的，这些方法均可用于本发明。所述的候选物质可以选自：肽、聚合肽、拟肽、非肽化合物、碳水化合物、脂、抗体或抗体片段、配体、有机小分子、无机小分子和核酸序列等。根据待筛选的物质的种类，本领域人员清楚如何选择适用的筛选方法。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
材料和方法
1、植物材料与载体构建
拟南芥Col-0生态型用作拟南芥野生型。
ham1(FLAG_239F03)ham2(SALK_150134)ham3(CS100299))三突变体材料参见Wang et al.，Mol Plant，2010(vol3)。
拟南芥miR171a成熟序列：TTGAGCCGCGCCAATATCTCA(SEQ ID NO:7)；
拟南芥miR171b或miR171c成熟序列：TTGAGCCGTGCCAATATCACG(SEQ ID NO:8)。
mir171a，mir171b和mir171a mir171b双突变体材料的构建：CRISPR/cas9系统(Mao et al.，Plant Biotech J，2016(vol 2)，同时靶标miR171a，miR171b，miR171c成熟序列 对应的颈环序列，目的是删除相对应的颈环序列，破坏成熟miRNA的功能。最后只得到mir171a mir171b双突变体材料，连续回交两次，最终得到mir171a，mir171b和mir171a mir171b突变体材料。
pHB-MIR171C载体构建：用PCR扩增拟南芥MIR171C的片段，克隆到pBSK载体的BamH I和Xba I位点之间。测序确认序列无误后，将MIR171C的片段用BamH I和Xba I切出，克隆到双元载体pHB载体中。该载体带有2x35S启动子。MIR171C的片段的基因序列如SEQ ID NO:9(其中第497～517位为miR171c成熟序列相应位点)。
p35S-MIR171A载体构建：用PCR扩增拟南芥MIR171A的片段，使用同源重组酶克隆到双元载体JW807表达载体Kpn I和Spe I之间，测序确认序列无误后用于后续实验。MIR171A的片段的基因序列如SEQ ID NO:10(其中第591～611位为miR171a成熟序列相应位点)。
p35S-MIR171B载体构建：用PCR扩增拟南芥MIR171B的片段，使用同源重组酶克隆到双元载体JW807表达载体(Tian-Qi,Zhang et al,The Plant Cell,Vol 27:349-360,Feb,2015)Kpn I和Spe I之间，测序确认序列无误后用于后续实验。MIR171B的片段的基因序列如SEQ ID NO:11(其中第522～542位为miR171b成熟序列相应位点)。
pER8-MIR171C载体构建：用PCR扩增拟南芥MIR171C的片段，克隆到pBSK载体的Xho I和Spe I位点之间。测序确认序列无误后，将MIR171C的片段用Xho I和Spe I切出，克隆到pER8载体(Zuo et al.，Plant J，2000(Volume 24,Issue 2))。pHB-MIR171A或pHB-MIR171B载体的构建也类同。
pHB-GFP-rHAM1和pHB-GFP-rHAM3载体的构建：用PCR扩增获得GFP的全长序列，克隆入pBSK载体的Pst I和Spe I位点之间，得到中间载体命名为pBS-GFP。rHAM1和rHAM3是抗miR171的突变体形式，在其编码区的miR171识别位点进行定点突变，保持编码的氨基酸序列不变，构建方法采用融合PCR的方法。将rHAM1(SEQ ID NO:12)和rHAM3(如SEQ ID NO:13)全长序列克隆入pBSK的Spe I和Xba I位点之间，获得pBSK-GFP-rHAM1和pBSK-GFP-rHAM3载体；将经测序后，GFP-rHAM1和GFP-rHAM3片段载体用PstI和Xba I切出，克隆到pHB载体的Pst I和Xba I位点之间，最终获得pHB-GFP-rHAM1和pHB-GFP-rHAM3表达载体。
2、拟南芥转化和筛选
(1)转化之前需要提前种植植物至开花，长日照条件大约生长30天。
(2)挑取1-2个农杆菌至5mL抗性LB中，28℃摇菌过夜。
(3)取700μL农杆菌加入300μL 50％甘油，-80℃保存菌种。按1:100比例大摇菌农杆菌，28℃过夜培养。
(4)一般菌液颜色呈现橘黄色，表明农杆菌菌量已适宜转化。
(5)收集菌液，4000rpm，室温离心15min。同时配制农杆菌转化溶液(Infiltration Buffer)：1L体系加入50g蔗糖和300μL silwet-77。
(6)弃上清，用Infiltration Buffer悬浮沉淀菌体。
(7)将拟南芥花序完全浸润在Infiltration Buffer中，大约1min之后，用吸水纸尽量吸干花序之外的菌液。
(8)温室，黑暗放置一天、第二天扶正植物即可。
(9)植物转基因的筛选一般有Basta，Kan和Hygro三种抗性标记，Basta的T0植物用1‰的Agrose悬浮之后，均匀种植在0.05％Basta土壤中。而Kan和Hygro的T0植物，无菌处理之后，涂布在含有50μg/mL Kan或40μg/mL Hygro抗性1/2MS培养基上。基因表达分析。
3、拟南芥再生芽再生实验
(1)：用20％漂水(加入一滴Triton)溶液无菌化处理种子，4℃低温处理2天。
(2)：将灭菌种子，点在含1/2MS培养基的方皿上。22℃黑暗条件下培养下胚轴7天。
(3)：配制CIM培养基，MS固体培养基中含2.2μM 2,4-dichlorophenoxyacetic acid(2,4-D)，0.2μM kinetin。
(4)：截取下胚轴组织，并置于CIM培养基中，22℃培养7天。
(5)：配制SIM培养基，MS固体培养基中含5.0μM 2-isopentenyladenine(2-IP)，0.9μM indole-3-acetic acid(IAA)。
(6)：将CIM培养7天的外植体愈伤组织移植到SIM培养基中，22℃诱导芽的分化，一般7-10天开始可见，21-30天统计再生能力。
4、拟南芥根再生实验
(1)用20％漂水(加入一滴Triton)溶液无菌化处理种子，4℃低温处理2天。
(2)将灭菌种子点在含1/2MS培养基的圆皿上。长日照条件生长16天。
(3)剪取第一片莲座叶，放置(伤口方向一致)于含B5培养基的方皿，光下垂直(伤口朝下)培养16天，每隔2天统计不定根再生事件。
5、拟南芥体细胞胚发生实验
(1)选取拟南芥后期果荚适量，加入1ml消毒液(2％次氯酸钠，0.1％Triton X-100)慢速旋转18min，无菌水冲洗5次。
(2)用1ml带针头注射器剖取子叶期绿色不成熟胚，置于含E5培养基(0.8％agar，5μM 2,4-D)的培养皿，每皿大约放置12个胚，光下培养15天。
(3)将经过E5培养基诱导的胚转移至MS培养基，光下培养10天，显微镜下考察体细胞胚发生数量，或者培养20天左右，统计小苗数量。
6、甜菜、烟草的遗传转化
(1)转基因甜菜的制备
70s::tDT表达载体构建：以70s启动子替换JW807表达载体中的35S启动子，将报告基因tDT(WO2019134884A1)插入到70s启动子之后，使用70s启动子驱动报告基因tDT的表达。
70s::miR171C表达载体构建：以70s启动子替换JW807表达载体中的35S启动子，使用70s启动子驱动miR171C(拟南芥来源，SEQ ID NO:9)的表达。
通过农杆菌侵染甜菜愈伤组织，通过愈伤组织芽从头再生的方式再生出转基因植株。甜菜转化及测定方法还可参见WO2019134884A1。
(2)转基因烟草的制备
构建载体35s::AtrHAM1-3xFLAG(用于过表达rHAM1)，其中AtrHAM1序列如SEQ ID NO:12。
将上述片段插入到JW807表达载体的35S启动子之后，获得35s::AtrHAM1-3xFLAG。
构建载体pRIBO::MIM171a(用于沉默miR171)：
AtIPS1(SEQ ID NO:14；MIM171a序列包含其中)：
Figure PCTCN2019121984-appb-000001
以RIBO启动子替换JW807表达载体中的35S启动子，将上述片段插入到的pRIBO启动子之后，获得pRIBO::MIM171a。RIBO启动子序列如下(SEQ ID NO:15)：
Figure PCTCN2019121984-appb-000002
Figure PCTCN2019121984-appb-000003
构建载体35s::AtMIR171B(用于上调miR171)：
将MIR171B(SEQ ID NO:11，拟南芥来源，即AtMIR171B)插入到JW807表达载体的35S启动子之后，获得35s::AtMIR171B。
载体构建完成后，利用烟草叶盘法转基因，拿到T2转基因植株。
6、再生率的统计分析
拟南芥的芽再生率：由每个处理组合中再生芽的总数除以外植体数目表征。
拟南芥的不定根再生率：由每个处理组合中再生出的不定根的总数除以外植体数目表征。
拟南芥的体细胞胚发生能力：由每个外植体(胚性愈伤组织)所再生出的体细胞胚数目表征。
实施例1、miR171及其靶基因HAM在所有植物中都保守
miR171的靶标基因是GRAS类转录因子，在拟南芥基因组中包括三个基因，分别是HAM1、HAM2和HAM3。
进化分析表明，miR171在所有陆生植物中保守，广泛存在于苔藓植物、蕨类植物、裸子植物和被子植物中，如图1。
同时，本发明人利用NCBI网站蛋白质序列“blast”功能(https://blast.ncbi.nlm.nih.gov/Blast.cgi？PROGRAM＝blastp&PAGE_TYPE＝BlastSearch&LINK_LOC＝blasthome)，检索拟南芥中HAM3的全长蛋白序列，得到若干不同物种中与拟南芥HAM3蛋白序列高度同源的蛋白质序列，将得到的序列信息通过MAFFT软件(https://mafft.cbrc.jp/alignment/software/)分析后得到了同源性信息，后截取了部分同源性信息展示如图2，其中图中黑框突出显示不同物种植物中被miR171所识别并剪切的核苷酸位点所对应的蛋白质序列，可以看到蛋白质高度保守。而且可以在这些物种中找到miR171的靶向序列。以上各图分别展示了不同植物中HAM同源蛋白部分序列的比对信息。综上可以得出结论，HAM在大部分陆生植物中都存在同源蛋白，而且被miR171所识别的位点高度保守。也即，miR171靶向于HAM这一机制在植物中高度保守。
实施例2、miR171调控拟南芥芽再生能力
拟南芥基因组中miR171共有三个编码基因，MIR171A、MIR171B、MIR171C；它们的编码产物即成熟序列分别为miR171a、miR171b、miR171c，其中miR171b与miR171c成熟序列相同。本发明人分别制备了mir171a，mir171b和mir171ab突变体，考察突变体的芽再生能力。
结果如图3所示，mir171b和mir171a b芽再生能力显著降低，提示miR171主要参与了芽再生能力的调控。
实施例3、过量表达miR171或失活突变HAM显著提高拟南芥的再生率
HAM为miR171在植物体内的唯一靶点，且其或其同源基因在各种植物中均保守存在。根据已有的数据显示，miR171可以剪切它的靶基因HAM1，HAM2和HAM3。所以在过表达miR171的植物中，HAM1，HAM2和HAM3的mRNA水平会显著的降低(Wang et al.，Mol Plant，2010vol3.和Llave,C.et al.，Science，2002.vol 297)。根据前述实施例2可见miR171通过靶向其唯一靶点HAM发挥了调控再生的功能。本发明人进一步研究过量表达miR171以及下调HAM的拟南芥的芽再生情况。
本发明人构建了拟南芥MIR171C过量表达植物(MIR171C-OX，将pHB-MIR171C载体转入拟南芥Col-0生态型而获得)，ham1 ham2 ham3三突变体和过量表达HAM1和HAM3的植物(35S::rHAM1和35S::rHAM3)。以下胚轴为外植体测试芽再生率。
如图4，结果显示，与野生型相比，MIR171C-OX和ham1 ham2 ham3三突变体的芽再生能力显著增强，而35S::rHAM1和35S::rHAM3的芽再生能力显著降低。提示HAM是植物细胞全能性的抑制因子，负调控芽再生率。
植物的愈伤组织可以来源于不同的植物器官和组织。常用的有植物的下胚轴、子叶、根和叶片。本发明人进一步研究miR171过量表达是否可以提高其它来源外植体的再生率，考察野生型(Col-0生态型)和MIR171C-OX植物材料根和叶片来源的愈伤组织再生芽的能力。结果如图5所示，两种植物材料根来源的愈伤再生芽能力都很强，而野生型叶片来源的愈伤芽再生能力远远低于MIR171C-OX材料叶片来源的愈伤。
实施例4、miR171-HAM途径促进芽再生标志基因的表达
1、WUS和CLV3在野生型和过量表达miR171植物中的表达模式
为了在分子水平验证miR171过量表达可以提高芽再生率，选取野生型和MIR171C-OX为材料，在SIM培养基上培养2小时至96小时的时间段内，用定量PCR的方法考察WUS和CLV3的表达量。
结果显示，与野生型相比，WUS和CLV3的表达均提前，这与MIR171C-OX芽再生能力提高相吻合，如图6所示。
2、靶向HAM放大芽建立的标志基因CUC信号途径
本发明人分析了拟南芥野生型Col-0及MIR171C-OX材料在芽从头再生过程中，芽再生标志基因CUC1和CUC2的表达量变化趋势。结果如图12，显示过量表达miR171可以通过靶向下调HAM，直接或间接放大芽建立的标志基因CUC信号途径。
实施例5、诱导表达miR171显著下调HAM的表达水平
为了考察miR171c诱导表达的作用，本发明人构建了诱导表达型载体PER8-MIR171C。该载体中miR171c可以被雌二醇(Estradiol，ES)诱导表达。将PER8-MIR171C转入拟南芥，获得转基因植株。
将获得的转基因PER8-MIR171C植物在SIM培养基上培养，以5微摩尔雌二醇/DMSO诱导处理该植物材料后，定量PCR分析pri-mir171c和HAM2的表达量变化；结果如图13，显示PER8-MIR171C植物中HAM2的mRNA水平被显著降低。
实施例6、诱导表达miR171同样能够提高植物芽再生率
为了考察miR171c在CIM时期的作用，取诱导表达型转基因PER8-MIR171C植物的外植体(下胚轴)分别置于CIM(10μM ES)和CIM(10μM DMSO)上培养7天，后转移至不含有ES的SIM上培养24天，考察芽从头再生能力。如图7所示，在CIM时期诱导表达MIR171C就可以显著促进拟南芥芽从头再生能力。
一般而言，拟南芥中外植体通过愈伤组织培养，可以大大提高细胞全能性和芽再生率，不经历愈伤组织培养阶段的外植体芽再生率极低。那么miR171高表达是否可以绕过愈伤组织培养阶段直接提高外植体的芽再生率呢？为此，本发明人选取在1/2MS上垂直培养7天的拟南芥幼苗根作为外植体，将PER8-MIR171C与野生型(WT)的根为外植体(1cm长)，直接置于含有DMSO(20μM，对照组)或ES(20μM，实验组)的SIM培养基上，考察芽再生能力。如图8所示，在SIM上诱导表达MIR171C可以直接显著增强外植体的芽再生能力，且外植体不需要再经历愈伤组织培养阶段。
实施例7、诱导表达miR171能够提高植物体细胞胚再生率
利用PER8-MIR171C材料进一步考察MIR171C对拟南芥体细胞胚发生能力的作用。
拟南芥体细胞胚发生实验结果如图9所示，诱导表达MIR171C可以提高拟南芥体细胞胚发生的能力。
实施例8、过量或诱导表达miR171能够提高植物根再生率
考察miR171c在不定根发生过程中的作用，使用野生型(Col-0)，MIR171C-OX(组成型表达)和PER8-MIR171C(诱导型表达)材料在1/2MS上生长12天后取第一片莲座叶分别转移到B5和含有20μM DMSO或20μM ES的B5培养基上，培养16天考察不定根发生能力。
结果如图10所示，诱导MIR171C高表达后不定根从头发生能力得以增强，且不定根发生速率较快。
实施例9、miR171-HAM途径促进高细胞分裂素条件下的再生
为了考察生长素和细胞分裂素在miR171c促进再生过程中的作用，本发明人分析了拟南芥野生型Col-0及MIR171C-OX材料在高生长素条件下(CIM)或高细胞分裂素条件下(SIM)，芽再生标志基因WUS和CLV3的表达量变化。结果如图11，在高细胞分裂素条件下(SIM)，MIR171C-OX植物中的WUS和CLV3基因表达量显著增加，提示miR171-HAM途径促进高细胞分裂素条件下的再生。
高等植物的再生一般通过两种途径：体细胞发生和器官从头再生。双子叶植物的再生一般选取幼苗的各个组织，例如子叶、根或下胚轴。这些外植体在高细胞分裂素的条件下通过器官从头再生通路实现再生。与双子叶植物不同，单子叶植物在胚后再生能力丧失，因此单子叶植物一般都选取幼胚作为外植体，通过体细胞胚通路实现再生，在此过程中高浓度的生长素是至关重要的。
上述实验结果表明，miR171-HAM途径在高细胞分裂素介导的器官从头再生过程中发挥作用，起到提高再生率的效果。因此可以推断，miR171-HAM途径可有效促进双子叶植物的再生。
实施例10、过量表达miR171可以提高甜菜和烟草的再生率
1、过表达miR171C提高甜菜的转基因效率
本发明人测定了miR171C(拟南芥来源)对双子叶经济作物甜菜(Sugar beet)进行农杆菌介导的转基因效率的影响。利用甜菜中遍在表达启动子驱动miR171C或tDT的质粒进行农杆菌介导的愈伤组织转化实验，或者混合两种菌液进行共转化，随后进行植物抗性筛选，获得阳性转基因植株，统计转化效率(Transformation rate)。测定方法参见WO2019134884A1。
甜菜的转化操作是通过农杆菌侵染甜菜愈伤组织(即转基因)，通过愈伤组织芽从头再生的方式再生出转基因植株。由于农杆菌侵染的效率非常高，而芽再生率相对很低， 是转化的限制性因素，因此最终的转化效率即直接反映了再生率，亦即愈伤组织的再生能力。
结果如图14，与对照相比，转化过表达miR171C的质粒或者共转过表达miR171C的质粒，可以显著提高甜菜的转化效率，即提高了甜菜的再生效率，说明过表达miR171C可以提高甜菜的再生能力。
2、过表达HAM、miR171C或下调表达miR171C对于双子叶植物烟草的作用
利用烟草叶盘法转基因，本发明获得了T2代烟草转基因植株，剪取叶片为小块(0.5cm×0.5cm)，放置MS0培养2天，随后转移至MS1培养，等待芽长出后，统计每块外植体再生芽的数量，并与野生型烟草进行比较。结果发现，过表达MIR171A/B可以提高转基因烟草的芽再生数目，而过表达rHAM1和过表达MIM171a会减少转基因烟草的芽再生数目。
实施例11、筛选方法
设置：
测试组：拟南芥细胞系(其中内源表达HAM)，并给予候选物质；
对照组：拟南芥细胞系(其中内源表达HAM)，不给予候选物质。
分别检测测试组和对照组中HAM的表达情况，并进行比较。如果测试组中HAM的表达在统计学上低于(如低30％以上)对照组，则表明该候选物质是提高植物再生能力的试剂。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (24)

  1. 一种提高植物再生能力的方法，其特征在于，所述方法包括下调植物中HAM；所述的HAM包括其同源物。
  2. 如权利要求1所述的方法，其特征在于，所述的下调HAM包括：在植物中敲除或沉默HAM基因，或抑制HAM蛋白的活性；较佳地，包括：以特异性干扰HAM基因表达的干扰分子来沉默HAM，以基因编辑方法敲除HAM基因，以同源重组方法敲除HAM基因，或以紫外胁迫抑制HAM表达。
  3. 如权利要求2所述的方法，其特征在于，所述的干扰分子是以HAM的编码基因或其转录本为抑制或沉默靶标的dsRNA、反义核酸、小干扰RNA、微小RNA，或能表达或形成所述dsRNA、反义核酸、小干扰RNA、微小RNA的构建物。
  4. 如权利要求3所述的方法，其特征在于，所述的干扰分子是以HAM的编码基因或其转录本为抑制或沉默靶标的微小RNA，其是miR171；较佳地，上调植物中miR171或其同源基因、或它们的编码基因或前体基因，从而下调HAM表达。
  5. 如权利要求4所述的方法，其特征在于，通过miR171上调剂进行上调，包括选自下组的miR171上调剂：
    (a)多核苷酸，所述的多核苷酸能被植物转录或加工成miR171或其同源基因、或它们的前体；较佳地，所述多核苷酸具有SEQ ID NO:9或SEQ ID NO:10所示的序列；
    (b)表达构建物，所述的表达构建物含有miR171或其同源基因、或它们的前体、或(a)中所述的多核苷酸；
    (c)miR171或其同源基因、或它们的编码基因或前体基因的激动剂。
  6. 如权利要求5所述的方法，其特征在于，将(a)～(c)任一所述的上调剂引入到植物中，从而实现所述的上调。
  7. 如权利要求4所述的方法，其特征在于，所述的miR171的核苷酸序列如SEQ ID NO:7或SEQ ID NO:8所示；或所述的miR171的同源基因与miR171的序列同源性≥80％，较佳地≥90％，更佳地≥95％，进一步更佳地≥99％。
  8. 如权利要求1所述的方法，其特征在于，所述的植物再生包括：芽再生，根再 生，细胞胚再生；或
    所述的植物再生包括：植物外植体为基础的再生，植物愈伤组织为基础的再生；较佳地，所述的植物外植体或愈伤组织包括来自下组植物组织的外植体或愈伤组织：下胚轴，子叶，根，叶片，胚胎，花器官。
  9. 如权利要求1所述的方法，其特征在于，所述的植物包括或所述的HAM或其同源物来自于：双子叶植物；较佳地所述的植物包括或所述的HAM或其同源物来自于选自下组的植物：禾本科，十字花科，茄科，豆科，藜科，杨柳科，锦葵科，椴树科，芸香科，菊科，葫芦科，番木瓜科，木棉科，梧桐科，鼠李科，大戟科，桑科，土瓶草科，胡麻科，木犀科，猕猴桃科，蔷薇科的植物；较佳地，所述的植物包括或所述的HAM或其同源物来自于选自下组的植物：拟南芥，二穗短柄草，稻，番茄，烟草，甜菜，大豆，白菜，玉米，棉花，马铃薯，麦，高山南芥，芥菜，亚麻芥，油菜，Eutrema saisugineum，黄麻(包括糙叶山黄麻，异色山黄麻，长溯黄麻)，杨树，克里曼丁橘，莴苣，番南瓜，番木瓜，西葫芦，向日葵，榴莲，青蒿，南瓜，可可豆，枣树，三叶橡胶，川桑，胡杨，土瓶草，木豆，Cynara cardunculus var.scolymus，芝麻，甜橙，蜜柑，枸橘，油橄榄，猕猴桃，月季；更佳地，所述的植物包括或所述的HAM或其同源物来自于：拟南芥，甜菜，大豆，白菜，棉花，油菜，番茄，烟草。
  10. 一种HAM的用途，用于作为下调靶点，以提高植物再生能力；或用于筛选靶向于HAM从而提高植物再生率的试剂。
  11. 一种HAM下调剂的用途，用于提高植物再生率；较佳地，所述的HAM下调剂通过下调HAM，提高芽再生标志基因的表达；更佳地，所述芽再生标志基因包括：WUS、CLV3、CUC1或CUC2。
  12. 如权利要求11所述的用途，其特征在于，所述的HAM下调剂包括：敲除或沉默HAM基因或抑制HAM蛋白活性的下调剂；较佳地，包括：特异性干扰HAM基因表达的干扰分子，敲除HAM基因的基因编辑试剂，基于同源重组的敲除HAM基因的试剂。
  13. 如权利要求12所述的用途，其特征在于，所述的干扰分子是以HAM的编码基因或其转录本为抑制或沉默靶标的dsRNA、反义核酸、小干扰RNA、微小RNA，或能表达或形成所述dsRNA、反义核酸、小干扰RNA、微小RNA的构建物。
  14. 如权利要求13所述的用途，其特征在于，所述的干扰分子是以HAM的编码基因或其转录本为抑制或沉默靶标的miR171或miR171上调剂，所述miR171上调剂包括：
    (a)多核苷酸，所述的多核苷酸能被植物转录或加工成miR171或其同源基因、或它们的编码基因或前体基因；较佳地，所述多核苷酸具有SEQ ID NO:9或SEQ ID NO:10所示的序列；
    (b)表达构建物，所述的表达构建物含有miR171或其同源基因、或它们的编码基因或前体基因、或(a)中所述的多核苷酸；
    (c)miR171或其同源基因、或它们的编码基因或前体基因的激动剂。
  15. 如权利要求10～14任一所述的用途，其特征在于，所述的植物再生包括：芽再生，根再生，细胞胚再生；或
    所述的植物再生包括：植物外植体为基础的再生，植物愈伤组织为基础的再生；较佳地，所述的植物外植体或愈伤组织包括来自下组植物组织的外植体或愈伤组织：下胚轴，子叶，根，叶片，胚胎，花器官。
  16. 如权利要求10～14任一所述的用途，其特征在于，所述的植物包括或所述的HAM或其同源物来自于：双子叶植物；较佳地所述的植物包括选自下组的植物：禾本科，十字花科，茄科，豆科，藜科，杨柳科，锦葵科，椴树科，芸香科，菊科，葫芦科，番木瓜科，木棉科，梧桐科，鼠李科，大戟科，桑科，土瓶草科，胡麻科，木犀科，猕猴桃科，蔷薇科的植物；较佳地，所述的植物包括或所述的HAM或其同源物来自于选自下组的植物：拟南芥，二穗短柄草，稻，番茄，烟草，甜菜，大豆，白菜，玉米，棉花，马铃薯，麦，高山南芥，芥菜，亚麻芥，油菜，Eutrema saisugineum，黄麻，杨树，克里曼丁橘，莴苣，番南瓜，番木瓜，西葫芦，向日葵，榴莲，青蒿，南瓜，可可豆，枣树，三叶橡胶，川桑，胡杨，土瓶草，木豆，Cynara cardunculus var.scolymus，芝麻，甜橙，蜜柑，枸橘，油橄榄，猕猴桃，月季；更佳地，所述的植物包括或所述的HAM或其同源物来自于：拟南芥，甜菜，大豆，白菜，棉花，油菜，番茄，烟草。
  17. 一种HAM的用途，用于作为鉴定再生能力的分子标记；所述的HAM包括其同源物。
  18. 一种miR171或其同源基因、或它们的编码基因或前体基因的用途，用于作为鉴定再生能力的分子标记。
  19. 一种定向选择再生能力增强的植物的方法，所述方法包括：鉴定待测植物中HAM的表达，若是该待测植物的HAM表达显著低于该类植物的HAM平均表达值，则其为再生能力增强的植物；所述的HAM包括其同源物。
  20. 一种定向选择再生能力增强的植物的方法，所述方法包括：鉴定待测植物中miR171或其同源基因、或它们的编码基因或前体基因的表达，若是该待测植物的miR171或其同源基因、或它们的编码基因或前体基因的表达显著高于该类植物的平均表达值，则其为再生能力增强的植物。
  21. 一种筛选提高植物再生能力的试剂的方法，所述方法包括：(1)将候选物质加入到含有HAM的体系中；(2)观测(1)的体系中HAM的表达或活性；若所述候选物质抑制HAM的表达或活性，则表明该候选物质是提高植物再生能力的试剂；所述的HAM包括其同源物。
  22. 一种筛选提高植物再生能力的试剂的方法，所述方法包括：(1)将候选物质加入到含有miR171或其同源基因、或它们的编码基因或前体基因的体系中；(2)观测(1)的体系中miR171或其同源基因、或它们的编码基因或前体基因的表达或活性；若所述候选物质提高miR171或其同源基因、或它们的编码基因或前体基因的表达或活性，则表明该候选物质是提高植物再生能力的试剂。
  23. 表达构建物或含有该表达构建物的试剂盒，所述表达构建物含有多核苷酸，所述的多核苷酸能被植物转录或加工成miR171或其同源基因、或它们的编码基因或前体基因；或，其含有miR171或其同源基因的编码基因或前体基因；较佳地，所述的表达构建物为表达载体。
  24. 一种植物细胞，其中含有权利要求23所述的表达构建物，或其中包含外源引入的miR171或其同源基因、或它们的编码基因或前体基因。
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