Application of H2A.Z1 protein and coding gene thereof in regulation and control of plant chromosome doubling

Abstract

The invention discloses an H2A. Z1 protein and application of a coding gene of the H2A. Z1 protein in regulation and control of plant chromosome doubling. According to the invention, a model species poplar of a woody plant is taken as a research object, and a transgenic poplar with reduced H2A.Z1 gene expression quantity is constructed. Experiments prove that compared with wild poplar, the plant height and the stem node number of the transgenic poplar with reduced H2A.Z1 gene expression quantity are reduced, and the stem node length is increased. In addition, cells in stem and leaf organs of the transgenic poplar with reduced H2A.Z1 gene expression quantity become larger, cell chromosomes are doubled, and diploid is changed into tetraploid. The invention has important significance for researching polyploid breeding work of plants such as vegetables, important flowers, fruit trees and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of H2A.Z1 protein and related biomaterials in regulating chromosome doubling in plants.

[0002] This application is a divisional application of application number 202510171023.7, filed on February 17, 2025, entitled "Application of H2A.Z1 protein and related biomaterials in regulating plant chromosome doubling". Background Technology

[0003] Polyploid breeding is an important method for cultivating new plant varieties. Compared with conventional breeding methods, it has significant advantages such as rapid results, short generation cycle, low dependence on seeds, and fewer adverse effects from reduced fertility. However, polyploid germplasm resources in nature are very limited, and the technology for breeding polyploid plant varieties still needs improvement. Therefore, it is necessary to explore new breeding approaches to generate new autopolyploid plants, thereby enriching plant genetic diversity and providing important technical support for the breeding of new varieties of economic plants such as vegetables, flowers, and fruit trees.

[0004] Currently, the main methods for plant polyploid breeding include screening of natural polyploids, somatic chromosome doubling, hybridization of different ploidies, and hybridization of unmeiotic gametes. Each method has its advantages and disadvantages, and these methods vary considerably between different species. The primary method for plant chromosome doubling is still treating tissue-cultured seedlings with low concentrations of colchicine to the leaf discs, petioles, and stem segments. While this method is quick, efficient, and widely applicable, it suffers from drawbacks such as poor safety, a tendency to produce aneuploids, and low sample survival rates. Therefore, it is essential to develop safe and efficient new methods for plant chromosome doubling. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to artificially regulate chromosome doubling in plant cells. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] To address the aforementioned technical problems, this invention first provides novel uses for H2A.Z1 protein or substances that regulate the content and / or activity of said H2A.Z1 protein.

[0007] This invention provides the use of H2A.Z1 protein or substances regulating the content and / or activity of said H2A.Z1 protein in any of the following A1)-A7): A1) Regulating plant height; A2) Regulating the number of plant stem nodes; A3) Regulating the length of plant stem nodes; A4) Regulates plant cell size; A5) Regulates the ploidy of plant cell chromosomes; A6) Cultivate transgenic plants with altered plant height and / or number of stem nodes and / or stem node length and / or cell size and / or chromosome ploidy; A7) Plant breeding; The H2A.Z1 protein is any one of the following (B1)-B4): B1) The amino acid sequence of the protein is shown in sequence 3; B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 3; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 3. B4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 3 and has the same function.

[0008] In the protein described in B2) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0009] In the protein described in B3) above, the substitution and / or deletion and / or addition of one or more amino acid residues is no more than 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acid residues.

[0010] In the protein described in B4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity calculation for a pair of amino acid sequences, the identity value (%) can then be obtained. The identity includes amino acid sequences that have 80% or higher, or 85% or higher, or 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with the amino acid sequence shown in Sequence 3 of this invention.

[0011] The proteins described in B1)-B4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0012] The substances that regulate the content and / or activity of H2A.Z1 protein mentioned above include substances that increase the content and / or activity of H2A.Z1 protein or substances that decrease the content and / or activity of H2A.Z1 protein.

[0013] Furthermore, the substance that enhances the activity of H2A.Z1 protein may be a protein, polypeptide, or small molecule compound that enhances or promotes the function of H2A.Z1 protein.

[0014] The substance that increases the H2A.Z1 protein content can be a substance that promotes H2A.Z1 protein synthesis, inhibits H2A.Z1 protein degradation, or overexpresses H2A.Z1 protein.

[0015] The substance that reduces the activity of H2A.Z1 protein can be a protein, polypeptide, or small molecule compound that inhibits the function of H2A.Z1 protein.

[0016] The substance that reduces the H2A.Z1 protein content may be a substance that inhibits H2A.Z1 protein synthesis, promotes H2A.Z1 protein degradation, or knocks down (reduces) or removes the H2A.Z1 protein encoding gene.

[0017] Furthermore, the substance that knocks down (reduces) the H2A.Z1 protein-coding gene can be any nucleic acid molecule that can inhibit or interfere with the expression of the H2A.Z1 protein-coding gene, such as gRNA (e.g., sgRNA), siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0018] Furthermore, the nucleic acid molecule that inhibits or interferes with the expression of the H2A.Z1 protein-coding gene is a miRNA that inhibits or interferes with the expression of the H2A.Z1 protein-coding gene.

[0019] In some embodiments, the nucleotide sequence of the miRNA that inhibits or interferes with the expression of the H2A.Z1 protein-coding gene is shown in Sequence 7.

[0020] To address the aforementioned problems, this invention provides new uses for biomaterials related to the H2A.Z1 protein.

[0021] This invention provides the application of biomaterials related to the H2A.Z1 protein in any of the following A1)-A7): A1) Regulating plant height; A2) Regulating the number of plant stem nodes; A3) Regulating the length of plant stem nodes; A4) Regulates plant cell size; A5) Regulates the ploidy of plant cell chromosomes; A6) Cultivate transgenic plants with altered plant height and / or number of stem nodes and / or stem node length and / or cell size and / or chromosome ploidy; A7) Plant breeding; The biomaterial is any one of the following E1) to E5): E1) The nucleic acid molecule encoding the H2A.Z1 protein mentioned above; E2) Nucleic acid molecules that inhibit or interfere with the expression of the H2A.Z1 protein-encoding genes mentioned above; E3) An expression cassette containing the nucleic acid molecules described in E1) or E2); E4) A recombinant vector containing the nucleic acid molecules described in E1) or E2); E5) Recombinant microorganisms containing the nucleic acid molecules described in E1) or E2).

[0022] In the above applications, the nucleic acid molecule described in E1) is any of the following: F1) The DNA molecule shown in sequence 1, sequence 2, or sequence 6; The nucleotide sequence defined by F2) has 75% or more identity with F1) and is a DNA molecule encoding the H2A.Z1 protein described above.

[0023] In the above applications, the nucleic acid molecule described in E2) is any of the following: G1) RNA molecules shown in sequence 5 or sequence 7; The nucleotide sequence defined by G2) has 75% or more identity with G1) and is a nucleic acid molecule that inhibits or interferes with the expression of the H2A.Z1 protein-coding gene.

[0024] Those skilled in the art can readily mutate the nucleotide sequence encoding the H2A.Z1 protein of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified sequences, having characteristics different from those obtained in this invention... H2A.Z1 Nucleotides with 75% or higher nucleotide sequence identity, provided they encode the aforementioned H2A.Z1 protein and have the same function, are derived from and equivalent to the nucleotide sequences of this invention. This identity refers to sequence similarity to natural nucleic acid sequences, including nucleotide sequences with 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher nucleotide sequence identity to proteins composed of the amino acid sequence shown in Sequence 3 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0025] The nucleic acid molecule mentioned in E2 above can be gRNA (such as sgRNA), siRNA, dsRNA, shRNA, miRNA or antisense RNA.

[0026] Any of the nucleic acid molecules mentioned above can be DNA, such as cDNA, genomic DNA, or recombinant DNA.

[0027] Any of the nucleic acid molecules mentioned above can be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0028] The expression cassette described above may include a promoter, the nucleic acid molecule described in E1) or E2) above, and a terminator. Promoters that can be used in this invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Furthermore, the expression cassette may also include an enhancer sequence.

[0029] The vector mentioned above refers to a vector capable of carrying the nucleic acid molecules described in E1) or E2) into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage, etc.), granules (i.e., Cos plasmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0030] The recombinant vector described above refers to a recombinant DNA molecule constructed by ligating the nucleic acid molecule described in E1) or E2) above with the vector in vitro. Recombinant vectors containing the nucleic acid molecule described in E1) or E2) above can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The plant expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing the polyadenylated signal and any other DNA fragments involved in mRNA processing or gene expression.

[0031] The microorganisms mentioned above can be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The cells are Agrobacterium, such as Agrobacterium tumefaciens GV3101.

[0032] The recombinant microorganisms mentioned above refer to those obtained by manipulating and modifying the genes of a target microorganism, thereby altering its function. For example, recombinant microorganisms obtained after introducing the aforementioned recombinant vector into a target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring may not necessarily be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0033] In any of the above applications, the regulation of plant height involves either increasing or decreasing plant height. The regulation method is positive regulation, meaning that when the content and / or activity of H2A.Z1 protein in the plant increases, the plant height increases; conversely, when the content and / or activity of H2A.Z1 protein in the plant decreases or is absent, the plant height decreases.

[0034] In some implementation schemes, when plants H2A.Z1 When gene expression levels decrease, plant height decreases.

[0035] In any of the above applications, the regulation of the number of plant stem nodes involves either increasing or decreasing the number of plant stem nodes. The regulation method is positive regulation, meaning that when the content and / or activity of H2A.Z1 protein in the plant increases, the number of plant stem nodes increases; conversely, when the content and / or activity of H2A.Z1 protein in the plant decreases or is absent, the number of plant stem nodes decreases.

[0036] In some implementation schemes, when plants H2A.Z1 When gene expression levels decrease, the number of stem nodes in a plant decreases.

[0037] In any of the above applications, the regulation of plant stem segment length involves either increasing or decreasing the stem segment length. The regulation method is negative regulation, meaning that when the content and / or activity of H2A.Z1 protein in the plant increases, the plant stem segment length decreases; conversely, when the content and / or activity of H2A.Z1 protein in the plant decreases or is absent, the plant stem segment length increases.

[0038] In some implementation schemes, when plants H2A.Z1 When gene expression levels decrease, the length of plant stem segments (such as the total length of stem segments from the first to the ninth stem segment) increases.

[0039] In any of the above applications, the regulation of plant cell size refers to either increasing or decreasing the size of plant cells. The regulation method is negative regulation, meaning that when the content and / or activity of H2A.Z1 protein in the plant increases, the plant cells become smaller; when the content and / or activity of H2A.Z1 protein in the plant decreases or is absent, the plant cells become larger.

[0040] In some implementation schemes, when plants H2A.Z1 When gene expression levels decrease, the area of ​​plant cells (stem epidermal cells, leaf epidermal cells) increases.

[0041] In any of the above applications, the regulation of plant cell chromosome ploidy refers to causing the plant cell chromosomes to double. Specifically, when the content and / or activity of H2A.Z1 protein in plants decreases or is absent, the plant cell chromosomes double.

[0042] In some implementation schemes, when plants H2A.Z1 When gene expression levels decrease, plant cell chromosomes double, changing from diploid to tetraploid.

[0043] In any of the above applications, the purpose of the plant breeding is to cultivate polyploid plants (such as tetraploid plants) or plants with reduced plant height and / or reduced number of stem nodes and / or increased stem node length and / or larger cells.

[0044] To address the aforementioned technical problems, the present invention also provides a method for cultivating transgenic plants with reduced plant height and / or reduced number of stem nodes and / or increased stem node length and / or larger cells and / or doubled chromosomes.

[0045] The method for cultivating transgenic plants with reduced plant height and / or reduced number of stem nodes and / or increased stem node length and / or larger cells and / or doubled chromosomes provided by the present invention includes the following steps: reducing the content and / or activity of the above-mentioned H2A.Z1 protein in the target plant to obtain transgenic plants with reduced plant height and / or reduced number of stem nodes and / or increased stem node length and / or larger cells and / or doubled chromosomes.

[0046] Furthermore, the chromosome doubling refers to changing the plant from diploid to tetraploid. This change from diploid to tetraploid is manifested in the following 1) or 2): 1) The percentages of cells in the apical buds of the target plant at the 2C stage and the 4C stage are 80% and 20%, respectively; the percentages of cells in the apical buds of the transgenic plant at the 2C stage and the 4C stage are 10% and 90%, respectively. 2) The number of chromosomes in the nucleus of the root tip cells of the target plant is 38 (2 sets of chromosomes), and the number of chromosomes in the nucleus of the root tip cells of the transgenic plant is 76 (4 sets of chromosomes).

[0047] Furthermore, the method for reducing the content and / or activity of the H2A.Z1 protein in the target plant is to introduce a substance that inhibits or interferes with the expression of the H2A.Z1 protein encoding gene into the target plant.

[0048] Furthermore, the substance that inhibits the expression of the H2A.Z1 protein-coding gene in the target plant is a miRNA that inhibits or interferes with the expression of the H2A.Z1 protein-coding gene.

[0049] In some embodiments, the nucleotide sequence of the miRNA is shown in Sequence 7.

[0050] In any of the applications or methods described above, the transgenic plant includes not only the first-generation transgenic plant obtained by transforming the target plant with a substance that inhibits or interferes with the expression of the H2A.Z1 protein-coding gene, but also its progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The transgenic plant includes seeds, callus tissue, intact plants, and cells.

[0051] In any of the above applications or methods, the plant may be a dicotyledonous or monocotyledonous plant, including grain crops such as rice, wheat, barley, corn, soybeans, potatoes, beans, oats, and millet; vegetable crops such as Arabidopsis thaliana, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, zucchini, leek, onion, and carrot; cash crops such as ginseng, tobacco, cotton, sesame, sugarcane, sugar beets, wild sesame, peanuts, and rapeseed; and other plants including apples, pears, dates, peaches, etc. Fruits including kiwifruit, grapes, oranges, persimmons, plums, apricots, and bananas; flowers including roses, gladioli, dandelion, carnations, chrysanthemums, lilies, and tulips; forage crops including ryegrass, red clover, orchardgrass, alfalfa, tall buttercup, and perennial ryegrass; and woody plants used for timber production, including poplar, willow, birch, locust, elm, dawn redwood, spruce, beech, maple, oak, chinaberry, ash, goldenrod, ironwood, rosewood, yellow sandalwood, teak, ash, and maple. Further, the plants are dicotyledonous plants. Even further, the dicotyledonous plants are dicotyledonous woody plants. In a specific embodiment of the invention, the dicotyledonous woody plant is poplar.

[0052] To address the aforementioned technical problems, this invention ultimately provides a miRNA.

[0053] The miRNA provided by this invention is either a1) or a2): a1) The RNA molecule shown in sequence 7; a2) An RNA molecule that has one or more nucleotides deleted, added, or altered from sequence 7, and has the same function as sequence 7.

[0054] This invention constructs H2A.Z1 Transgenic poplar trees with reduced gene expression levels. Experiments have shown that, compared to wild-type poplar trees, [the following is likely a separate, unrelated sentence:] ... H2A.Z1 Transgenic poplars with reduced gene expression levels exhibited decreased plant height and number of stem nodes, but increased stem node length. Furthermore, H2A.Z1 In transgenic poplar trees with reduced gene expression, cells in the stems and leaves become larger, and chromosomes double, changing from diploid to tetraploid. This invention is of great significance for elucidating the regulatory molecular mechanisms of new organogenesis or formation in forest trees and for the polyploid breeding of poplar. Attached Figure Description

[0055] Picture 1 Wild-type poplar seedlings grown in soil pots for two months and H2A.Z1 Phenotypic observation and statistical graph of transgenic poplar seedlings. A represents wild-type poplar (WT) and seedlings grown in soil pots for 2 months. H2A.Z1The phenotype of transgenic poplar lines Ami-57, Ami-58, and Ami-203 was interfered with. B represents wild-type poplar seedlings and... H2A.Z1 Interference with transgenic poplar lines Ami-57, Ami-58, and Ami-203 H2A.Z1 Gene expression level detection results. C represents wild-type poplar seedlings and H2A.Z1 The plant height of the transgenic poplar lines Ami-57, Ami-58, and Ami-203 was interfering with the growth. D represents wild-type poplar seedlings and... H2A.Z1 The number of stem nodes in the transgenic poplar lines Ami-57, Ami-58, and Ami-203 was interfering with the growth. E represents wild-type poplar seedlings and... H2A.Z1 Interference was studied in the internode length of the transgenic poplar lines Ami-57, Ami-58, and Ami-203. An internode refers to the stem segment between two consecutive leaves, and the internode length refers to the total length from the first to the ninth internode. Specifically, the first internode from the terminal bud was defined as the first internode, the second internode as the second internode, and so on, with the ninth internode defined as the ninth internode. Five replicates were recorded for each lineage; identical letters indicated no significant difference.

[0056] Picture 2 Wild-type poplar trees grown in soil pots for two months and H2A.Z1 Cytological analysis diagram of transgenic poplar trees subjected to interference. A represents wild-type poplar and... H2A.Z1 Image of stem and leaf epidermal cells of the transgenic poplar Ami-203. B represents wild-type poplar and... H2A.Z1 Statistical analysis of stem epidermal cell area in the transgenic poplar Ami-203. C represents wild-type poplar and... H2A.Z1 Statistical analysis of leaf epidermal cell area in the transgenic poplar Ami-203.

[0057] Picture 3 wild-type poplar and H2A.Z1 Images showing the DNA content and chromosome number in the nuclei of transgenic poplar cells. A represents wild-type poplars grown in soil pots for two months and... H2A.Z1 Image showing DNA content detection in the apical cells of transgenic poplar shoots. B represents wild-type poplars grown in soil pots for two months and... H2A.Z1 Statistical graph of DNA content in the apical cells of transgenic poplar shoots after interference. C represents wild-type poplar after 10 days of tissue culture and... H2A.Z1 Chromosome diagram of root tip cells from transgenic poplar trees. D represents wild-type poplar trees cultured for 10 days and... H2A.Z1 A statistical chart of chromosome number in root tip cells of transgenic poplar trees. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0060] The PGWB2 vector in the following examples is described in the literature “Xu J, Lee YJ, Liu B. (2019) Establishment of a mitotic model system by transient expression of the D-typecyclin in differentiated leaf cells of tobacco (Nicotiana benthamiana).[J].New Phytol, 226(4):1213-1220.”.

[0061] The Agrobacterium tumefaciens strain GV3101 in the following examples is described in the literature “Zheng, S., et al. (2020). "Two MADS-box genes regulate vascular cambium activity and secondary growth via modulating auxin homeostasis in Populus." Plant Communications.”

[0062] The wild-type poplar “nanlin895” in the following examples is described in the literature “Chao Q., et al. (2019). The developmental dynamics of the Populus stem transcriptome.” PlantBiotechnol J 17(1): 206-219.”.

[0063] The pENTR / D-TOPO® vector used in the following examples is described in the literature “Shuman, S. (1994). Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA Topoisomerase. J. Biol. Chem. 269, 32678-32684.”.

[0064] Example 1: Obtaining the H2A.Z1 protein and its encoding gene 1. Take the whole "nanlin 895" poplar plant that has been growing in a tissue culture bottle for one month, freeze it in liquid nitrogen, grind it and extract total RNA, and reverse transcribe the total RNA to obtain poplar cDNA.

[0065] 2. Using the obtained cDNA as a template, PCR amplification was performed with 5'-ATGGCTGGAAAAGGAGG-3' as the forward primer and 5'-TCACTCTTTGGTGGTTTTGTTG-3' as the reverse primer to obtain the amplification product.

[0066] 3. After agarose gel electrophoresis, the amplified product was separated and purified into a DNA fragment of approximately 414 bp. This fragment was then ligated into pEASY. ® - The Blunt Simple cloning vector was used, and the vector ligated with the target fragment was sequenced. Because "nanlin 895" is a hybrid of American black poplar and European poplar, its genome is relatively complex, and the same gene locus may have more than one sequence. Two sequences were obtained through PCR amplification. H2A.Z1 The coding region sequence of a gene.

[0067] Sequencing results showed that in poplar tree “nanlin 895” H2A.Z1 The coding region sequence of the gene is shown in Sequence 1 and Sequence 2; Sequence 1 and Sequence 2 are shown in... H2A.Z1 The amino acid sequences encoded by the gene coding region of the H2A.Z1 protein are identical, as shown in Sequence 3.

[0068] Example 2: Construction of recombinant vector and recombinant Agrobacterium I. Construction of recombinant interference vector and recombinant interfering Agrobacterium 1. Construction of the recombinant interference vector PGWB2-Ami (1) Shown in sequence 1 and sequence 2 H2A.Z1The common coding sequence 5'-AGGGGCTTCTGGCAACGAAAA-3' of the gene coding region was used as the target sequence. Primers were designed on the primer design website http: / / wmd3.weigelworld.org / cgi-bin / webapp.cgi. The primer sequences are as follows: H2-I miR-s: 5'-gaTTTTCGTTGCCAGAAGCGCGTtctctcttttgtattcc-3'; H2-II miR-a: 5'-gaACGCGCTTCTGGCAACGAAAAtcaaagagaatcaatga-3'; H2-III miR*s: 5'-gaACACGCTTCTGGCTACGAAATtcacaggtcgtgatatg-3'; H2-IV miR*a: 5'-gaATTTCGTAGCCAGAAGCGTGTtctacatatattcct-3'.

[0069] (2) Using the pRS300 (miR319a) vector as a template, the artificial microRNA gene fragment was amplified by performing one round of conventional PCR and one round of overlap PCR using the four primer sequences in step (1) (for specific methods, refer to Rebecca Schwab, MPI for Developmental Biology, Tuebingen, 2005). The nucleotide sequence of the artificial microRNA gene fragment is shown in Sequence 4.

[0070] (3) The artificial microRNA gene fragment amplified in step (2) is ligated into the pENTR / D-TOPO® vector to obtain the pENTR / D-TOPO®-Ami vector; then, the pENTR / D-TOPO® vector is ligated into the pENTR / D-TOPO® vector via LR homologous recombination. ® The artificial microRNA gene fragment from the Ami vector was ligated into the PGWB2 vector to obtain the recombinant interference vector PGWB2-Ami. The nucleotide sequence expressed by the recombinant interference vector PGWB2-Ami is the RNA molecule of sequence 5. This RNA molecule contains the nucleotide sequence of the mature miRNA and the vector backbone sequence. The nucleotide sequence of the mature miRNA is shown in sequence 7.

[0071] 2. Transformation of recombinant Agrobacterium interfering PGWB2-Ami / GV3101 The recombinant interference vector PGWB2-Ami was transformed into Agrobacterium tumefaciens GV3101 using the Agrobacterium-mediated transformation method. After PCR detection, the recombinant interfering Agrobacterium PGWB2-Ami / GV3101 was obtained.

[0072] Example 3 H2A.Z1 Obtaining and identifying transgenic poplar trees with interference I. Obtaining Genetically Modified Poplar Trees Recombinant Agrobacterium interferon was used to infect the young leaves of tissue-cultured poplar seedlings "nanlin895" using the leaf disc method. After a series of callus induction, bud induction, and rooting induction processes, transgenic poplar seedlings were obtained. The specific steps are as follows: 1. The recombinant Agrobacterium interferingense PGWB2-Ami / GV3101 single clone obtained in Example 2 was first cultured in 5 mL of liquid YEB medium (liquid YEB medium is obtained by mixing 1 g yeast extract, 5 g tryptone, 5 g beef extract, 5 g sucrose, 1.954 g anhydrous magnesium sulfate and 1 L water, pH=7.0) for 12 hours. Then, 1 mL of the culture was transferred to 100 mL of liquid YEB medium and incubated at 28°C until OD reached. 600nm =0.8, then add 100 μm acetylsuccinone.

[0073] 2. Take a tender leaf from a poplar seedling that has been growing for one month in a tissue culture bottle. Use a scalpel to cut off the edges of the leaf, leaving about 1 cm of the leaf near the midrib. 2 Take a leaf, make 1-2 small cuts on the main vein, place it in a shaker of Agrobacterium, and gently shake it for 30 minutes to infect it.

[0074] 3. Remove the leaves and place them face down in a co-culture medium (the co-culture medium is a basal medium containing 0.2 mg / L kinetin, 0.75 mg / L 2,4-D, and 100 μm acetylsylgenone; the solvent of the basal medium is water, and the solutes and their concentrations are shown in Table 1) and co-culture at 28°C for two days.

[0075] 4. Transfer the leaves to the callus-inducing selection medium (the selection medium is a basal medium containing 0.2 mg / L kinetin, 0.75 mg / L 2,4-D, 50 mg / L kanamycin, 250 mg / L Cefotaxime sodium, and 300 mg / L Timentin; the solvent of the basal medium is water, and the solutes and their concentrations are shown in Table 1) and culture in the dark. Subculture once every 14 days until spherical callus tissue grows.

[0076] 5. Place the cut callus in a shoot-inducing differentiation medium (the differentiation medium is a basal medium containing 1 mg / L 6-BA, 0.05 mg / L NAA, 50 mg / L kanamycin, 250 mg / L Cefotaxime sodium, and 300 mg / L Timentin; the solvent of the basal medium is water, and the solutes and their concentrations are shown in Table 1) and culture under light. Subculture once every 15 days until shoots emerge.

[0077] 6. Cut off the buds and culture them independently in a rooting medium (the rooting medium is a basal medium containing 50 mg / L kanamycin, 250 mg / L Cefotaxime sodium, and 300 mg / L Timentin; the solvent of the basal medium is water, and the solutes and their concentrations are shown in Table 1) until roots are formed, thus obtaining transgenic poplar seedlings. The rooted transgenic poplar seedlings can be continuously propagated asexually.

[0078] Table 1. Solutes and their concentrations in the basal media of different culture media

[0079] II. Identification of Genetically Modified Poplar 1. PCR identification DNA was extracted from transgenic poplar seedlings for PCR identification, specifically including the following steps: DNA was extracted from all transgenic poplar seedlings using the SLS method, and PCR amplification was performed using PGWB2-Ami-F and PGWB2-Ami-R to obtain PCR products. Wild-type poplar DNA was used as a control. Primer sequences are as follows: PGWB2-Ami-F: 5'-GGGGACTCTAGAGTTATCAAC-3'; PGWB2-Ami-R: 5'-CTAAGCGCTGTTATCAACCAC-3'.

[0080] The PCR products can be detected by electrophoresis to preliminarily identify positive transgenic plants. A PCR product of 822 bp in size is considered a positive transgenic plant.

[0081] 2. H2A.Z1 Interference with the acquisition of genetically modified poplar For positive transgenic plants, expression levels were detected following these steps: Leaves from positive transgenic plants grown for one month and wild-type plants under the same growth conditions were collected. Total RNA was extracted from the leaves using Megan's Plant RNA Mini-Extraction Kit. cDNA was then synthesized using Invitrogen's Reverse Transcription Kit with Oligo d(T) as primers. H2A.Z1Gene-specific primers were used for real-time quantitative PCR detection, with Actin as an internal reference gene. The primers are as follows: qH2A.Z1F: 5'-GACAAGGACAAGAAGAGGCC-3'; qH2A.Z1R: 5'-TGAAGCCAAGTAGACAGCAGC-3'; qPdeActinF: 5'-GCAGTCTTCCCCAGTATTGTT-3'; qPdeActinR: 5'-TCCCCACATAGCATCTTTC-3'.

[0082] The results are as follows Picture 1 As shown, the results indicate that compared with wild-type poplar seedlings, H2A.Z1 Interference in transgenic poplar seedlings Ami-57, Ami-58 and Ami-203 H2A.Z1 The relative expression levels of the genes were downregulated by 39%, 37%, and 35%, respectively.

[0083] Example 4 H2A.Z1 Genes significantly influence the formation of new organs in poplar trees. Test material: Wild-type poplar “nanlin895” (WT) H2A.Z1 Interference with transgenic poplar lines Ami-57, Ami-58, and Ami-203.

[0084] Experimental method: Wild-type poplar seedlings grown in tissue culture bottles for 20 days and H2A.Z1 The transgenic poplar tissue culture seedlings were transferred to soil-grown pots in the culture room. After growing in the soil-grown pots for two months, the effects on wild-type poplar seedlings and... H2A.Z1 Phenotypic observation and quantitative trait statistics were conducted on the tree shape of the transgenic poplar seedlings, including plant height, number of stem nodes, and stem node length (first to ninth stem nodes).

[0085] The results are as follows Picture 1 As shown. The results show that compared with wild-type poplar seedlings, H2A.Z1 Interference significantly reduced the height of transgenic poplar seedlings, significantly decreased the number of stem nodes, and significantly increased the length of stem nodes. Among these, wild-type poplar seedlings... H2A.Z1 The average heights of the interference-transgenic poplar seedlings Ami-57, Ami-58, and Ami-203 were 62.7 cm, 41.9 cm, 48.3 cm, and 50.1 cm, respectively; the wild-type poplar seedlings, H2A.Z1 The average number of stem nodes in the interference transgenic poplar seedlings Ami-57, Ami-58, and Ami-203 were 36, 20, 21, and 22, respectively; wild-type poplar seedlings, H2A.Z1The stem segment lengths (first to ninth stem segments) of the interference transgenic poplar seedlings Ami-57, Ami-58, and Ami-203 were 116.1 mm, 229.5 mm, 231.3 mm, and 234.9 mm, respectively.

[0086] Example 5 H2A.Z1 Genes significantly affect the size of poplar cells Test material: Wild-type poplar “nanlin895” (WT) H2A.Z1 Interference with the transgenic poplar line Ami-203.

[0087] Experimental method: Wild-type poplar seedlings grown in tissue culture bottles for 20 days and H2A.Z1 Interference was caused by transferring transgenic poplar tissue culture seedlings to soil-grown pots in the culture room. Wild-type poplar seedlings that had grown in the soil-grown pots for two months were then compared with... H2A.Z1 A sampling experiment was conducted on transgenic poplar seedlings Ami-203 that interfered with the sampling of stem nodes at the same growth time (i.e., the first stem node was marked after 45 days of growth in soil-grown pots, and the marked stem node was considered the same growth time node after another 15 days of growth). Wild-type poplar seedlings and H2A.Z1 The stem nodes at the same growth time of the interference transgenic poplar seedling Ami-203 were the ninth stem node (IN9) and the fifth stem node (IN5). Cryo-scanning electron microscopy was performed on the stem epidermal cells of the same growth time nodes and their corresponding leaf lower epidermal cells, and the area of ​​stem epidermal cells and leaf epidermal cells was counted.

[0088] The results are as follows Picture 2 As shown. The results show that compared with wild-type poplar seedlings, H2A.Z1 Interference with transgenic poplar seedlings increased the area of ​​stem epidermal cells by 124.9% and the area of ​​lower leaf epidermal cells by 45.8%. H2A.Z1 A moderate reduction in gene expression significantly increased the area of ​​poplar epidermal cells.

[0089] Example 6 H2A.Z1 Genes significantly affect the ploidy of poplar cell chromosomes. Test material: Wild-type poplar “nanlin895” (WT) H2A.Z1 Interference with the transgenic poplar line Ami-203.

[0090] Experimental method: Wild-type poplar seedlings grown in tissue culture bottles for 20 days and H2A.Z1 Interference-transgenic poplar tissue culture seedlings were transferred to soil-grown pots in the culture room. After two months of growth in the soil-grown pots, flow cytometry was used to determine the difference between wild-type and transgenic poplar seedlings. H2A.Z1DNA content in cells of the Ami-203 transgenic poplar seedling strain was analyzed, and the proportion of cells at stage 2C and stage 4C was determined. Wild-type poplar seedlings grown in tissue culture flasks for 10 days and... H2A.Z1 Interference was applied to the root tips of transgenic poplar tissue culture seedlings up to 1 cm in diameter, followed by DAPI treatment of wild-type poplar seedlings and... H2A.Z1 The nuclei of root tip cells from the Ami-203 line of transgenic poplar seedlings were stained, and the number of chromosomes in individual cells was counted.

[0091] The results are as follows Picture 3 As shown in the figure. The results showed that the proportions of cells in the 2C and 4C stages in wild-type poplar seedlings were 80% and 20%, respectively, while H2A.Z1 The percentages of cells in the 2C and 4C stages of the transgenic poplar seedlings were 10% and 90%, respectively; the number of chromosomes in the nuclei of root tip cells of wild-type poplar seedlings was 38. H2A.Z1 The number of chromosomes in the root tip cells of the transgenic poplar seedlings was 76. These results indicate... H2A.Z1 A moderate decrease in gene expression levels leads to an increase in the ploidy of poplar chromosomes, transforming them from diploid poplars into tetraploid poplars.

[0092] In summary, H2A.Z1 Genes can regulate chromosome doubling, which in turn affects the occurrence and formation of new organs in poplar trees. This discovery can provide new ideas for cultivating polyploid poplar trees.

[0093] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of H2A.Z1 protein or substances regulating the content and / or activity of said H2A.Z1 protein in any of the following A1)-A7): A1) Regulating plant height; A2) Regulating the number of plant stem nodes; A3) Regulating the length of plant stem nodes; A4) Regulates plant cell size; A5) Regulates the ploidy of plant cell chromosomes; A6) Cultivate transgenic plants with altered plant height and / or number of stem nodes and / or stem node length and / or cell size and / or chromosome ploidy; A7) Plant breeding; The H2A.Z1 protein is any one of the following (B1)-B4): B1) The amino acid sequence of the protein is shown in sequence 3; B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 3; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 3. B4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 3 and has the same function.

2. Use of biomaterials related to the H2A.Z1 protein of claim 1 in any of the following A1)-A7): A1) Regulating plant height; A2) Regulating the number of plant stem nodes; A3) Regulating the length of plant stem nodes; A4) Regulates plant cell size; A5) Regulates the ploidy of plant cell chromosomes; A6) Cultivate transgenic plants with altered plant height and / or number of stem nodes and / or stem node length and / or cell size and / or chromosome ploidy; A7 plant breeding; The biomaterial is any one of the following E1) to E5): E1) A nucleic acid molecule encoding the H2A.Z1 protein as described in claim 1; E2) Inhibit or interfere with the expression of the H2A.Z1 protein-encoding gene as described in claim 1; E3) An expression cassette containing the nucleic acid molecules described in E1) or E2); E4) A recombinant vector containing the nucleic acid molecules described in E1) or E2); E5) Recombinant microorganisms containing the nucleic acid molecules described in E1) or E2).

3. The application according to claim 2, characterized in that: E1) The nucleic acid molecule is any one of the following: F1) The DNA molecule shown in sequence 1, sequence 2, or sequence 6; The nucleotide sequence defined by F2) has 75% or more identity with F1) and is a DNA molecule encoding the H2A.Z1 protein.

4. The application according to claim 2, characterized in that, E2) The nucleic acid molecule is any one of the following: G1) RNA molecules shown in sequence 5 or sequence 7; The nucleotide sequence defined by G2) has 75% or more identity with G1) and is an RNA molecule that inhibits or interferes with the expression of the H2A.Z1 protein-coding gene.

5. The application according to any one of claims 1-4, characterized in that: The regulation of ploidy in plant cell chromosomes is to double the number of chromosomes in the plant cell.

6. A method for cultivating transgenic plants with reduced plant height and / or reduced number of stem nodes and / or increased stem node length and / or larger cells and / or doubled chromosomes, comprising the following steps: reducing the content and / or activity of the H2A.Z1 protein as described in claim 1 in the target plant to obtain transgenic plants with reduced plant height and / or reduced number of stem nodes and / or increased stem node length and / or larger cells and / or doubled chromosomes.

7. The method according to claim 6, characterized in that: The chromosome doubling is to change the plant from diploid to tetraploid.

8. The method according to claim 7, characterized in that: The method for reducing the content and / or activity of the H2A.Z1 protein in the target plant as described in claim 1 is to introduce a substance that inhibits or interferes with the expression of the H2A.Z1 protein encoding gene as described in claim 1 into the target plant; Alternatively, the substance that inhibits or interferes with the expression of the H2A.Z1 protein-coding gene in claim 1 may be a miRNA that inhibits or interferes with the expression of the H2A.Z1 protein-coding gene in claim 1; Alternatively, the nucleotide sequence of the miRNA is shown in Sequence 7.

9. The application according to any one of claims 1-5 or the method according to any one of claims 6-8, characterized in that: The plant is a dicotyledonous plant or a monocotyledonous plant.

10. miRNA, wherein the miRNA is an RNA molecule that has one or more nucleotides of sequence 7 deleted, added, or altered, and has the same function as sequence 7.

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