Application of H2A.Z1 protein and related biological materials thereof in regulation and control of plant chromosome doubling

Abstract

The invention discloses an application of H2A. Z1 protein and related biological materials thereof 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 a wild type material, 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] The present invention relates to the field of biotechnology, and particularly to the application of H2A.Z1 protein and related biological materials in regulating chromosome doubling in plants. Background Art

[0002] Polyploid breeding is an important means for cultivating new plant varieties. Compared with conventional breeding methods, it has significant advantages such as quick results, short generation cycles, low dependence on seeds, and few adverse effects caused by reduced fertility. However, the polyploid germplasm resources in nature are very limited, and the technology for cultivating plant polyploid varieties still needs to be improved. Therefore, it is necessary to explore new breeding approaches to generate new autopolyploid plants, thereby enriching the genetic diversity of plants and providing important technical support for the cultivation of new varieties of economic plants such as vegetables, flowers, and fruit trees.

[0003] At present, the main methods of plant polyploid breeding include screening natural polyploids, doubling somatic chromosomes, crossing different ploidy bodies, crossing unreduced gametes, etc. These methods have their own advantages and disadvantages and vary greatly among different species. The main method used for plant chromosome doubling is still to treat leaf discs, petioles, stem segments, etc. of tissue culture seedlings with low-concentration colchicine. Although this method is fast, efficient, and widely applicable, it has disadvantages such as poor safety, easy generation of aneuploids, and low sample survival rate. Therefore, it is highly necessary to develop a new safe and efficient mode for plant chromosome doubling. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to artificially regulate chromosome doubling in plant cells. The technical problems to be solved are not limited to the described technical theme, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0005] To solve the above technical problems, the present invention first provides a new use of H2A.Z1 protein or a substance that regulates the content and / or activity of the H2A.Z1 protein.

[0006] The present invention provides the application of H2A.Z1 protein or a substance that regulates the content and / or activity of the H2A.Z1 protein in any one of the following A1)-A7): A1) Regulating the plant height; A2) Regulating the number of plant stem nodes; A3) Regulating the length of plant stem nodes; A4) Regulating the size of plant cells; A5) Regulating the ploidy of plant cell chromosomes; A6) Cultivating transgenic plants with altered plant height and / or number of stem nodes and / or length of stem nodes and / or size of cells and / or chromosome ploidy; A7) Plant breeding; The H2A.Z1 protein is any one of the following B1)-B4): B1) A protein with an amino acid sequence shown in SEQ ID NO: 3; B2) A fusion protein with the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 3; B3) A protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO: 3; B4) A protein with 80% or more identity to the amino acid sequence shown in SEQ ID NO: 3 and having the same function.

[0007] In the protein described in B2) above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology for the purpose of facilitating the expression, detection, tracing, and / or purification of the target protein. The tags include, but are not limited to: GST (glutathione S-transferase) 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 (monomeric red fluorescent protein), or AviTag tag protein.

[0008] In the protein described in B3) above, the substitution and / or deletion and / or addition of one or several 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 residue substitution and / or deletion and / or addition.

[0009] 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 web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by 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 Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search, the identity of a pair of amino acid sequences can be calculated, and then the identity value (%) can be obtained. The identity includes an amino acid sequence having 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 the present invention.

[0010] The protein described in B1)-B4) above can be artificially synthesized, or its coding gene can be synthesized first and then biologically expressed.

[0011] Any of the substances described above for regulating the content and / or activity of the H2A.Z1 protein includes a substance that increases the content and / or activity of the H2A.Z1 protein or a substance that decreases the content and / or activity of the H2A.Z1 protein.

[0012] Furthermore, the substance that increases the activity of the H2A.Z1 protein can be a protein, polypeptide, or small molecule compound that enhances or promotes the function of the H2A.Z1 protein.

[0013] The substance that increases the content of the H2A.Z1 protein can be a substance that promotes the synthesis of the H2A.Z1 protein, inhibits the degradation of the H2A.Z1 protein, or overexpresses the H2A.Z1 protein.

[0014] The substance that decreases the activity of the H2A.Z1 protein can be a protein, polypeptide, or small molecule compound that inhibits the function of the H2A.Z1 protein.

[0015] The substance that decreases the content of the H2A.Z1 protein can be a substance that inhibits the synthesis of the H2A.Z1 protein, promotes the degradation of the H2A.Z1 protein, or knocks down (knocks down) or knocks out the coding gene of the H2A.Z1 protein.

[0016] Furthermore, the substance for knocking down (knocking down) the H2A.Z1 protein-coding gene can be any nucleic acid molecule capable of inhibiting or interfering with the expression of the above-mentioned H2A.Z1 protein-coding gene, such as gRNA (such as sgRNA), siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0017] Furthermore, the nucleic acid molecule for inhibiting or interfering with the expression of the above-mentioned H2A.Z1 protein-coding gene is miRNA for inhibiting or interfering with the expression of the above-mentioned H2A.Z1 protein-coding gene.

[0018] In some embodiments, the nucleotide sequence of the miRNA for inhibiting or interfering with the expression of the above-mentioned H2A.Z1 protein-coding gene is as shown in Sequence 7.

[0019] To solve the above problems, the present invention also provides a new use of a biological material related to the H2A.Z1 protein.

[0020] The present invention provides the application of a biological material related to the H2A.Z1 protein in any one of the following A1)-A7): A1) Regulating plant plant height; A2) Regulating the number of plant stem nodes; A3) Regulating the length of plant stem nodes; A4) Regulating plant cell size; A5) Regulating the ploidy of plant cell chromosomes; A6) Cultivating transgenic plants with altered plant height and / or number of stem nodes and / or length of stem nodes and / or cell size and / or chromosome ploidy; A7) Plant breeding; The biological material is any one of the following E1) to E5): E1) A nucleic acid molecule encoding the above-mentioned H2A.Z1 protein; E2) A nucleic acid molecule for inhibiting or interfering with the expression of the above-mentioned H2A.Z1 protein-coding gene; E3) An expression cassette containing the nucleic acid molecule described in E1) or E2); E4) A recombinant vector containing the nucleic acid molecule described in E1) or E2); E5) A recombinant microorganism containing the nucleic acid molecule described in E1) or E2).

[0021] In the above application, the nucleic acid molecule in E1) is any one of the following: F1) The DNA molecule shown in Sequence 1 or Sequence 2 or Sequence 6; F2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in F1) and encoding the above-mentioned H2A.Z1 protein.

[0022] In the above application, the nucleic acid molecule E2) is any one of the following: G1) RNA molecule shown in SEQ ID NO: 5 or SEQ ID NO: 7; G2) A nucleic acid molecule that has 75% or more identity with the nucleotide sequence defined in G1) and inhibits or interferes with the expression of the gene encoding the H2A.Z1 protein.

[0023] A person skilled in the art can easily adopt known methods, such as directed evolution and point mutation, to mutate the nucleotide sequence encoding the above H2A.Z1 protein of the present invention. H2A.Z1 Nucleotides with 75% or higher identity to the nucleotide sequence are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above-mentioned H2A.Z1 protein and have the same function. The identity refers to the sequence similarity with the natural nucleic acid sequence, including nucleotide sequences with 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity to the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 3 of the present invention. The identity can be evaluated by the naked eye or 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.

[0024] The nucleic acid molecule described in E2) above may be gRNA (such as sgRNA), siRNA, dsRNA, shRNA, miRNA or antisense RNA.

[0025] Any of the above-mentioned nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA.

[0026] Any of the above-mentioned nucleic acid molecules can be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0027] Any of the above-mentioned expression cassettes may include a promoter, the nucleic acid molecule described in E1) or E2) above, and a terminator. Promoters that can be used in the present 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.

[0028] Any of the above-mentioned vectors refers to a vector that can transport the nucleic acid molecule described in E1) or E2) above into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0029] Any of the above-mentioned recombinant vectors refers to a recombinant DNA molecule constructed by ligating the nucleic acid molecule described in E1) or E2) above with the vector in vitro. Existing plant expression vectors can be used to construct recombinant vectors containing the nucleic acid molecule described in E1) or E2) above. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant particle bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (from CAMBIA company), etc. The plant expression vector may also contain the 3´ untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression.

[0030] Any of the above-mentioned microorganisms can be bacteria, fungi, actinomycetes, protozoa, algae or viruses. The cell is Agrobacterium, such as Agrobacterium tumefaciens GV3101.

[0031] Any of the above-mentioned recombinant microorganisms refers to the recombinant microorganisms obtained by operating and modifying the genes of the target microorganisms, so that the functions are changed. Such as the recombinant microorganisms obtained after introducing the above-mentioned recombinant vector into the target microorganisms. The recombinant microorganisms can be understood as not only referring to specific recombinant microorganisms, but also referring to the offspring of such cells. And due to natural, accidental or intentional mutations and / or alterations, the offspring may not be exactly the same as the original parental cells, but are still included in the scope of recombinant microorganisms.

[0032] In any of the above-mentioned applications, the regulation of plant plant height is to increase or decrease the plant plant height. The regulation method is positive regulation, that is, when the content and / or activity of H2A.Z1 protein in the plant increases, the plant plant height increases, and when the content and / or activity of H2A.Z1 protein in the plant decreases or is absent, the plant plant height decreases.

[0033] In some embodiments, when H2A.Z1 the gene expression level in the plant decreases, the plant plant height decreases.

[0034] In any of the above applications, the regulation of the number of plant stem segments is to increase or decrease the number of plant stem segments. The regulation method is positive regulation, that is, when the content and / or activity of H2A.Z1 protein in the plant increases, the number of plant stem segments increases, and when the content and / or activity of H2A.Z1 protein in the plant decreases or is absent, the number of plant stem segments decreases.

[0035] In some embodiments, when H2A.Z1 the gene expression level in the plant decreases, the number of plant stem segments decreases.

[0036] In any of the above applications, the regulation of the length of plant stem segments is to increase or decrease the length of plant stem segments. The regulation method is negative regulation, that is, when the content and / or activity of H2A.Z1 protein in the plant increases, the length of plant stem segments decreases, and when the content and / or activity of H2A.Z1 protein in the plant decreases or is absent, the length of plant stem segments increases.

[0037] In some embodiments, when H2A.Z1 the gene expression level in the plant decreases, the length of plant stem segments (such as the total length of the first to the ninth stem segments) increases.

[0038] In any of the above applications, the regulation of the size of plant cells is to make plant cells larger or smaller. The regulation method is negative regulation, that is, when the content and / or activity of H2A.Z1 protein in the plant increases, plant cells become smaller, and when the content and / or activity of H2A.Z1 protein in the plant decreases or is absent, plant cells become larger.

[0039] In some embodiments, when H2A.Z1 the gene expression level in the plant decreases, the area of plant cells (such as stem epidermal cells and leaf epidermal cells) becomes larger.

[0040] In any of the above applications, the regulation of the ploidy of plant cell chromosomes is to double the chromosomes of plant cells. Specifically, when the content and / or activity of H2A.Z1 protein in the plant decreases or is absent, the chromosomes of plant cells double.

[0041] In some embodiments, when H2A.Z1 the gene expression level in the plant decreases, the chromosomes of plant cells double, changing from diploid to tetraploid.

[0042] In any of the above applications, the purpose of plant breeding is to cultivate polyploid plants (such as tetraploid plants) or plants with reduced plant height and / or reduced number of stem segments and / or increased stem segment length and / or larger cells.

[0043] To solve the above 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 enlarged cells and / or chromosome doubling.

[0044] 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 enlarged cells and / or chromosome doubling 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 enlarged cells and / or chromosome doubling.

[0045] Furthermore, the chromosome doubling is to change the plant from diploid to tetraploid. The change of the plant from diploid to tetraploid is manifested as follows 1) or 2): 1) The proportions of cells in the 2C stage and 4C stage in the apical bud cells of the target plant are 80% and 20% respectively, and the proportions of cells in the 2C stage and 4C stage in the apical bud cells of the transgenic plant are 10% and 90% respectively; 2) The chromosome content in the nucleus of the root tip cells of the target plant is 38 (2 chromosome sets), and the chromosome content in the nucleus of the root tip cells of the transgenic plant is 76 (4 chromosome sets).

[0046] Still further, the method for reducing the content and / or activity of the above-mentioned 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.

[0047] Even further, the substance that inhibits the expression of the H2A.Z1 protein-encoding gene in the target plant is an miRNA that inhibits or interferes with the expression of the above-mentioned H2A.Z1 protein-encoding gene.

[0048] In some embodiments, the nucleotide sequence of the miRNA is as shown in Sequence 7.

[0049] In any of the above-mentioned applications or methods, the transgenic plants not only include the first-generation transgenic plants obtained by transforming the target plant with a substance that inhibits or interferes with the expression of the H2A.Z1 protein-encoding gene, but also include their progeny. For transgenic plants, the gene can be propagated in this species, or the gene can be transferred into other varieties of the same species, especially including commercial varieties, by conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

[0050] In any of the above applications or methods, the plant can be a dicotyledon or a monocotyledon, including food crops such as rice, wheat, barley, corn, soybean, potato, bean, oat and millet; vegetable crops such as Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, zucchini, leek, onion and carrot; economic crops such as ginseng, tobacco, cotton, sesame, sugarcane, beet, wild sesame, peanut and rapeseed; and cash crops such as apple, pear, jujube, peach, Fruits including kiwi, grape, orange, persimmon, plum, apricot and banana; flowers including rose, gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; fodder crops including ryegrass, red clover, orchard grass, alfalfa, tall butter grass and perennial ryegrass; woody plants for making wood including poplar, willow, birch, locust, elm, metasequoia, spruce, beech, maple, oak, chinaberry, ash, nasturtium, iron wire, red sandalwood, yellow sandalwood, teak, ash, maple, etc. Further, the plant is a dicotyledon. Further, the dicotyledon is a dicotyledonous woody plant. In a specific embodiment of the present invention, the dicotyledonous woody plant is poplar.

[0051] In order to solve the above technical problems, the present invention finally provides a miRNA.

[0052] The miRNA provided by the present invention is as follows a1) or a2): a1) RNA molecule shown in sequence 7; a2) An RNA molecule with the same function as sequence 7, wherein one or several nucleotides are deleted, added or changed.

[0053] The present invention constructs H2A.Z1 Transgenic poplars with reduced gene expression. Experiments have shown that compared with wild-type poplars, H2A.Z1 The transgenic poplars with reduced gene expression had reduced plant height and number of nodes, and increased node length. H2A.Z1 The cells in the stem and leaf organs of the transgenic poplar with reduced gene expression become larger, and the chromosomes of the cells double, changing from diploid to tetraploid. The present invention is of great significance for revealing the regulatory molecular mechanism of the occurrence or formation of new organs of forest trees and poplar polyploid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Two-month-old wild-type poplar seedlings were grown in soil pots. H2A.Z1 Phenotypic observation and statistical diagram of the interference transgenic poplar seedlings. A is a wild-type poplar (WT) and H2A.Z1The phenotypes of the transgenic poplar lines Ami-57, Ami-58, and Ami-203 were interfered with. B is a wild-type poplar seedling and H2A.Z1 Interference in transgenic poplar lines Ami-57, Ami-58, and Ami-203 H2A.Z1 Gene expression detection results. C is wild-type poplar seedlings and H2A.Z1 The plant height of the transgenic poplar lines Ami-57, Ami-58, and Ami-203 was interfered. D is the height of wild-type poplar seedlings and H2A.Z1 The number of stem nodes of the transgenic poplar lines Ami-57, Ami-58, and Ami-203 was interfered. E is the number of stem nodes of wild-type poplar seedlings and H2A.Z1 Interference transgenic poplar strains Ami-57, Ami-58, Ami-203 stem node length. The stem node refers to the stem segment between two consecutive leaves, and the stem node length here refers to the total length of the first stem node to the ninth stem node. Among them, the first stem node from the terminal bud is defined as the first stem node, the second stem node is defined as the second stem node, and so on, the ninth stem node is defined as the ninth stem node. Each strain system counted 5 replicates, the same letters: no significant difference.

[0055] Figure 2 Wild-type poplars grown in soil pots for two months H2A.Z1 Cytological analysis of the interference transgenic poplar. A is a wild-type poplar and H2A.Z1 Figure 1 shows the stem epidermal cells and leaf epidermal cells of the Ami-203 transgenic poplar. B shows the stem epidermal cells and leaf epidermal cells of the Ami-203 transgenic poplar. H2A.Z1 Statistical analysis of stem epidermal cell area of ​​Ami-203 transgenic poplar. C is a graph showing the area of ​​stem epidermal cells of wild-type poplar and H2A.Z1 Statistical analysis of leaf epidermal cell area of ​​the interference transgenic poplar Ami-203.

[0056] Figure 3 For wild-type poplar and H2A.Z1 Figure 2. DNA content and chromosome number detection in the nucleus of the interfering transgenic poplar. A is a wild-type poplar grown in a soil pot for two months. H2A.Z1 Figure 2. DNA content detection of the top cells of the stems of the interfering transgenic poplars. B is a picture of the wild-type poplars and H2A.Z1 Statistical diagram of DNA content in the stem apical cells of the interfering transgenic poplar. C is a wild-type poplar and H2A.Z1 Chromosome map of root tip cells of interference transgenic poplar. D is the wild type poplar and H2A.Z1 Statistical diagram of chromosome number in root tip cells of interference transgenic poplar. DETAILED DESCRIPTION

[0057] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0058] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0059] The PGWB2 vector in the following embodiments is described in the literature "Xu J, Lee YJ, Liu B. (2019) Establishment of a mitotic model system by transient expression of the D-type cyclin in differentiated leaf cells of tobacco (Nicotiana benthamiana).[J].New Phytol, 226(4):1213-1220."

[0060] The Agrobacterium tumefaciens GV3101 strain in the following embodiments 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."

[0061] The wild-type poplar "nanlin895" in the following embodiments is described in the literature "Chao Q., et al. (2019). "The developmental dynamics of the Populus stem transcriptome." Plant Biotechnol J 17(1): 206-219."

[0062] The pENTR / D-TOPO® vector 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."

[0063] Example 1. Obtaining of H2A.Z1 Protein and Its Encoding Gene 1. The whole plant of "nanlin 895" poplar growing in tissue culture flasks for one month was frozen in liquid nitrogen, ground, and total RNA was extracted. Then the total RNA was reverse transcribed to obtain poplar cDNA.

[0064] 2. Using the obtained cDNA as a template, with 5'-ATGGCTGGAAAAGGAGG-3' as the forward primer and 5'-TCACTCTTTGGTGGTTTTGTTG-3' as the reverse primer for PCR amplification to obtain an amplification product.

[0065] 3. After detecting the amplification product by agarose gel electrophoresis, a DNA fragment of about 414 bp was separated and purified, and then this fragment was ligated to the pEASY ® -Blunt Simple cloning vector, and the vector ligated with the target fragment was sequenced. Since "nanlin 895" is a variety obtained by crossing Populus deltoides and Populus euramericana, its genome is relatively complex, and there may be more than one sequence information at the same gene locus. Two H2A.Z1 coding region sequences of the gene were obtained by PCR amplification.

[0066] The sequencing results showed that in poplar "nanlin 895", H2A.Z1 the coding region sequences of the gene are as shown in Sequence 1 and Sequence 2; the amino acid sequences of the H2A.Z1 proteins encoded by the coding region sequences shown in Sequence 1 and Sequence 2 are the same, both as shown in Sequence 3. H2A.Z1

[0067] Example 2. Construction of Recombinant Vectors and Recombinant Agrobacterium I. Construction of Recombinant Interference Vectors and Recombinant Interference Agrobacterium 1. Construction of the recombinant interference vector PGWB2-Ami (1) Using the H2A.Z1 ​The common coding sequence 5'-AGGGGCTTCTGGCAACGAAAA-3' in the gene coding region is used as the target sequence, and primers are 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'-gaATTTCGTAGCCAGAAGCGTGTtctacatatatattcct-3'.

[0068] (2) Using the pRS300 (miR319a) vector as a template, a round of conventional PCR and a round of overlap PCR are performed with the four primer sequences in step (1) (for the specific method, refer to Rebecca Schwab, MPI for Developmental Biology, Tuebingen, 2005) to amplify the artificial microRNA gene fragment. The nucleotide sequence of the artificial microRNA gene fragment is shown in Sequence 4.

[0069] (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, through the LR homologous recombination reaction, the artificial microRNA gene fragment in the pENTR / D-TOPO ® -Ami vector is ligated into the vector PGWB2 to obtain the recombinant interference vector PGWB2-Ami. The recombinant interference vector PGWB2-Ami expresses an RNA molecule with the nucleotide sequence of Sequence 5, and 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.

[0070] 2. Transformation of recombinant interference Agrobacterium PGWB2-Ami / GV3101 The recombinant interference vector PGWB2-Ami was transferred into Agrobacterium tumefaciens GV3101 by the Agrobacterium-mediated transformation method. After PCR detection, the recombinant interference Agrobacterium PGWB2-Ami / GV3101 was obtained.

[0071] Example 3 H2A.Z1 Obtaining and identification of interference transgenic poplars I. Obtaining of transgenic poplars The young leaves of tissue-cultured poplar seedlings "nanlin895" were infected with the recombinant interference Agrobacterium by the leaf disc method. After a series of callus induction, bud induction and rooting induction, transgenic poplar seedlings were obtained. The specific steps are as follows: 1. The monoclonal of the recombinant interference Agrobacterium PGWB2-Ami / GV3101 obtained in Example 2 was first cultured in 5 mL of liquid YEB medium (the liquid YEB medium was obtained by mixing 1 g of yeast extract, 5 g of tryptone, 5 g of beef extract, 5 g of sucrose, 1.954 g of anhydrous magnesium sulfate and 1 L of water, pH = 7.0) for 12 hours. Then, 1 mL of the cultured bacterial solution was taken into 100 mL of liquid YEB medium and cultured at 28 °C with shaking until OD 600nm = 0.8, and then 100 μm of acetosyringone was added.

[0072] 2. The young leaves of poplar seedlings grown in tissue culture bottles for one month were taken, and the four sides of the leaves were cut off with a surgical blade, leaving the leaves with a size of about 1 cm near the main vein 2 . One or two small wounds were gently cut on the main vein, and the leaves were placed in the well-cultured Agrobacterium and gently shaken for 30 min for infection.

[0073] 3. The leaves were fished out and placed with the back side down on the co-culture medium (the co-culture medium was the basal medium containing 0.2 mg / L of kinetin, 0.75 mg / L of 2,4-D, 100 μm of acetosyringone; the solvent of the basal medium was water, and the solutes and their concentrations were shown in Table 1) and co-cultured at 28 °C for two days.

[0074] 4. The leaves were transferred to the screening medium for callus induction (the screening medium was the basal medium containing 0.2 mg / L of kinetin, 0.75 mg / L of 2,4-D, 50 mg / L of kanamycin, 250 mg / L of cefotaxime sodium, 300 mg / L of timentin; the solvent of the basal medium was water, and the solutes and their concentrations were shown in Table 1) and cultured in the dark, and subcultured every 14 days until spherical callus tissues grew out.

[0075] 5. Cut the callus and place it on the differentiation medium for bud induction (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). Culture it under light and subculture every 15 days until buds emerge.

[0076] 6. Cut the small buds and culture them independently on the 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 grow, obtaining transgenic poplar seedlings. The transgenic poplar seedlings after rooting can be continuously propagated asexually.

[0077] Table 1. Solutes and their concentrations in the basal media of different media

[0078] II. Identification of transgenic poplars 1. PCR identification Extract the DNA of transgenic poplar seedlings for PCR identification, which specifically includes the following steps: Extract the DNA of all transgenic poplar seedlings using the SLS method, perform PCR amplification using PGWB2-Ami-F and PGWB2-Ami-R to obtain PCR products. At the same time, use wild-type poplar DNA as a control. The primer sequences are as follows: PGWB2-Ami-F: 5'-GGGGACTCTAGAGTTATCAAC-3'; PGWB2-Ami-R: 5'-CTAAGCGCTGTTATCAACCAC-3'.

[0079] The PCR products are detected by electrophoresis, and preliminary positive transgenic plants can be obtained. The PCR product with a size of 822 bp amplified is a positive transgenic plant.

[0080] 2. H2A.Z1 Obtaining of interfering transgenic poplars For positive transgenic plants, perform expression level detection according to the following steps: Take the leaves of positive transgenic plants grown for one month and wild-type plants under the same growth conditions, extract the total RNA of the leaves using the plant RNA small extraction kit from Megan Company, then use the reverse transcription kit from Invitrogen Company to synthesize cDNA with Oligo d(T) as the primer, and use H2A.Z1Gene-specific primers were used for real-time quantitative PCR detection, with Actin as the 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'.

[0081] The results are as follows Figure 1 The results showed 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.

[0082] Embodiment 4, H2A.Z1 Gene significantly affects the formation of new organs in poplar trees Test materials: wild-type poplar "nanlin895" (WT), H2A.Z1 Interference with transgenic poplar lines Ami-57, Ami-58, and Ami-203.

[0083] Experimental method: Wild-type poplar tissue culture seedlings grown in tissue culture bottles for 20 days and H2A.Z1 The transgenic poplar tissue culture seedlings were transferred to the soil pots in the culture room. After growing in the soil pots for two months, the wild-type poplar seedlings and H2A.Z1 The tree shape of the transgenic poplar seedlings was disturbed, including plant height, number of nodes and node length (first to ninth nodes), for phenotypic observation and statistics of quantitative traits.

[0084] The results are as follows Figure 1 The results showed that compared with wild-type poplar seedlings, H2A.Z1 The height of the transgenic poplar seedlings was significantly reduced, the number of stem nodes was significantly reduced, and the length of the stem nodes was significantly increased. H2A.Z1 The average plant heights of the genetically modified 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 average plant heights of the wild-type poplar seedlings, H2A.Z1 The average number of stem nodes of the transgenic poplar seedlings Ami-57, Ami-58, and Ami-203 was 36, 20, 21, and 22, respectively; the average number of stem nodes of the wild-type poplar seedlings, H2A.Z1The stem segment lengths (the first to the ninth stem segments) of the transgenic poplar seedlings Ami-57, Ami-58, and Ami-203 were interfered with, and were 116.1 mm, 229.5 mm, 231.3 mm, and 234.9 mm respectively.

[0085] Example 5, H2A.Z1 The gene significantly affects the size of poplar cells Test materials: Wild-type poplar "nanlin895" (WT), H2A.Z1 The transgenic poplar Ami-203 strain with interference.

[0086] Experimental method: Transfer the wild-type poplar tissue culture seedlings grown in tissue culture flasks for 20 days and H2A.Z1 the transgenic poplar tissue culture seedlings with interference to soil culture pots in the culture room for cultivation. After growing in the soil culture pots for two months, sample the stem segments of the wild-type poplar seedlings and H2A.Z1 the transgenic poplar seedlings Ami-203 with the same growth time (i.e., mark at the first stem segment after growing in the soil culture pot for 45 days, and the stem segment at the marked position after growing for another 15 days is the stem segment with the same growth time) for experiments. The stem segments with the same growth time of the wild-type poplar seedlings and H2A.Z1 the transgenic poplar seedlings Ami-203 with interference are the ninth stem segment (IN9) and the fifth stem segment (IN5) respectively. Observe the stem epidermal cells and their corresponding leaf lower epidermal cells of the stem segments with the same growth time by cryo-scanning electron microscopy, and count the areas of the stem epidermal cells and leaf epidermal cells.

[0087] The results are as Figure 2 shown. The results show that: compared with the wild-type poplar seedlings, H2A.Z1 the area of the stem epidermal cells of the transgenic poplar seedlings with interference increased by 124.9%, and the area of the leaf lower epidermal cells increased by 45.8%; H2A.Z1 After the gene expression level was moderately reduced, the area of the poplar epidermal cells increased significantly.

[0088] Example 6, H2A.Z1 The gene significantly affects the chromosome ploidy of poplar cells Test materials: Wild-type poplar "nanlin895" (WT), H2A.Z1 The transgenic poplar Ami-203 strain with interference.

[0089] Experimental method: Transfer the wild-type poplar tissue culture seedlings grown in tissue culture flasks for 20 days and H2A.Z1 the transgenic poplar tissue culture seedlings with interference to soil culture pots in the culture room for cultivation. After growing in the soil culture pots for two months, use a flow cytometer to measure the wild-type poplar seedlings and H2A.Z1Interfere with the DNA content in the cells of the transgenic poplar seedling line Ami-203, and count the proportions of cells in the 2C stage and 4C stage. Take the wild-type poplar tissue culture seedlings that have grown in tissue culture flasks for 10 days and H2A.Z1 Take 1 cm of the root tips of the transgenic poplar tissue culture seedlings with interference, and then use DAPI to stain the cell nuclei of the root tips of the wild-type poplar seedlings and H2A.Z1 the transgenic poplar seedling line Ami-203 with interference, and count the number of chromosomes in a single cell.

[0090] The results are as Figure 3 shown. The results show that the proportions of cells in the 2C stage and 4C stage in the wild-type poplar seedling cells are 80% and 20% respectively, while H2A.Z1 the proportions of cells in the 2C stage and 4C stage in the transgenic poplar seedling cells with interference are 10% and 90% respectively; the number of chromosomes in the cell nuclei of the root tips of the wild-type poplar seedlings is 38, H2A.Z1 and the number of chromosomes in the cell nuclei of the root tips of the transgenic poplar seedlings with interference is 76. The above results indicate that H2A.Z1 a moderate decrease in gene expression level increases the chromosome ploidy of poplar, changing from diploid poplar to tetraploid poplar.

[0091] In summary, H2A.Z1 genes can regulate chromosome doubling, thereby affecting the occurrence and formation of new organs in poplar. This discovery can provide new ideas for cultivating polyploid poplars.

[0092] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including changes made by using conventional techniques known in the art that are outside the scope disclosed in this application. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. Use of H2A.Z1 protein or a substance that regulates the content and / or activity of the H2A.Z1 protein in any of the following A1)-A7): A1) Regulate plant height; A2) Regulate the number of plant stem nodes; A3) Regulate the length of plant stem nodes; A4) Regulate plant cell size; A5) Regulates the ploidy of plant cell chromosomes; A6) Cultivating transgenic plants with altered plant height and / or number of nodes and / or 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 is the protein shown in SEQ ID NO:3; B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 3; B3) A protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 3; B4) A protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 3 and has the same function.

2. Use of biological materials related to the H2A.Z1 protein described in claim 1 in any of the following A1)-A7): A1) Regulate plant height; A2) Regulate the number of plant stem nodes; A3) Regulate the length of plant stem nodes; A4) Regulate plant cell size; A5) Regulates the ploidy of plant cell chromosomes; A6) Cultivating transgenic plants with altered plant height and / or number of nodes and / or node length and / or cell size and / or chromosome ploidy; A7 Plant breeding; The biological material is any one of the following E1) to E5): E1) a nucleic acid molecule encoding the H2A.Z1 protein of claim 1; E2) a nucleic acid molecule that inhibits or interferes with the expression of the gene encoding the H2A.Z1 protein of claim 1; E3) an expression cassette containing the nucleic acid molecule described in E1) or E2); E4) a recombinant vector containing the nucleic acid molecule described in E1) or E2); E5) A recombinant microorganism containing the nucleic acid molecule described in E1) or E2).

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

4. The use according to claim 2, characterized in that: E2) The nucleic acid molecule is any of the following: G1) RNA molecule shown in SEQ ID NO: 5 or SEQ ID NO: 7; G2) is an RNA molecule that has 75% or more identity with the nucleotide sequence defined in G1) and inhibits or interferes with the expression of the H2A.Z1 protein encoding gene.

5. The use according to any one of claims 1 to 4, characterized in that: The regulating the ploidy of plant cell chromosomes is to double the plant cell chromosomes.

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 enlarged cells and / or doubled chromosomes, comprising the following steps: reducing the content and / or activity of the H2A.Z1 protein 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 enlarged 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 of claim 1 in the target plant is to introduce a substance that inhibits or interferes with the expression of the gene encoding the H2A.Z1 protein of claim 1 into the target plant; Or, the substance that inhibits or interferes with the expression of the gene encoding the H2A.Z1 protein in claim 1 is a miRNA that inhibits or interferes with the expression of the gene encoding the H2A.Z1 protein in claim 1; Alternatively, the nucleotide sequence of the miRNA is as shown in Sequence 7.

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

10. miRNA, wherein the miRNA is as follows a1) or a2): a1) RNA molecule shown in sequence 7; a2) An RNA molecule with the same function as sequence 7, wherein one or several nucleotides are deleted, added or changed.

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