Application of h2a.z1 protein and related biological materials in regulating plant chromosome doubling

Abstract

The application discloses H2A.Z1 protein and related biomaterials in regulating plant chromosome doubling. H2A.Z1 The application takes a model species, poplar, as a research object, and constructs a transgenic poplar with reduced gene expression. H2A.Z1 Experiments prove that, compared with wild type materials, the transgenic poplar with reduced gene expression has reduced plant height and stem node number and increased stem node length. H2A.Z1 In addition, the cells in the stem and leaf organs of the transgenic poplar with reduced gene expression are large, and the cell chromosomes are doubled, changing from diploid to tetraploid. The application has important significance for researching polyploid breeding of vegetables, important flowers and fruit trees.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of H2A.Z1 protein and related biological materials in regulating plant chromosome doubling. BACKGROUND

[0002] Polyploidy breeding is an important means of cultivating new plant varieties. Compared with conventional breeding methods, it has the advantages of fast effect, short generation cycle, low dependence on seeds, and less adverse effects of reduced fertility. However, the resources of polyploid germplasm in nature are very limited, and the technology of breeding plant polyploid varieties still needs to be improved. Therefore, it is necessary to explore new breeding methods to produce new homologous polyploid plants and enrich the genetic diversity of plants, thereby 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 polyploidy breeding are screening of natural polyploids, doubling of somatic chromosomes, crossing of different ploidy, and crossing of unreduced gametes. These methods have their own advantages and disadvantages, and differ greatly between different species. The main method used for plant chromosome doubling is still the treatment of leaf discs, petioles, and stem segments of tissue culture seedlings with low concentration of autumn water acyl. Although this method is fast, efficient, and widely applicable, it has the disadvantages of poor safety, easy production of aneuploids, and low sample survival rate. Therefore, it is necessary to develop a new model of safe and efficient plant chromosome doubling. SUMMARY

[0004] The technical problem to be solved by the present application is how to artificially regulate chromosome doubling in plant cells. The technical problem to be solved is not limited to the technical subject described, and other technical subjects not mentioned in this article can be clearly understood by those skilled in the art through the following description.

[0005] To solve the above technical problem, the present application 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 application provides the use of H2A.Z1 protein or a substance that regulates the content and / or activity of the H2A.Z1 protein in any one of A1) to A7):

[0007] A1) regulating plant height;

[0008] A2) regulating the number of plant stem nodes;

[0009] A3) regulating the length of plant stem nodes;

[0010] A4) regulating plant cell size;

[0011] A5) modulating ploidy of chromosomes in plant cells;

[0012] A6) breeding 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;

[0013] A7) plant breeding;

[0014] The H2A.Z1 protein is any one of the following B1) to B4):

[0015] B1) a protein having an amino acid sequence as shown in SEQ ID NO: 3;

[0016] B2) a fusion protein having the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid sequence as shown in SEQ ID NO: 3;

[0017] B3) a protein having the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence as shown in SEQ ID NO: 3;

[0018] B4) a protein having 80% or more identity to the amino acid sequence as shown in SEQ ID NO: 3 and having the same function.

[0019] In the protein of B2) above, the tag refers to a polypeptide or protein that is expressed in fusion with the protein of interest using DNA recombination technology in vitro, so as to facilitate expression, detection, tracking and / or purification of the protein of interest. The tag includes but is not limited to a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein) or an AviTag tag protein.

[0020] In the protein of B3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is substitution and / or deletion and / or addition of 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.

[0021] The identity in the above B4) refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and performing a search in the high-level BLAST 2.1, and then the value of the identity (%) can be obtained. The identity includes an amino acid sequence having 80% or more, or having 85% or more, or having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 3 of the present application.

[0022] The proteins in the above B1) to B4) can be artificially synthesized, or a gene encoding the same can be synthesized first and then expressed biologically.

[0023] The substance for regulating the content and / or activity of the H2A.Z1 protein includes a substance for increasing the content and / or activity of the H2A.Z1 protein or a substance for decreasing the content and / or activity of the H2A.Z1 protein.

[0024] Further, the substance for increasing the activity of the H2A.Z1 protein can be a protein, a polypeptide, or a small molecule compound that enhances or promotes the function of the H2A.Z1 protein.

[0025] The substance for increasing the content of the H2A.Z1 protein can be a substance that promotes the synthesis of the H2A.Z1 protein, or inhibits the degradation of the H2A.Z1 protein, or overexpresses the H2A.Z1 protein.

[0026] The substance for decreasing the activity of the H2A.Z1 protein can be a protein, a polypeptide, or a small molecule compound that inhibits the function of the H2A.Z1 protein.

[0027] The substance for decreasing the content of the H2A.Z1 protein can be a substance that inhibits the synthesis of the H2A.Z1 protein, or promotes the degradation of the H2A.Z1 protein, or knocks down (knocks down) or knocks out the gene encoding the H2A.Z1 protein.

[0028] Further, the substance for knocking down (knocking down) the H2A.Z1 protein-encoding gene can be any nucleic acid molecule capable of inhibiting or interfering with the expression of the H2A.Z1 protein-encoding gene, such as gRNA (such as sgRNA), siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0029] Further, the nucleic acid molecule for inhibiting or interfering with the expression of the H2A.Z1 protein-encoding gene is a miRNA for inhibiting or interfering with the expression of the H2A.Z1 protein-encoding gene.

[0030] In some embodiments, the nucleotide sequence of the miRNA for inhibiting or interfering with the expression of the H2A.Z1 protein-encoding gene is as shown in SEQ ID NO: 7.

[0031] To solve the above problems, the present application further provides a new use of a biological material related to H2A.Z1 protein.

[0032] The present application provides the use of a biological material related to H2A.Z1 protein in any one of A1) to A7) below:

[0033] A1) regulating plant height;

[0034] A2) regulating the number of stem nodes of a plant;

[0035] A3) regulating the length of stem nodes of a plant;

[0036] A4) regulating cell size of a plant;

[0037] A5) regulating the ploidy of chromosomes of a plant cell;

[0038] A6) breeding transgenic plants with altered plant height and / or the number of stem nodes and / or the length of stem nodes and / or cell size and / or chromosome ploidy;

[0039] A7) plant breeding;

[0040] The biological material is any one of E1) to E5) below:

[0041] E1) a nucleic acid molecule encoding the H2A.Z1 protein;

[0042] E2) a nucleic acid molecule for inhibiting or interfering with the expression of the H2A.Z1 protein-encoding gene;

[0043] E3) an expression cassette containing the nucleic acid molecule of E1) or E2);

[0044] E4) a recombinant vector containing the nucleic acid molecule of E1) or E2);

[0045] E5) Recombinant microorganisms containing the nucleic acid molecules described in E1) or E2).

[0046] In the above applications, the nucleic acid molecule described in E1) is any of the following:

[0047] F1) The DNA molecule shown in sequence 1, sequence 2, or sequence 6;

[0048] 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.

[0049] In the above applications, the nucleic acid molecule described in E2) is any of the following:

[0050] G1) RNA molecules shown in sequence 5 or sequence 7;

[0051] 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.

[0052] 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.

[0053] The nucleic acid molecule mentioned in E2 above can be gRNA (such as sgRNA), siRNA, dsRNA, shRNA, miRNA or antisense RNA.

[0054] Any of the nucleic acid molecules mentioned above can be DNA, such as cDNA, genomic DNA, or recombinant DNA.

[0055] Any of the nucleic acid molecules mentioned above can be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0056] Any of the above-described expression cassettes can include a promoter, the nucleic acid molecule of E1) or E2) above, and a terminator. Promoters useful in the present application include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Further, the expression cassette can also include an enhancer sequence.

[0057] Any of the above-described vectors refers to a vector capable of carrying the nucleic acid molecule of 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, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0058] Any of the above-described recombinant vectors refers to a recombinant DNA molecule constructed by in vitro ligation of the nucleic acid molecule of E1) or E2) above with the vector. A recombinant vector containing the nucleic acid molecule of E1) or E2) above can be constructed using existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors useful for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA), etc. The plant expression vector can also contain a 3' untranslated region of a foreign gene, i.e., a polyadenylation signal and any other DNA segment involved in mRNA processing or gene expression.

[0059] Any of the above-described microorganisms can be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The cell is Agrobacterium, such as Agrobacterium tumefaciens GV3101.

[0060] Any of the above-described recombinant microorganisms refers to a recombinant microorganism obtained by manipulating and modifying the genes of the target microorganism, so that the function of the recombinant microorganism is changed. For example, a recombinant microorganism obtained by introducing the above-described recombinant vector into the target microorganism. The recombinant microorganism can be understood not only as a specific recombinant microorganism, but also as the progeny of such a cell, and due to natural, accidental or intentional mutations and / or changes, the progeny can not necessarily be completely identical to the original parent cell, but is still included in the scope of the recombinant microorganism.

[0061] 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.

[0062] In some implementation schemes, when plants H2A.Z1 When gene expression levels decrease, plant height decreases.

[0063] 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.

[0064] In some implementation schemes, when plants H2A.Z1 When gene expression levels decrease, the number of stem nodes in a plant decreases.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In some implementation schemes, when plantsH2A.Z1 When the expression amount of the gene is reduced, the chromosome of the plant cell is doubled, and the plant cell changes from diploid to tetraploid.

[0071] In any of the above applications, the purpose of the plant breeding is to breed a polyploid plant (such as a tetraploid plant) or to breed a plant with reduced plant height and / or reduced number of stem nodes and / or increased length of stem nodes and / or enlarged cells.

[0072] To solve the above technical problems, the present application also provides a method for breeding a transgenic plant with reduced plant height and / or reduced number of stem nodes and / or increased length of stem nodes and / or enlarged cells and / or chromosome doubling.

[0073] The method for breeding a transgenic plant with reduced plant height and / or reduced number of stem nodes and / or increased length of stem nodes and / or enlarged cells and / or chromosome doubling provided by the present application comprises the following steps: reducing the content and / or activity of the H2A.Z1 protein in the target plant to obtain a transgenic plant with reduced plant height and / or reduced number of stem nodes and / or increased length of stem nodes and / or enlarged cells and / or chromosome doubling.

[0074] Further, the chromosome doubling is to change the plant from diploid to tetraploid. The change of the plant from diploid to tetraploid is embodied as the following 1) or 2):

[0075] 1) The proportion of cells in the 2C phase and the 4C phase in the stem apical bud cells of the target plant is 80% and 20% respectively, and the proportion of cells in the 2C phase and the 4C phase in the stem apical bud cells of the transgenic plant is 10% and 90% respectively;

[0076] 2) The content of chromosomes in the cell nucleus of the root tip cells of the target plant is 38 (2 chromosome sets), and the content of chromosomes in the cell nucleus of the root tip cells of the transgenic plant is 76 (4 chromosome sets).

[0077] Further, 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 coding gene into the target plant.

[0078] Further, the substance that inhibits the expression of the H2A.Z1 protein coding gene in the target plant is an miRNA that inhibits or interferes with the expression of the H2A.Z1 protein coding gene.

[0079] In some embodiments, the nucleotide sequence of the miRNA is shown in SEQ ID NO: 7.

[0080] 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.

[0081] 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.

[0082] To address the aforementioned technical problems, this invention ultimately provides a miRNA.

[0083] The miRNA provided by this invention is either a1) or a2):

[0084] a1) The RNA molecule shown in sequence 7;

[0085] 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.

[0086] 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.Z1In 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 polyploid breeding of poplar trees. Attached Figure Description

[0087] Figure 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.Z1 The 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.

[0088] Figure 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.

[0089] Figure 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.Z1Figure 1. DNA content detection of stem tip cells of transgenic poplar interfering with. B is wild type poplar grown in soil pots for two months and H2A.Z1 Figure 2. Statistical chart of DNA content of stem tip cells of transgenic poplar interfering with. C is wild type poplar cultured in tissue for 10 days and H2A.Z1 Figure 3. Chromosome staining of root tip cells of transgenic poplar interfering with. D is wild type poplar cultured in tissue for 10 days and H2A.Z1 Figure 4. Statistical chart of chromosome number of root tip cells of transgenic poplar interfering with. DETAILED DESCRIPTION

[0090] The present application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.

[0091] The experimental methods in the following examples are all routine methods, unless otherwise specified, according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0092] 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-type cyclin in differentiated leaf cells of tobacco (Nicotiana benthamiana). [J]. New Phytol, 226(4): 1213-1220."

[0093] The Agrobacterium tumefaciens GV3101 strain 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."

[0094] 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." Plant Biotechnol J 17(1): 206-219."

[0095] 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."

[0096] Example 1, obtaining of H2A.Z1 protein and its encoding gene

[0097] 1. The whole plant of "nanlin 895" poplar grown in a tissue culture bottle for one month was frozen in liquid nitrogen, and total RNA was extracted after grinding, and then the total RNA was reverse transcribed to obtain poplar cDNA.

[0098] 2. The obtained cDNA was used as a template, 5'-ATGGCTGGAAAAGGAGG-3' was used as a forward primer, and 5'-TCACTCTTTGGTGGTTTTGTTG-3' was used as a reverse primer for PCR amplification to obtain an amplification product.

[0099] 3. The amplification product was detected and separated and purified by agarose gel electrophoresis to obtain a DNA fragment of about 414 bp, and then the fragment was connected to the pEASY ® -Blunt Simple cloning vector, and the vector connected with the target fragment was sequenced. Since "nanlin 895" is a variety obtained by crossing Populus deltoids and Populus tremula, its genome is relatively complex, and there may be more than one sequence information at the same genetic locus. Two coding region sequences of the gene were obtained by PCR amplification. H2A.Z1

[0100] The sequencing results show that the coding region sequence of the gene in the poplar "nanlin 895" is as shown in Sequence 1 and Sequence 2; the amino acid sequence of the H2A.Z1 protein encoded by the coding region sequence of the gene as shown in Sequence 1 and Sequence 2 is the same, and is as shown in Sequence 3. H2A.Z1 H2A.Z1 The sequencing results show that the coding region sequence of the gene in the poplar "nanlin 895" is as shown in Sequence 1 and Sequence 2; the amino acid sequence of the H2A.Z1 protein encoded by the coding region sequence of the gene as shown in Sequence 1 and Sequence 2 is the same, and is as shown in Sequence 3.

[0101] ​​Example 2, Construction of Recombinant Vectors and Recombinant Agrobacterium

[0102] I. Construction of Recombinant Interference Vector and Recombinant Interference Agrobacterium

[0103] 1. Construction of Recombinant Interference Vector PGWB2-Ami

[0104] (1) The common coding sequence 5'-AGGGGCTTCTGGCAACGAAAA-3' of the coding region of the genes shown in Sequence 1 and Sequence 2 was taken as the target sequence, and primers were designed on the primer design website http: / / wmd3.weigelworld.org / cgi-bin / webapp.cgi. The primer sequences are as follows: H2A.Z1

[0105] H2-I miR-s: 5'-gaTTTTCGTTGCCAGAAGCGCGTtctctcttttgtattcc-3';

[0106] H2-II miR-a: 5'-gaACGCGCTTCTGGCAACGAAAAtcaaagagaatcaatga-3';

[0107] H2-III miR*s: 5'-gaACACGCTTCTGGCTACGAAATtcacaggtcgtgatatg-3';

[0108] H2-IV miR*a: 5'-gaATTTCGTAGCCAGAAGCGTGTtctacatatatattcct-3'.

[0109] (2) The pRS300 (miR319a) vector was taken as the template, and four primer sequences in step (1) were used for one round of conventional PCR and one round of overlap PCR (for specific methods, 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.

[0110] (3) The artificial microRNA gene fragment amplified in step (2) was connected into the pENTR / D-TOPO® vector to obtain the pENTR / D-TOPO®-Ami vector; then the pENTR / D-TOPO ® ​The artificial microRNA gene fragment in the Ami vector is connected to the vector PGWB2 to obtain the recombinant interference vector PGWB2-Ami. The nucleotide sequence expressed by the recombinant interference vector PGWB2-Ami is an RNA molecule of sequence 5, which comprises a mature miRNA nucleotide sequence as shown in sequence 7 and a vector backbone sequence.

[0111] 2. Transformation of the recombinant interference Agrobacterium PGWB2-Ami / GV3101

[0112] The recombinant interference vector PGWB2-Ami is transformed into Agrobacterium tumefaciens GV3101 by Agrobacterium transformation method, and the recombinant interference Agrobacterium PGWB2-Ami / GV3101 is obtained by PCR detection.

[0113] Example 3, H2A.Z1 Obtaining and identification of the interference transgenic poplar

[0114] I. Obtaining of the transgenic poplar

[0115] The recombinant interference Agrobacterium is used to infect the tender leaves of the tissue culture poplar seedling "nanlin895" by the leaf disc method, and a transgenic poplar seedling is obtained through a series of callus induction, bud induction and rooting induction. The specific steps are as follows:

[0116] 1. The recombinant interference Agrobacterium PGWB2-Ami / GV3101 obtained in Example 2 is first cultured in 5 mL of liquid YEB medium (the liquid YEB medium is 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 is taken into 100 mL of liquid YEB medium, and the culture is shaken at 28°C until OD 600nm = 0.8, and then 100 μm of acetosyringone is added.

[0117] 2. The tender leaves of the poplar seedling grown in the tissue culture bottle for one month are taken, and the leaves are cut around the four sides with a scalpel, leaving the main vein near the size of about 1 cm 2 of the leaves. 1-2 small wounds are cut on the main vein, and they are placed in the shaken Agrobacterium and gently shaken for 30 min.

[0118] 3. The leaves are taken out and placed in the co-culture medium (the co-culture medium is a basic medium containing 0.2 mg / L of kinetin, 0.75 mg / L of 2,4-D and 100 μm of acetosyringone; the solvent of the basic medium is water, and the solutes and their concentrations are shown in Table 1) with the back down at 28°C for two days of co-culture.

[0119] 4. The leaves are transferred to the screening medium (the screening medium is the basic medium containing 0.2 mg / L kinetin, 0.75 mg / L 2,4-D, 50 mg / L kanamycin, 250 mg / L Cefotaxime sodium, 300 mg / L Timentin; the solvent of the basic medium is water, and the solutes and their concentrations are shown in Table 1) for dark culture, and subcultured every 14 days until the round globular callus is grown.

[0120] 5. The callus is cut and placed in the differentiation medium (the differentiation medium is the basic medium containing 1 mg / L 6-BA, 0.05 mg / L NAA, 50 mg / L kanamycin, 250 mg / L Cefotaxime sodium, 300 mg / L Timentin; the solvent of the basic medium is water, and the solutes and their concentrations are shown in Table 1) for light culture, and subcultured every 15 days until the sprouting.

[0121] 6. The small sprouts are cut and independently cultured in the rooting medium (the rooting medium is the basic medium containing 50 mg / L kanamycin, 250 mg / L Cefotaxime sodium, 300 mg / L Timentin; the solvent of the basic medium is water, and the solutes and their concentrations are shown in Table 1) until the rooting, and the transgenic poplar seedlings are obtained. The transgenic poplar seedlings after rooting can be continuously asexually propagated.

[0122] Table 1. The solutes and their concentrations in the basic medium of different culture media

[0123]

[0124] II. Identification of the transgenic poplar

[0125] 1. PCR identification

[0126] The DNA of the transgenic poplar seedlings is extracted for PCR identification, which specifically includes the following steps: the DNA of all the transgenic poplar seedlings is extracted by SLS method, and PGWB2-Ami-F and PGWB2-Ami-R are used for PCR amplification to obtain the PCR product. Meanwhile, the wild-type poplar DNA is used as a control. The primer sequences are as follows:

[0127] PGWB2-Ami-F: 5'-GGGGACTCTAGAGTTATCAAC-3';

[0128] PGWB2-Ami-R: 5'-CTAAGCGCTGTTATCAACCAC-3'.

[0129] PCR products were detected by electrophoresis, and positive transgenic plants were preliminarily obtained. The PCR product with a size of 822 bp was obtained by amplification, which was a positive transgenic plant.

[0130] 2、 H2A.Z1 Interference of transgenic poplar

[0131] For the positive transgenic plants, the expression amount was detected according to the following steps: the leaves of the positive transgenic plants and wild type plants grown for one month under the same growth conditions were taken, the total RNA of the leaves was extracted by using the plant RNA small amount extraction kit of Megan company, then the cDNA was synthesized by using the reverse transcription kit of Invitrogen company with Oligo d(T) as a primer, and the real-time quantitative PCR detection was performed by using the gene specific primer, with Actin as an internal reference gene. The primers were as follows: H2A.Z1

[0132] qH2A.Z1F: 5'-GACAAGGACAAGAAGAGGCC-3';

[0133] qH2A.Z1R: 5'-TGAAGCCAAGTAGACAGCAGC-3';

[0134] qPdeActinF: 5'-GCAGTCTTCCCCAGTATTGTT-3';

[0135] qPdeActinR: 5'-TCCCCAACATAGGCATCTTTC-3'.

[0136] The results are shown in Table 1. Figure 1 The results show that, compared with the wild type poplar seedlings, H2A.Z1 the relative expression amount of the gene in the interference transgenic poplar seedlings Ami-57, Ami-58 and Ami-203 is down-regulated by 39%, 37% and 35% respectively. H2A.Z1

[0137] Example 4, H2A.Z1 Gene significantly affects the formation of new organs of poplar

[0138] Test materials: wild type poplar "nanlin895" (WT), interference transgenic poplar strains Ami-57, Ami-58 and Ami-203. H2A.Z1 Test materials: wild type poplar "nanlin895" (WT), interference transgenic poplar strains Ami-57, Ami-58 and Ami-203.

[0139] Experimental method: the wild type poplar tissue culture seedlings grown in tissue culture bottles for 20 days and the interference transgenic poplar tissue culture seedlings grown in tissue culture bottles for 20 days were taken. 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).

[0140] The results are as follows Figure 1 As shown. The results show that compared with wild-type poplar seedlings, H2A.Z1 Interference with transgenic poplar seedlings significantly reduced plant height, significantly decreased the number of stem nodes, and significantly increased stem node length. 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.Z1 The 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.

[0141] Example 5 H2A.Z1 Genes significantly affect the size of poplar cells

[0142] Test material: Wild-type poplar “nanlin895” (WT) H2A.Z1 Interference with the transgenic poplar line Ami-203.

[0143] 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.

[0144] The results are as follows Figure 2 As shown. The results show that compared with wild-type poplar seedlings,H2A.Z1 The stem epidermal cell area of the transgenic poplar was increased by 124.9%, and the leaf lower epidermal cell area was increased by 45.8%; H2A.Z1 The moderate decrease in the gene expression amount significantly increased the poplar epidermal cell area.

[0145] Example 6, H2A.Z1 The gene significantly affects the poplar cell chromosome ploidy

[0146] Test material: wild-type poplar "nanlin895" (WT), H2A.Z1 The transgenic poplar Ami-203 strain was interfered.

[0147] Experimental method: The wild-type poplar tissue culture seedlings grown in the tissue culture bottles for 20 days and the transgenic poplar tissue culture seedlings were transferred to the soil culture pots in the culture room for culture. After growing in the soil culture pots for two months, the DNA content in the cells of the wild-type poplar seedlings and the transgenic poplar seedlings Ami-203 strain was determined by flow cytometry, and the proportion of cells in the 2C and 4C phases was counted. The root tips of the wild-type poplar tissue culture seedlings grown in the tissue culture bottles for 10 days and the transgenic poplar tissue culture seedlings were taken 1 cm, and then the root tip cell nuclei of the wild-type poplar seedlings and the transgenic poplar seedlings Ami-203 strain were stained with DAPI, and the number of chromosomes in a single cell was counted. H2A.Z1 H2A.Z1 H2A.Z1 H2A.Z1

[0148] The results are shown in Figure 3 The results show that the proportion of cells in the 2C and 4C phases in the wild-type poplar seedlings was 80% and 20%, respectively, while the proportion of cells in the 2C and 4C phases in the transgenic poplar seedlings was 10% and 90%, respectively; the number of chromosomes in the root tip cell nuclei of the wild-type poplar seedlings was 38, H2A.Z1 H2A.Z1 H2A.Z1 The moderate decrease in the gene expression amount increased the poplar chromosome ploidy, and the diploid poplar was transformed into a tetraploid poplar.

[0149] In summary, H2A.Z1 The gene can regulate chromosome doubling, thereby affecting the occurrence and formation of new organs of poplar, which provides a new idea for cultivating polyploid poplar.

[0150] ​​​​​​The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Use of a substance reducing the content of H2A.Z1 protein in any one of A1 ) to A7) below: A1 ) reducing the plant height; A2) reducing the number of stem nodes; A3) increasing the length of stem nodes; A4) increasing the cell size; A5) doubling the chromosomes of plant cells; A6) breeding a transgenic plant with reduced plant height and / or reduced number of stem nodes and / or increased length of stem nodes and / or enlarged cells and / or doubled chromosomes; A7) breeding plants; said H2A.Z1 protein is any one of B1 ) to B2) below: B1 ) a protein with the 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- and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 3; said substance reducing the content of H2A.Z1 protein is a miRNA inhibiting or interfering with the expression of a gene encoding H2A.Z1 protein; said plant is a poplar; said doubling of chromosomes is from diploid to tetraploid.

2. Use according to claim 1, characterized in that: said miRNA is the RNA molecule shown in SEQ ID NO: 5 or SEQ ID NO:

7.

3. A method for breeding a transgenic plant with reduced plant height and / or reduced number of stem nodes and / or increased length of stem nodes and / or enlarged cells and / or doubled chromosomes, comprising the step of reducing the content of H2A.Z1 protein in a plant of interest to obtain a transgenic plant with reduced plant height and / or reduced number of stem nodes and / or increased length of stem nodes and / or enlarged cells and / or doubled chromosomes; said H2A.Z1 protein is any one of B1 ) to B2) below: B1 ) a protein with the 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- and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 3; said plant is a poplar; said doubling of chromosomes is from diploid to tetraploid.

4. The method of claim 3, wherein: said method for reducing the content of H2A.Z1 protein in a plant of interest is introducing a substance inhibiting or interfering with the expression of a gene encoding H2A.Z1 protein into the plant of interest.

5. The method of claim 4, wherein: said substance inhibiting or interfering with the expression of a gene encoding H2A.Z1 protein is a miRNA inhibiting or interfering with the expression of a gene encoding H2A.Z1 protein.

6. The method of claim 5, wherein: said miRNA has the nucleotide sequence shown in SEQ ID NO:

7.

7. A miRNA, which is the RNA molecule shown in SEQ ID NO: 7.

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