Application of PdeAPL protein and related biological materials in regulating poplar chromosome doubling

Abstract

The application discloses PdeAPL protein and related biomaterials in the application of regulating poplar chromosome doubling. The application constructs PdeAPL overexpression transgenic poplar and PdeAPL interference transgenic poplar. Experiments prove that, compared with wild type poplar, the proportion of 2C cell types of the PdeAPL overexpression transgenic poplar is significantly increased; and the PdeAPL interference transgenic poplar does not detect 2C cells, and 4C cells are the main cell types. The application first finds that the pdeAPL gene can regulate poplar chromosome doubling, and has important significance for revealing the regulation molecular mechanism of forest tree chromosome doubling, improving forest tree shape, growth speed and wood quality, and directionally cultivating forest trees with shapes or material qualities meeting actual production requirements.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of PdeAPL protein and related biomaterials in regulating chromosome doubling in poplar trees. Background Technology

[0002] Poplar (Populus spp.) is one of the most widely distributed fast-growing tree species globally, renowned for its rapid growth, adaptability, and wide range of timber uses. Poplars play a vital role not only in ecological conservation, carbon sequestration, and biodiversity maintenance, but also have significant economic value in forestry production, pulp and paper industries. Therefore, improving poplar varieties to enhance their growth rate and timber quality is crucial for sustainable forestry development and timber supply security.

[0003] Chromosome doubling, or polyploidization, is an important phenomenon in plant genetics and breeding. Polyploid plants typically exhibit stronger stress resistance, higher biomass, and better growth stability. In poplar, chromosome doubling can lead to changes in plant architecture, growth rate, and wood properties, potentially enhancing its commercial value and adaptability. However, naturally occurring chromosome doubling events are rare and uncontrollable; therefore, inducing chromosome doubling in poplar through genetic engineering has become a research hotspot. Recent studies have shown that specific gene expression and regulatory mechanisms play a crucial role in plant chromosome doubling. By regulating these genes, chromosome doubling can be artificially induced, thereby accelerating the breeding process and creating new varieties with superior characteristics. In poplar, identifying and utilizing these regulatory genes has significant scientific and applied value for achieving chromosome doubling and variety improvement. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate the ploidy of plant cell chromosomes. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, the present invention first provides a novel use for PdeAPL protein or substances that regulate the content and / or activity of said PdeAPL protein.

[0006] This invention provides the use of PdeAPL protein or a substance that regulates the content and / or activity of said PdeAPL protein in any of the following A1)-A3):

[0007] A1) Regulates the ploidy of plant cell chromosomes;

[0008] A2) Cultivating transgenic plants with altered chromosome ploidy;

[0009] A3) Plant breeding;

[0010] The PdeAPL protein is PdeAPL-1 protein and / or PdeAPL-2 protein;

[0011] The PdeAPL-1 protein is any one of the following B1)-B4):

[0012] B1) The amino acid sequence is that of the protein shown in sequence 3;

[0013] B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 3;

[0014] B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 3.

[0015] B4) Proteins that have 80% or more of the same amino acid sequence as those shown in Sequence 3 and have the same function;

[0016] The PdeAPL-2 protein is any one of the following C1)-C4):

[0017] C1) The amino acid sequence is the protein shown in sequence 4;

[0018] C2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 4;

[0019] C3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 4.

[0020] C4) is a protein that has 80% or more of the same amino acid sequence as the one shown in Sequence 4 and has the same function.

[0021] In the proteins described in B2) or C2) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0022] In the protein described in B3) or C3) above, the substitution and / or deletion and / or addition of one or more amino acid residues is as follows: substitution and / or deletion and / or addition of no more than 10 amino acid residues; substitution and / or deletion and / or addition of no more than 9 amino acid residues; substitution and / or deletion and / or addition of no more than 8 amino acid residues; substitution and / or deletion and / or addition of no more than 7 amino acid residues; substitution and / or deletion and / or addition of no more than 6 amino acid residues; substitution and / or deletion and / or addition of no more than 5 amino acid residues; substitution and / or deletion and / or addition of no more than 4 amino acid residues; substitution and / or deletion and / or addition of no more than 3 amino acid residues; substitution and / or deletion and / or addition of no more than 2 amino acid residues; or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0023] In the proteins described in B4) or C4) above, the identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, 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 an identity calculation for a pair of amino acid sequences, the identity value (%) can be obtained. The identity includes amino acid sequences that have 80% or higher, or 85% or higher, or 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with the amino acid sequences shown in Sequence 3 or Sequence 4 of this invention.

[0024] The proteins described in B1)-B4) or C1)-C4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0025] The substances that regulate the content and / or activity of PdeAPL protein mentioned above can be substances that regulate the content and / or activity of PdeAPL-1 protein and / or substances that regulate the content and / or activity of PdeAPL-2 protein.

[0026] The substances that regulate the content and / or activity of PdeAPL-1 protein include substances that increase the content and / or activity of PdeAPL-1 protein or substances that decrease the content and / or activity of PdeAPL-1 protein.

[0027] Furthermore, the substance that enhances the activity of PdeAPL-1 protein may be a protein, polypeptide, or small molecule compound that enhances or promotes the function of PdeAPL-1 protein.

[0028] The substance that increases the content of PdeAPL-1 protein may be a substance that promotes the synthesis of PdeAPL-1 protein, inhibits the degradation of PdeAPL-1 protein, or overexpresses PdeAPL-1 protein.

[0029] The substance that reduces the activity of PdeAPL-1 protein can be a protein, polypeptide, or small molecule compound that inhibits the function of PdeAPL-1 protein.

[0030] The substance that reduces PdeAPL-1 protein content may be a substance that inhibits PdeAPL-1 protein synthesis, promotes PdeAPL-1 protein degradation, or knocks down (reduces) or eliminates the PdeAPL-1 protein encoding gene.

[0031] Furthermore, the substance that knocks down (reduces) the PdeAPL-1 protein-coding gene can be any substance that can inhibit or interfere with the expression of the PdeAPL-1 protein-coding gene, such as gRNA (e.g., sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0032] Furthermore, the substance that inhibits or interferes with the expression of the PdeAPL-1 protein-coding gene is a miRNA that inhibits or interferes with the expression of the PdeAPL-1 protein-coding gene.

[0033] In some embodiments, the nucleotide sequence of the miRNA that inhibits or interferes with the expression of the PdeAPL-1 protein-coding gene is shown in Sequence 6.

[0034] The substances mentioned above that regulate the content and / or activity of PdeAPL-2 protein include substances that increase the content and / or activity of PdeAPL-2 protein or substances that decrease the content and / or activity of PdeAPL-2 protein.

[0035] Furthermore, the substance that enhances the activity of PdeAPL-2 protein may be a protein, polypeptide, or small molecule compound that enhances or promotes the function of PdeAPL-2 protein.

[0036] The substance that increases the content of PdeAPL-2 protein may be a substance that promotes the synthesis of PdeAPL-2 protein, inhibits the degradation of PdeAPL-2 protein, or overexpresses PdeAPL-2 protein.

[0037] The substance that reduces the activity of PdeAPL-2 protein can be a protein, polypeptide, or small molecule compound that inhibits the function of PdeAPL-2 protein.

[0038] The substance that reduces PdeAPL-2 protein content may be a substance that inhibits PdeAPL-2 protein synthesis, promotes PdeAPL-2 protein degradation, or knocks down (reduces) or eliminates the PdeAPL-2 protein encoding gene.

[0039] Furthermore, the substance that knocks down (reduces) the PdeAPL-2 protein-coding gene can be any substance that can inhibit or interfere with the expression of the PdeAPL-2 protein-coding gene, such as gRNA (e.g., sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0040] Furthermore, the substance that inhibits or interferes with the expression of the PdeAPL-2 protein-coding gene is a miRNA that inhibits or interferes with the expression of the PdeAPL-2 protein-coding gene.

[0041] In some embodiments, the nucleotide sequence of the miRNA that inhibits or interferes with the expression of the PdeAPL-2 protein-coding gene is shown in Sequence 6.

[0042] To address the aforementioned problems, this invention provides new uses for biomaterials related to PdeAPL proteins.

[0043] This invention provides the use of biomaterials related to the PdeAPL protein in any of the following A1)-A3):

[0044] A1) Regulates the ploidy of plant cell chromosomes;

[0045] A2) Cultivating transgenic plants with altered chromosome ploidy;

[0046] A3) Plant breeding;

[0047] The biomaterial is any one of the following E1) to E5):

[0048] E1) The nucleic acid molecule that encodes the PdeAPL protein mentioned above;

[0049] E2) Knock down or knock out the nucleic acid molecules of the PdeAPL protein-encoding gene mentioned above;

[0050] E3) An expression cassette containing the nucleic acid molecules described in E1) or E2);

[0051] E4) A recombinant vector containing the nucleic acid molecules described in E1) or E2);

[0052] E5) Recombinant microorganisms containing the nucleic acid molecules described in E1) or E2).

[0053] In the above applications, the nucleic acid molecule described in E1) is any of the following:

[0054] F1) DNA molecules shown in sequence 1 or sequence 2;

[0055] The nucleotide sequences defined by F2 and F1) have 75% or more identity and encode the aforementioned PdeAPL-1 or PdeAPL-2 protein DNA molecules.

[0056] In the above applications, the nucleic acid molecule described in E2) is any of the following:

[0057] The RNA molecule shown in sequence 6 of G1);

[0058] Nucleic acid molecules that have 75% or more identity with the nucleotide sequences defined by G2 and G1, and that inhibit the expression of the genes encoding the PdeAPL-1 protein and / or PdeAPL-2 protein.

[0059] Those skilled in the art can readily mutate the nucleotide sequences encoding the PdeAPL-1 or PdeAPL-2 proteins of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides having 75% or higher identity with the PdeAPL-1 or PdeAPL-2 nucleotide sequences isolated according to the present invention, provided they encode the PdeAPL-1 or PdeAPL-2 proteins and have the same function, are nucleotide sequences derived from and equivalent to those of the present invention. This identity refers to sequence similarity to natural nucleic acid sequences, including nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequences of proteins constituting the amino acid sequences shown in Sequence 3 or Sequence 4 of the present 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.

[0060] The nucleic acid molecule mentioned in E2 above can be gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, or antisense RNA.

[0061] Any of the nucleic acid molecules mentioned above can be DNA, such as cDNA, genomic DNA, or recombinant DNA.

[0062] Any of the nucleic acid molecules mentioned above can be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0063] The expression cassette described above may include a promoter, the nucleic acid molecule described in E1) or E2) above, and a terminator. Promoters that can be used in this invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Furthermore, the expression cassette may also include an enhancer sequence.

[0064] The vector mentioned above refers to a vector capable of carrying the nucleic acid molecules described in E1) or E2) into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage, etc.), granules (i.e., Cos plasmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0065] The recombinant vector described above refers to a recombinant DNA molecule constructed by in vitro ligation of the nucleic acid molecule described in E1) or E2) with the vector. Recombinant vectors containing the nucleic acid molecule described in E1) or E2) can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The plant expression vector may also contain the 3′ untranslated region of the exogenous gene, i.e., containing the polyadenylated signal and any other DNA fragments involved in mRNA processing or gene expression.

[0066] The microorganisms mentioned above may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses.

[0067] The recombinant microorganisms mentioned above refer to those obtained by manipulating and modifying the genes of a target microorganism, thereby altering its function. For example, recombinant microorganisms obtained after introducing the aforementioned recombinant vector into a target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring may not necessarily be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0068] In any of the above applications, the regulation of plant cell chromosome ploidy refers to the doubling of plant cell chromosomes. Specifically, when the content and / or activity of PdeAPL protein in plants decreases or is absent, plant cell chromosomes double.

[0069] In some implementations, when the expression levels of the PdeAPL-1 and / or PdeAPL-2 genes in plants decrease, the plant cell chromosomes double, changing from diploid to tetraploid.

[0070] In any of the above applications, the purpose of the plant breeding is to cultivate polyploid plants (such as tetraploid plants).

[0071] To address the aforementioned technical problems, the present invention also provides a method for cultivating transgenic plants with doubled chromosomes.

[0072] The method for obtaining a chromosome-doubled transgenic plant provided by the present invention includes the following steps: reducing the content and / or activity of the above-mentioned PdeAPL protein in the target plant to obtain a chromosome-doubled transgenic plant.

[0073] In the above method, chromosome doubling refers to changing the plant from diploid to tetraploid. This change from diploid to tetraploid is manifested in either 1) or 2) below:

[0074] 1) The proportion of 4C cells in the transgenic plant is higher than that in the target plant, and the proportion of 2C cells is lower than that in the target plant (4C cells are the main cell type in the transgenic plant, and 2C cells are the main cell type in the target plant);

[0075] 2) The nuclear replication index of the transgenic plant is significantly higher than that of the target plant.

[0076] In some embodiments, the target plant contains 87.7% 2C cells and 12.3% 4C cells. In the transgenic plant, the proportion of 2C cells is 0, and the proportion of 4C cells is 93.4%.

[0077] In some embodiments, the target plant contains 87.7% 2C cells and 12.3% 4C cells. In the transgenic plant, the proportion of 2C cells is 0, and the proportion of 4C cells is 94.7%.

[0078] In the above method, the method for reducing the content and / or activity of the PdeAPL protein in the target plant is to introduce a substance that inhibits or interferes with the expression of the PdeAPL protein-coding gene or a substance that knocks out the PdeAPL protein-coding gene into the target plant.

[0079] Furthermore, the substance that inhibits the expression of the PdeAPL protein-coding gene in the target plant is a miRNA that inhibits or interferes with the expression of the PdeAPL protein-coding gene.

[0080] Furthermore, the nucleotide sequence of the miRNA is shown in Sequence 6.

[0081] 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 PdeAPL 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, particularly commercial varieties, using conventional breeding techniques. The transgenic plant includes seeds, callus tissue, intact plants, and cells.

[0082] 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 (e.g., nanlin895).

[0083] To address the aforementioned technical problems, this invention ultimately provides a miRNA.

[0084] The miRNA provided by this invention is either a1) or a2):

[0085] a1) The RNA molecule shown in sequence 6;

[0086] a2) An RNA molecule that has one or more nucleotides deleted, added, or altered from sequence 6, and has the same function as sequence 6.

[0087] This invention constructed PdeAPL-overexpressing transgenic poplar and PdeAPL-interference transgenic poplar. Experiments demonstrated that, compared to wild-type poplar, the PdeAPL-overexpressing transgenic poplar showed a significantly increased proportion of 2C cell types; while the PdeAPL-interference transgenic poplar showed no detectable 2C cells, with 4C cells being the predominant cell type. This invention is of great significance for elucidating the regulatory molecular mechanisms of chromosome doubling in forest trees, improving tree shape, growth rate, and timber quality, and for the targeted cultivation of trees with shapes or wood properties that meet actual production needs.

[0088] The beneficial effects of this invention are as follows: This invention is the first to discover that the pdeAPL gene can regulate chromosome doubling in poplar trees. By precisely regulating the expression of the target gene pdeAPL through RNA interference technology, artificial chromosome doubling in poplar trees can be achieved. This invention will help cultivate new poplar varieties that grow faster, are more resistant to adverse conditions, and have superior timber quality. This will not only meet the market demand for high-quality timber but also promote the sustainable development of forestry. Furthermore, the chromosome doubling technology achieved through gene regulation can also be applied to the breeding of other tree species, possessing broad market prospects. Attached Figure Description

[0089] Figure 1 The results show the expression levels of the PdeAPL gene in PdeAPL-interference transgenic poplar lines. WT represents wild-type poplar, while mi-L229, mi-L6, and mi-L21 are three different transgenic poplar lines incorporating the recombinant interference vector PGWB2-R.

[0090] Figure 2 The results show the expression level of PdeAPL-1 overexpressing transgenic poplar seedlings.

[0091] Figure 3Phenotypic observations and statistical graphs of wild-type poplar seedlings (WT), PdeAPL-interference transgenic poplar seedlings (mi-L6 and mi-L21 lines), and PdeAPL-overexpressing transgenic poplar seedlings (OE30 line) were obtained by flow cytometry. AD represents the ploidy level of cells in the first diploid (including the apical bud) of WT, mi-L6, mi-L21, and OE30 plants, analyzed by flow cytometry. 2C represents the DNA content in G1 phase; 4C represents the DNA content in G2 phase and the DNA content during nuclear replication; 8C represents the DNA content during nuclear replication. E represents the ploidy distribution of cells at different stages of the cell cycle in WT, mi-L6, mi-L21, and OE30 plants, n>5. F represents the nuclear replication index of the first diploid (including the apical bud) of WT, mi-L6, mi-L21, and OE30 plants. The error bars in the figure represent standard deviations, and different letters indicate significant differences determined by the Duncan test at a significance level of p < 0.05. Detailed Implementation

[0092] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0093] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0094] The PGWB2 vector in the following embodiments is described in the literature "Figueiredo J,P". Lahaye T, et al. (2011) Agrobacterium-mediated transient expression in citrus leaves: a rapidtool for gene expression and functional gene assay. [J]. Plant Cell Reports, 30(7):1339-1345."

[0095] The PGWB2-5×MYC carrier in the following embodiments is a carrier obtained by linking a 5×MYC tag into a PGWB2 carrier.

[0096] The Agrobacterium tumefaciens strain GV3101 in the following examples is described in the literature “Zheng, S., et al. (2020). Two MADS-box genes regulate vascular cambium activity and secondary growth via modulating auxin homeostasis in Populus. Plant Communications.”

[0097] The wild-type poplar “nanlin895” in the following examples is described in the literature “Zhu,Y., et al. (2018). AHD-ZIP III gene,PtrHB4, is required for interfascicular cambium development in Populus.”Plant Biotechnol J 16(3):808-817.”

[0098] pENTR / D- in the following embodiments The vector 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."

[0099] In the following examples, "C" represents the DNA content in a haploid cell, that is, the total amount of DNA in a set of chromosomes.

[0100] In the following examples, 2C cells refer to cells with a DNA content of 2C, where 2C refers to the total amount of DNA in a set of chromosomes in a diploid cell.

[0101] In the following examples, 4C cells refer to cells with a DNA content of 4C, where 4C refers to the total amount of DNA in the two sets of chromosomes in a diploid cell.

[0102] In the following examples, 8C cells refer to cells with a DNA content of 8C, meaning that during interphase of mitosis, DNA replicates, causing the DNA content in the cell to double from 4C to 8C.

[0103] Example 1: Obtaining the PdeAPL protein and its encoding gene

[0104] 1. Take the whole "nanlin 895" poplar plant that has been growing in a tissue culture bottle for one month, freeze it in liquid nitrogen, grind it and extract total RNA, and reverse transcribe the total RNA to obtain poplar cDNA.

[0105] 2. Using the obtained cDNA as a template, PCR amplification was performed with 5'-ATGTTCCATACCAAGAAACC-3' as the forward primer and 5'-TCAACCAAATGGTGAGTTTC-3' as the reverse primer to obtain the amplification product.

[0106] 3. After the amplification product was detected by agarose gel electrophoresis, a DNA fragment of about 1000 bp was separated and purified. Then, this PCR product was ligated into the cloning vector peasy-Blunt simple, and the ligated vector was sequenced.

[0107] Sequencing results showed that two PdeAPL coding region sequences exist in poplar “nanlin 895”, as shown in Sequence 1 and Sequence 2 respectively. The gene shown in Sequence 1 is named PdeAPL-1, and the amino acid sequence of the PdeAPL-1 protein it encodes is shown in Sequence 3. The gene shown in Sequence 2 is named PdeAPL-2, and the amino acid sequence of the PdeAPL-2 protein it encodes is shown in Sequence 4.

[0108] Example 2: Construction of recombinant vector and recombinant Agrobacterium

[0109] I. Construction of recombinant interference vector and recombinant interfering Agrobacterium

[0110] 1. Construction of the recombinant interference vector PGWB2-R

[0111] (1) Primers were designed using the common coding sequence 5'-TAACGCAGATCGTCGGGCCAA-3' of the PdeAPL gene coding region shown in Sequences 1 and 2 as the target sequence, on the primer design website http: / / wmd3.weigelworld.org / cgi-bin / webapp.cgi?page=Home;project=stdwmd. The primer sequences are as follows:

[0112] I miR-s: gaTAACGCAGATCGTCGGGCCAAtctctcttttgtattcc;

[0113] II miR-a: gaTTGGCCCGACGATCTGCGTTAtcaaagagaatcaatga;

[0114] III miR*s: gaTTAGCCCGACGATGTGCGTTTtcacaggtcgtgatatg;

[0115] IV miR*a: gaAAACGCACATCGTCGGGCTAAtctacatatattcct.

[0116] (2) Using the pRS300 (MIR319a Arabidopsis thaliana) vector as a template, artificial microRNA was synthesized by two rounds of nested PCR using the four primer sequences in step (1) (for specific methods, refer to Rebecca Schwab, MPI for Developmental Biology, Tuebingen, 2005). The nucleotide sequence of the artificial microRNA is shown in Sequence 5.

[0117] (3) Link the artificial microRNA synthesized in step (2) into pENTR / D- In the vector, pENTR / D- -artificial microRNA vector; then pENTR / D- is carried out via LR homologous recombination reaction. Artificial microRNA is ligated into vector PGWB2 to obtain the recombinant interference vector PGWB2-R. The recombinant interference vector PGWB2-R can express a mature miRNA for interfering with the expression of the PdeAPL gene, the nucleotide sequence of which is shown in Sequence 6.

[0118] 2. Construction of recombinant Agrobacterium interfering PGWB2-R / GV3101

[0119] The recombinant interference vector PGWB2-R was transformed into Agrobacterium tumefaciens GV3101 using the Agrobacterium-mediated transformation method. After PCR detection, the recombinant interfering Agrobacterium PGWB2-R / GV3101 was obtained.

[0120] II. Construction of Recombinant Overexpression Vector and Recombinant Overexpression Agrobacterium

[0121] 1. Construction of the recombinant overexpression vector p35S::pdeAPL-1-5×MYC

[0122] The DNA fragment shown in sequence 1 (PdeAPL-1CDS sequence) was ligated to pENTR / D- via homologous recombination. In the carrier, Vector; then pENTR / D- is transferred via LR homologous recombination reaction. The PdeAPL-1CDS sequence from the APL-1 vector was ligated into the PGWB2-5×MYC vector to obtain the recombinant overexpression vector p35S::pdeAPL-1-5×MYC.

[0123] 2. Construction of recombinant Agrobacterium p35S::pdeAPL-1-5×MYC / GV3101 overexpression

[0124] The recombinant overexpression vector p35S::pdeAPL-1-5×MYC was transformed into Agrobacterium tumefaciens GV3101 using the Agrobacterium transformation method. After PCR detection, recombinant overexpressing Agrobacterium p35S::pdeAPL-1-5×MYC / GV3101 containing the recombinant overexpression vector p35S::pdeAPL-1-5×MYC was obtained.

[0125] Example 3: Obtaining and Identifying Transgenic Poplar Trees

[0126] I. Obtaining Genetically Modified Poplar Trees

[0127] All the recombinant Agrobacterium bacteria constructed in Example 2 were used to infect the young leaves of tissue-cultured poplar seedlings "nanlin895" using the leaf disc method. After a series of callus induction, bud induction, and rooting induction processes, transgenic poplar seedlings were obtained. The specific steps are as follows:

[0128] 1. The Agrobacterium monoclonal strain obtained in Example 2 was first cultured in 5 mL of liquid YEB medium (liquid YEB medium is obtained by mixing 1 g yeast extract, 5 g tryptone, 5 g beef extract, 5 g sucrose, 1.954 g anhydrous magnesium sulfate and 1 L water, pH = 7.0) for 12 hours. Then, 1 mL of the cultured bacterial solution was transferred to 100 mL of liquid YEB medium and incubated at 28 °C until OD reached. 600nm =0.8, then add 100μm acetylsuccinone.

[0129] 2. Take a tender leaf from a poplar seedling that has been growing for one month in a tissue culture bottle. Use a scalpel to cut off the edges of the leaf, leaving about 1 cm of the leaf near the midrib. 2 Take a leaf, make 3-4 small cuts on the main vein, place it in a shaker of Agrobacterium, and gently shake it for 30 minutes to infect it.

[0130] 3. Remove the leaves and place them face down on the co-culture medium (the co-culture medium is a basal medium containing 0.5 mg / L kinetin, 0.75 mg / L 2-4-D, and 100 μm acetylsylgenone; the solvent of the basal medium is water, and the solutes and their concentrations are shown in Table 1) and co-culture at 28°C for two days.

[0131] 4. Transfer the leaves to the callus-inducing selection medium (the selection medium is a basal medium containing 0.5 mg / L kinetin, 0.75 mg / L 2-4-D, 50 mg / L kan, 250 mg / L carb, 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 in the dark, and subculture every 14 days until spherical callus tissue grows.

[0132] 5. Cut off the callus and place it on differentiation medium (differentiation medium is a basal medium containing 0.2 mg / L thidiazuron, 50 mg / L kan, 250 mg / L carb, and 300 mg / L Timentin; the solvent of the basal medium is water, and the solutes and their concentrations are shown in Table 1) and culture it under light. Subculture once every 15 days until buds emerge.

[0133] 6. Cut off the buds and culture them independently on rooting medium (the rooting medium is a basal medium containing 50 mg / L kan, 250 mg / L carb, and 300 mg / L Timentin; the solvent of the basal medium is water, and the solutes and their concentrations are shown in Table 1) until roots are formed, thus obtaining transgenic poplar seedlings. The rooted transgenic poplar seedlings can be continuously propagated.

[0134] Table 1. Solutes and their concentrations in the basal media of different culture media

[0135] Medicines / Reagents co-culture medium Screening culture medium Differentiation culture medium Rooting medium sucrose 20g / L 20g / L 20g / L 20g / L WPM 2.5g / L 2.5g / L 2.5g / L 2.5g / L MES 0.5g / L 0.5g / L 0.5g / L 0.5g / L Plant agar 7.3g / L 7.3g / L 7.3g / L 7.3g / L

[0136] II. Identification of Genetically Modified Poplar

[0137] 1. PCR identification

[0138] DNA was extracted from transgenic poplar seedlings for PCR identification, which included the following steps: DNA was extracted from all transgenic poplar seedlings using the SLS method, and primers were designed at both ends of the above recombinant vector to amplify the DNA of each plant by PCR, while wild-type poplar DNA was used as a control.

[0139] The primer sequences designed for the PGWB2-R vector are as follows:

[0140] 5'-GGGGACTCTAGAGTTATCAAC-3';

[0141] 5'-CTAAGCGCTGTTATCAACCAC-3'.

[0142] The primer sequences designed for the PGWB2-5×MYC vector are as follows:

[0143] 5'-GGGGACTCTAGAGTTATCAAC-3';

[0144] 5'-GCATGTCTTGCGTTGATGAAGC-3'.

[0145] After electrophoresis, the PCR products can be used to initially identify positive transgenic plants. For the pGWB2-R vector, a PCR product of 822 bp indicates a positive transgenic plant. For the PGWB2-5×MYC vector, a PCR product of 1639 bp indicates a positive transgenic plant.

[0146] 2. Obtaining transgenic poplar trees through interference and overexpression.

[0147] For positive transgenic plants, expression levels were detected as follows: Leaves from one-month-old positive transgenic plants and wild-type plants under the same growth conditions were collected. Total RNA was extracted from the leaves using Megan's Plant RNA Mini-Extraction Kit. cDNA was then synthesized using Invitrogen's Reverse Transcription Kit with Oligo d(T) primers. Real-time quantitative PCR was performed using pdeAPL gene-specific primers, with Actin as an internal control gene. Primers are as follows:

[0148] QpdeAPL-1-F: 5'-TTAAGGACCGAGGCTCAAGG-3';

[0149] QpdeAPL-1-R: 5'-TCCTGGTATGGGTGCAATGAA-3';

[0150] QpdeAPL-2-F: 5'-AGATCCAGATTGCACCACCA-3';

[0151] QpdeAPL-2-R: 5'-GGCCAGCTTGACCTTCTACA-3';

[0152] QpdeActinF: 5'-AAACTGTAATGGTCCTCCCTCCG-3';

[0153] QpdeActinR: 5'-GCATCATCACAATCACTCTCCGA-3'.

[0154] The expression levels of pdeAPL-1 and pdeAPL-2 genes in different interference transgenic poplar trees are as follows: Figure 1As shown in the figure. The mi-L6 and mi-L21 lines of pdeAPL-interference transgenic poplar, which showed significantly lower expression levels of both pdeAPL-1 and pdeAPL-2 genes compared to wild-type poplar, were selected for subsequent research experiments.

[0155] The expression levels of the pdeAPL-1 gene in different overexpression transgenic poplar trees are as follows: Figure 2 As shown in the figure. The OE-30 transgenic poplar line overexpressing pdeAPL-1, which showed the most significant upregulation of pdeAPL-1 gene expression compared to wild-type poplar, was selected for subsequent research experiments.

[0156] Example 4: The pdeAPL gene significantly affects the chromosome ploidy of poplar cells.

[0157] Test materials: wild-type poplar “nanlin895” (WT), pdeAPL interference transgenic poplar lines mi-L6 and mi-L21, and pdeAPL-1 overexpression transgenic poplar line OE30.

[0158] Experimental Methods: Cell ploidy analysis was performed on the young tissue (first and second segments) from the stem apex to the second segment of the test material, and the nuclear replication index was calculated. The nuclear replication index was calculated according to the method in the reference "DING AM, XU CT, XIE Q, et al. 2022. ERF4 interacts with and antagonizes TCP15 in regulating endoreduplication and cell growth in Arabidopsis. J Integr Plant Biol[J],64:1673-1689." The specific calculation formula is as follows: Nuclear replication index = (2C cell percentage × 1) + (4C cell percentage × 2) + (8C cell percentage × 3). The specific steps are as follows:

[0159] 1. After 25 days of growth in tissue culture medium, the tissue culture seedlings were transferred to soil culture pots. After one month of growth in the culture room, approximately 50 mg of the portion above the second stem node (including the stem tip, stem, and leaves) of the poplar seedlings was taken as a sample. The sample was chopped and placed in 1 mL of LB011 buffer (solvent was water, and the solutes and their concentrations were: 15 mM Tris, 2 mM EDTA, 80 mM KCl, 0.5 mM spermine tetrahydrochloride, 20 mM NaCl, 0.1% Triton X-100, 15 mM β-mercaptoethanol, pH = 7.5) to obtain the cell culture medium.

[0160] 2. Gently collect the cell culture medium and filter it through a 400-mesh sieve. Collect the filtrate below the sieve to obtain a cell suspension.

[0161] 3. The cell suspension was stained in ice water with PI (propiodine) at a concentration of 100 μg / mL and RNase at a concentration of 100 μg / mL for 10 min to obtain the stained sample.

[0162] 4. The stained samples were analyzed using a Beckman MoFlo-XDP flow cytometer with a 70 μm ceramic nozzle at a sheath flow pressure of 60 psi.

[0163] 5. The fluorescence of PI was excited using a solid-state laser with a wavelength of 488nm, and the fluorescence of the cell nucleus was detected by an HQ bandpass filter of 625-626nm.

[0164] 6. To improve data accuracy, FL3-Height and SSC-Height gating methods were used to exclude debris, cell fragments, and dead cells from the samples. The ratio of FL3-Height to FL3-Area was used to distinguish between single cells and aggregated cells.

[0165] The results are as follows Figure 3 As shown, the results indicate that, as a diploid species, poplar exhibits predominantly 2C cells (87.7% of total cells) in the wild-type (WT) population, with approximately 12.3% of 4C cells undergoing nuclear replication. In mi-L6 and mi-L21, no 2C cells were detected, while 4C cells were the predominant cell type, accounting for approximately 93.4% and 94.7% of total cells, respectively, with 6.6% and 5.3% of 8C cells detected, respectively. In OE30, similar to the wild-type, 2C cells remained the predominant cell type, with a small number of 4C cells undergoing nuclear replication. However, unlike the wild-type, OE30 showed a statistically significant difference: approximately 91.4% of cells were 2C cells, and approximately 8.6% were 4C cells. Furthermore, compared to WT, the nuclear replication index was significantly increased in mi-L6 and mi-L21, and significantly decreased in OE30. Among them, the kernel replication index of WT was 112.32, the kernel replication indices of mi-L6 and mi-L21 were 213.15 and 210.62 respectively, and the kernel replication index of OE30 was 108.63.

[0166] In conclusion, the pdeAPL gene significantly affects poplar chromosomes, and this discovery can provide new ideas for cultivating high-quality, fast-growing poplars with good tree shape.

[0167] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of PdeAPL protein or a substance regulating the content and / or activity of said PdeAPL protein in any of the following A1)-A3): A1) Regulates the ploidy of plant cell chromosomes; A2) Cultivating transgenic plants with altered chromosome ploidy; A3) Plant breeding; The PdeAPL protein is PdeAPL-1 protein and / or PdeAPL-2 protein; The PdeAPL-1 protein is any one of the following B1)-B4): B1) The amino acid sequence is that of the protein shown in sequence 3; B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 3; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 3. B4) Proteins that have 80% or more of the same amino acid sequence as those shown in Sequence 3 and have the same function; The PdeAPL-2 protein is any one of the following C1)-C4): C1) The amino acid sequence is the protein shown in sequence 4; C2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 4; C3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 4. C4) is a protein that has 80% or more of the same amino acid sequence as the one shown in Sequence 4 and has the same function.

2. Use of biomaterials related to the PdeAPL protein of claim 1 in any of the following A1)-A3): A1) Regulates the ploidy of plant cell chromosomes; A2) Cultivating transgenic plants with altered chromosome ploidy; A3) Plant breeding; The biomaterial is any one of the following E1) to E5): E1) A nucleic acid molecule encoding the PdeAPL protein as described in claim 1; E2) Knock down or knock out the nucleic acid molecule of the PdeAPL protein-encoding gene as described in claim 1; E3) An expression cassette containing the nucleic acid molecules described in E1) or E2); E4) A recombinant vector containing the nucleic acid molecules described in E1) or E2); E5) Recombinant microorganisms containing the nucleic acid molecules described in E1) or E2).

3. The application according to claim 2, characterized in that: E1) The nucleic acid molecule is any one of the following: F1) DNA molecules shown in sequence 1 or sequence 2; The nucleotide sequences defined by F2 and F1) have 75% or more identity and encode the DNA molecule of the PdeAPL protein.

4. The application according to claim 2, characterized in that, E2) The nucleic acid molecule is any one of the following: The RNA molecule shown in sequence 6 of G1); The nucleotide sequence defined by G2) has 75% or more identity with G1) and is a nucleic acid molecule that inhibits the expression of the PdeAPL protein-coding gene.

5. The application according to any one of claims 1-4, characterized in that: The regulation of ploidy of plant cell chromosomes is intended to change the plant from diploid to tetraploid.

6. A method for cultivating a transgenic plant with doubled chromosomes, comprising the following steps: reducing the content and / or activity of the PdeAPL protein as described in claim 1 in the target plant to obtain a transgenic plant with 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 PdeAPL 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 PdeAPL protein of claim 1 into the target plant.

9. The method according to claim 8, characterized in that: The substance that inhibits or interferes with the expression of the PdeAPL protein-coding gene in claim 1 is a miRNA that inhibits or interferes with the expression of the PdeAPL protein-coding gene in claim 1. Alternatively, the nucleotide sequence of the miRNA is shown in Sequence 6.

10. miRNA, wherein the miRNA is either a1) or a2): a1) The RNA molecule shown in sequence 6; a2) An RNA molecule that has one or more nucleotides deleted, added, or altered from sequence 6, and has the same function as sequence 6.

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