Use of pdeapl protein and gene encoding same during regulation of wood properties of poplar

Abstract

Provided is the use of a PdeAPL protein and a gene encoding same during the regulation of wood properties of poplar. A transgenic poplar overexpressing PdeAPL and a transgenic poplar having a reduced PdeAPL gene expression level are constructed. It is verified through experiments that compared with a control, the transgenic poplar overexpressing PdeAPL exhibits significantly increased plant height, internode number and internode length, but decreased leaf size; whereas the transgenic poplar having a reduced PdeAPL gene expression level exhibits decreased plant height and internode number, and increased internode length and leaf size. In addition, the transgenic poplar having a reduced PdeAPL gene expression level exhibits a decrease in xylem width, and an increase in the areas of xylem vessel cells, xylem fiber cells and phloem cells.

Description

Application of PdeAPL protein and its encoding gene in regulating poplar wood quality

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202411099235.0, filed on August 12, 2024, the entire contents of which are hereby incorporated by reference. TECHNICAL FIELD

[0003] The present application relates to the field of biotechnology, in particular to the application of PdeAPL protein and its encoding gene in regulating poplar wood quality. BACKGROUND

[0004] Wood is used in a variety of applications, including construction and structural engineering, furniture manufacturing, decoration and interior design, pulp and papermaking, packaging materials, energy and fuel, and horticulture and landscape design. Wood is of great importance in various fields due to its multifunctionality, renewability, and environmental friendliness. Wood quality encompasses density and weight, moisture content, strength and hardness, toughness and elasticity, stability, durability, processability, and decorative properties. These characteristics determine the performance and suitability of wood in different applications.

[0005] Poplar is a model plant for forest tree research and is an important source of wood supply in China. Therefore, cultivating poplar trees with high-quality wood quality has important application value, not only meeting people's actual needs, but also playing a positive role in sustainable development.

[0006] The development of wood in forest tree stems is produced by vascular tissue development. Vascular cells mainly include two cell types, xylem and phloem. Xylem is responsible for water transport, and phloem is mainly responsible for transporting photosynthetic products from mature leaves to developing tissues. Among them, leaves are the main organs of photosynthesis, and the development of mesophyll cells is crucial for the production and transport of photosynthetic products. Therefore, exploring genes involved in regulating vascular tissue development is of great significance for revealing the molecular mechanisms of forest tree stem vascular development and improving forest tree growth rate and wood quality.

[0007] Generally, the width and cell size of xylem will affect the density and structural uniformity of wood, and the thickness of secondary wall and lignification degree of xylem cell are directly related to the wood quality and property of poplar (Hao, Y., Lu, F., Pyo, S., Kim, M., Ko, J., Yan, X., Ralph, J., & Li, Q. (2024). PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar. Journal of integrative plant biology.), while the size of phloem cell may be related to the growth rate of tree and the annual ring width of wood.

[0008] SUMMARY

[0009] The technical problem to be solved by the present application is how to regulate the development of plant vascular tissue to achieve the directional cultivation of tree shape or wood quality and property meeting the actual production needs.

[0010] To solve the above technical problems, the present application first provides a new use of PdeAPL protein or a substance for regulating the content and / or activity of the PdeAPL protein.

[0011] The present application provides the use of PdeAPL protein or a substance for regulating the content and / or activity of the PdeAPL protein in any one of A1) to A10) as follows:

[0012] A1) regulating the plant height;

[0013] A2) regulating the number of plant stem nodes;

[0014] A3) regulating the length of plant stem nodes;

[0015] A4) regulating the width of plant xylem;

[0016] A5) regulating the size of plant xylem cells;

[0017] A6) regulating the size of plant phloem cells;

[0018] A7) regulating the size of plant leaves;

[0019] A8) cultivating transgenic plants with changed plant height and / or the number of stem nodes and / or the length of stem nodes and / or the width of xylem and / or the size of xylem cells and / or the size of phloem cells and / or the size of leaves;

[0020] A9) improving plant tree shape or wood quality or wood properties;

[0021] A10) plant breeding;

[0022] The PdeAPL protein is a PdeAPL-1 protein and / or a PdeAPL-2 protein;

[0023] The PdeAPL-1 protein is any one of the following B1) to B4):

[0024] B1) a protein having an amino acid sequence of SEQ ID No. 3;

[0025] 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 described in B1);

[0026] 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 described in B1);

[0027] B4) a protein having 80% or more identity to the amino acid sequence described in B1) and having the same function;

[0028] The PdeAPL-2 protein is any one of the following C1) to C4):

[0029] C1) a protein having an amino acid sequence of SEQ ID No. 4;

[0030] C2) 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 described in C1);

[0031] C3) 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 described in C1);

[0032] C4) a protein having 80% or more identity to the amino acid sequence described in C1) and having the same function.

[0033] In the above-mentioned protein of B2) or C2), the tag refers to a polypeptide or protein which is fused and expressed with the protein of interest by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the protein of interest. The tag includes but is not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0034] In the above-mentioned protein of B3) or C3), the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues, or a substitution and / or deletion and / or addition of no more than 9 amino acid residues, or a substitution and / or deletion and / or addition of no more than 8 amino acid residues, or a substitution and / or deletion and / or addition of no more than 7 amino acid residues, or a substitution and / or deletion and / or addition of no more than 6 amino acid residues, or a substitution and / or deletion and / or addition of no more than 5 amino acid residues, or a substitution and / or deletion and / or addition of no more than 4 amino acid residues, or a substitution and / or deletion and / or addition of no more than 3 amino acid residues, or a substitution and / or deletion and / or addition of no more than 2 amino acid residues, or a substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0035] The identity in the above-mentioned protein of B4) or C4) 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 website. For example, the identity (%) can be obtained by calculating the identity of a pair of amino acid sequences 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 Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and then performing a search. The identity includes an amino acid sequence having 80% or more, or having 81% or more, or having 82% or more, or having 83% or more, or having 84% or more, or having 85% or more, or having 86% or more, or having 87% or more, or having 88% or more, or having 89% 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 or SEQ ID No. 4 of the present application.

[0036] The above-mentioned protein of B1) to B4) or C1) to C4) can be artificially synthesized, or a gene encoding the same can be synthesized first and then expressed biologically.

[0037] The above-mentioned substance for regulating the content and / or activity of the PdeAPL protein can be a substance for regulating the content and / or activity of PdeAPL-1 protein and / or a substance for regulating the content and / or activity of PdeAPL-2 protein.

[0038] The substance for regulating the content and / or activity of PdeAPL-1 protein includes a substance for increasing the content and / or activity of PdeAPL-1 protein or a substance for decreasing the content and / or activity of PdeAPL-1 protein.

[0039] Further, the substance for increasing the activity of PdeAPL-1 protein can be a protein, a polypeptide or a small molecule compound that enhances or promotes the function of PdeAPL-1 protein.

[0040] The substance for increasing the content of PdeAPL-1 protein can be a substance that promotes the synthesis of PdeAPL-1 protein, or inhibits the degradation of PdeAPL-1 protein, or overexpresses PdeAPL-1 protein.

[0041] The substance that reduces the activity of PdeAPL-1 protein can be a protein, a polypeptide or a small molecule compound that inhibits the function of PdeAPL-1 protein.

[0042] The substance that reduces the content of PdeAPL-1 protein can be a substance that inhibits the synthesis of PdeAPL-1 protein or promotes the degradation of PdeAPL-1 protein or knocks down (knocks down) or knocks out the PdeAPL-1 protein coding gene.

[0043] Further, the substance that knocks down (knocks down) the PdeAPL-1 protein coding gene can be any nucleic acid molecule that can inhibit or interfere with the expression of the above-mentioned PdeAPL-1 protein coding gene, such as gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0044] The substance that knocks out the PdeAPL-1 protein coding gene can be a substance that achieves in any way that the host cell does not produce a functional protein product of the PdeAPL-1 gene, specific ways such as removing all or part of the coding gene sequence, introducing mutations so that no functional protein is produced, removing or changing the regulatory components (for example, promoter editing) so that the coding gene sequence is not transcribed, preventing translation by binding to mRNA, etc. Generally, the knockout is carried out at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout.

[0045] Further, the nucleic acid molecule that inhibits or interferes with the expression of the above-mentioned PdeAPL-1 protein coding gene is a miRNA that inhibits or interferes with the expression of the above-mentioned PdeAPL-1 protein coding gene.

[0046] In some embodiments, the miRNA that inhibits or interferes with the expression of the above-mentioned PdeAPL-1 protein coding gene is a RNA molecule with a nucleotide sequence of SEQ ID No. 6.

[0047] The substance that regulates the content and / or activity of PdeAPL-2 protein includes a substance that increases the content and / or activity of PdeAPL-2 protein or a substance that reduces the content and / or activity of PdeAPL-2 protein.

[0048] Further, the substance that increases the activity of PdeAPL-2 protein can be a protein, a polypeptide or a small molecule compound that enhances or promotes the function of PdeAPL-2 protein.

[0049] The substance that increases the content of PdeAPL-2 protein can be a substance that promotes the synthesis of PdeAPL-2 protein or inhibits the degradation of PdeAPL-2 protein or overexpresses PdeAPL-2 protein.

[0050] The substance that reduces the activity of PdeAPL-2 protein can be a protein, a polypeptide or a small molecule compound that inhibits the function of PdeAPL-2 protein.

[0051] The substance that reduces the content of PdeAPL-2 protein can be a substance that inhibits the synthesis of PdeAPL-2 protein or promotes the degradation of PdeAPL-2 protein or knocks down (knocks down) or knocks out the PdeAPL-2 protein coding gene.

[0052] Further, the substance that knocks down (knocks down) the PdeAPL-2 protein coding gene can be any nucleic acid molecule that can inhibit or interfere with the expression of the above-mentioned PdeAPL-2 protein coding gene, such as gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0053] The substance that knocks out the PdeAPL-2 protein coding gene can be a substance that achieves in any way that the host cell does not produce a functional protein product of the PdeAPL-2 gene, such as removing all or part of the coding gene sequence, introducing mutations so that no functional protein is produced, removing or changing the regulatory components (such as promoter editing) so that the coding gene sequence is not transcribed, preventing translation by binding to mRNA, etc. Generally, the knockout is carried out at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout.

[0054] Further, the nucleic acid molecule that inhibits or interferes with the expression of the above-mentioned PdeAPL-2 protein coding gene is a miRNA that inhibits or interferes with the expression of the above-mentioned PdeAPL-2 protein coding gene.

[0055] In some embodiments, the miRNA that inhibits or interferes with the expression of the above-mentioned PdeAPL-2 protein coding gene is a RNA molecule with a nucleotide sequence of SEQ ID No. 6.

[0056] In order to solve the above-mentioned problems, the present application further provides a new use of biological materials related to PdeAPL protein.

[0057] The present application provides the use of biological materials related to PdeAPL protein in any one of the following A1) to A10):

[0058] A1) regulating plant height;

[0059] A2) regulating the number of plant stem nodes;

[0060] A3) regulating the length of plant stem nodes;

[0061] A4) regulating the width of plant xylem;

[0062] A5) modulating plant xylem cell size;

[0063] A6) modulating plant phloem cell size;

[0064] A7) modulating plant leaf size;

[0065] A8) breeding transgenic plants with altered stem height and / or stem node number and / or stem node length and / or xylem width and / or xylem cell size and / or phloem cell size and / or leaf size;

[0066] A9) improving plant tree shape or wood quality or wood property;

[0067] A10) plant breeding;

[0068] The biological material is any one of the following E1) to E5):

[0069] E1) a nucleic acid molecule encoding the above-mentioned PdeAPL protein;

[0070] E2) a nucleic acid molecule knocking down or knocking out the gene encoding the above-mentioned PdeAPL protein;

[0071] E3) an expression cassette containing the nucleic acid molecule of E1) or E2);

[0072] E4) a recombinant vector containing the nucleic acid molecule of E1) or E2);

[0073] E5) a recombinant microorganism containing the nucleic acid molecule of E1) or E2).

[0074] In the above-mentioned applications, the nucleic acid molecule of E1) is any one of the following:

[0075] F1) a DNA molecule with a nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2;

[0076] F2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in F1) and encoding the PdeAPL protein.

[0077] Among them, the DNA molecule with a nucleotide sequence of SEQ ID No. 1 encodes the above-mentioned PdeAPL-1 protein, and the DNA molecule with a nucleotide sequence of SEQ ID No. 2 encodes the above-mentioned PdeAPL-2 protein.

[0078] In the above-mentioned applications, the nucleic acid molecule of E2) is any one of the following:

[0079] G1) an RNA molecule with a nucleotide sequence of SEQ ID No. 6;

[0080] G2) a nucleic acid molecule having 75% or more identity to the nucleotide sequence defined in G1) and inhibiting the expression of the PdeAPL protein-encoding gene.

[0081] The skilled person can easily mutate the nucleotide sequence encoding the PdeAPL-1 protein or PdeAPL-2 protein of the present application using known methods, such as methods of directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the PdeAPL-1 or PdeAPL-2 nucleotide sequence isolated from the present application, as long as they encode the PdeAPL-1 protein or PdeAPL-2 protein and have the same function, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application. The identity refers to the sequence similarity to the natural nucleic acid sequence, including the nucleotide sequence having 75% or more identity to the nucleotide sequence encoding the protein consisting of the amino acid sequence shown in SEQ ID No. 3 or SEQ ID No. 4 of the present application. The identity can be evaluated by naked eyes or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.

[0082] The 75% or more identity can be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0083] The nucleic acid molecule of E2) above can be gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA or antisense RNA.

[0084] Any of the nucleic acid molecules described above can be DNA, such as cDNA, genomic DNA or recombinant DNA.

[0085] Any of the nucleic acid molecules described above can be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0086] Any of the expression cassettes described above can comprise a promoter, the nucleic acid molecule of E1) or E2) above and a terminator. The 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 further comprise an enhancer sequence.

[0087] The vector as described above is a vector capable of carrying the nucleic acid molecule as described in E1) or E2) into a host cell for amplification and expression, which can be a cloning vector or an expression vector, including but not limited to: plasmid, bacteriophage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e. cos plasmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.).

[0088] The recombinant vector as described above is a recombinant DNA molecule constructed by connecting the nucleic acid molecule as described in E1) or E2) with the vector in vitro. The recombinant vector containing the nucleic acid molecule as described in E1) or E2) can be constructed using existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company), etc. The plant expression vector can also contain the 3' untranslated region of the foreign gene, that is, it contains a polyadenyl signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenyl signal can guide the addition of polyadenyl to the 3' end of the mRNA precursor, such as the untranslated region of the 3' end of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nos gene of nopaline synthase), the 3' end of the plant gene (such as the soybean storage protein gene) transcription. When using the gene of the present application to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent regions of start codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is wide, which can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants to produce color-changing enzymes or luminescent compounds (GUS gene, luciferase gene, etc.), marker genes of antibiotics (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, and dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate), or anti-chemical agent marker genes (such as herbicide-resistant genes), mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0089] The microorganism can be a bacterium, a fungus, an actinomycete, a protozoan, an alga, or a virus. The bacterium can be from the genus Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but is not limited thereto. For example, the bacterium can be Escherichia coli, Bacillus subtilis, or Bacillus pumilus. The fungus can be a yeast from the genus Saccharomyces sp. (e.g., Saccharomyces cerevisiae), Kluyveromyces sp. (e.g., Kluyveromyces lactis), Pichia sp. (e.g., Pichia pastoris), Schizosaccharomyces sp. (e.g., Schizosaccharomyces pombe), Hansenula sp. (e.g., Hansenula polymorpha), etc., but is not limited thereto. The fungus can also be from the genus Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., Cephalosporium sp., etc., but is not limited thereto. The actinomycete can be from the genus Streptomyces sp., Nocardia sp., Micromonospora sp., Streptosporangium sp., Actinoplanes sp., Thermoactinomyces sp., etc., but is not limited thereto. The alga can be from the genus Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., etc., but is not limited thereto. The virus can be a rotavirus, a herpes virus, an influenza virus, an adenovirus, etc., but is not limited thereto.

[0090] The recombinant microorganism as described in any of the above refers to a recombinant microorganism obtained by manipulating and modifying the genes of a target microorganism, so that the function of the recombinant microorganism is changed. For example, the recombinant microorganism obtained after introducing the above-mentioned 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 cells, and due to natural, accidental or intentional mutations and / or changes, the progeny can not necessarily be completely consistent with the original parent cell, but is still included in the scope of the recombinant microorganism.

[0091] In any of the above-mentioned applications, the plant height is increased or decreased. The regulation mode is positive regulation, that is, when the content and / or activity of PdeAPL protein in the plant is increased, the plant height is increased, and when the content and / or activity of PdeAPL protein in the plant is reduced or deleted, the plant height is decreased.

[0092] In some embodiments, when the expression amount of PdeAPL-1 gene or PdeAPL-2 gene in the plant is increased, the plant height is increased.

[0093] In some embodiments, when the expression amount of PdeAPL-1 gene and PdeAPL-2 gene in the plant is reduced, the plant height is decreased.

[0094] In any of the above-mentioned applications, the number of stem nodes of the plant is increased or decreased. The regulation mode is positive regulation, that is, when the content and / or activity of PdeAPL protein in the plant is increased, the number of stem nodes of the plant is increased, and when the content and / or activity of PdeAPL protein in the plant is reduced or deleted, the number of stem nodes of the plant is decreased.

[0095] In some embodiments, when the expression amount of PdeAPL-1 gene or PdeAPL-2 gene in the plant is increased, the number of stem nodes of the plant is increased.

[0096] In some embodiments, when the expression amount of PdeAPL-1 gene and PdeAPL-2 gene in the plant is reduced, the number of stem nodes of the plant is decreased.

[0097] In any of the above-mentioned applications, the length of the stem node of the plant is increased, specifically: when the content and / or activity of PdeAPL protein in the plant is increased, the length of the stem node of the plant is increased, and when the content and / or activity of PdeAPL protein in the plant is reduced or deleted, the length of the stem node of the plant is increased.

[0098] In some embodiments, when the expression amount of PdeAPL-1 gene or PdeAPL-2 gene in the plant is increased, the length of the stem node of the plant (the total length of the fifth stem node to the eighth stem node) is increased.

[0099] In some embodiments, when the expression level of PdeAPL-1 gene and PdeAPL-2 gene in the plant is reduced, the stem node length (total stem node length from the first stem node to the fifth stem node) of the plant is increased.

[0100] In any of the above-mentioned applications, the regulation of the plant xylem width is to increase the plant xylem width or to decrease the plant xylem width. The regulation mode is positive regulation, that is, when the PdeAPL protein content and / or activity in the plant is increased, the plant xylem width is increased, and when the PdeAPL protein content and / or activity in the plant is reduced or deleted, the plant xylem width is decreased.

[0101] In some embodiments, when the expression level of PdeAPL-1 gene and PdeAPL-2 gene in the plant is reduced, the xylem width of the plant is decreased.

[0102] In any of the above-mentioned applications, the regulation of the plant xylem cell size is to make the plant xylem cell larger or to make the plant xylem cell smaller. The regulation mode is negative regulation, that is, when the PdeAPL protein content and / or activity in the plant is increased, the plant xylem cell is smaller, and when the PdeAPL protein content and / or activity in the plant is reduced or deleted, the plant xylem cell is larger.

[0103] In some embodiments, when the expression level of PdeAPL-1 gene and PdeAPL-2 gene in the plant is reduced, the area of xylem vessel cells and fiber cells of the plant is increased.

[0104] In any of the above-mentioned applications, the regulation of the plant phloem cell size is to make the plant phloem cell larger or to make the plant phloem cell smaller. The regulation mode is negative regulation, that is, when the PdeAPL protein content and / or activity in the plant is increased, the plant phloem cell is smaller, and when the PdeAPL protein content and / or activity in the plant is reduced or deleted, the plant phloem cell is larger.

[0105] In some embodiments, when the expression level of PdeAPL-1 gene and PdeAPL-2 gene in the plant is reduced, the area of phloem cells of the plant is larger.

[0106] In any of the above-mentioned applications, the regulation of the plant leaf size is to make the plant leaf larger or to make the plant leaf smaller. The regulation mode is negative regulation, that is, when the PdeAPL protein content and / or activity in the plant is increased, the plant leaf is smaller, and when the PdeAPL protein content and / or activity in the plant is reduced or deleted, the plant leaf is larger.

[0107] In some embodiments, when the expression level of PdeAPL-1 gene and PdeAPL-2 gene in the plant is reduced, the leaf area and leaf width of the plant are increased.

[0108] In some embodiments, when the expression amount of PdeAPL-1 gene and PdeAPL-2 gene in the plant is increased, the plant leaf area, leaf length and leaf width are reduced.

[0109] To solve the above technical problems, the present application also provides a method for cultivating a transgenic plant with increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves.

[0110] The method for cultivating a transgenic plant with increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves can also be a method for producing a transgenic plant with increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves.

[0111] The method for cultivating a transgenic plant with increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves provided by the present application comprises the following steps: increasing the content and / or activity of the above-mentioned PdeAPL protein in the target plant, so as to obtain a transgenic plant with increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves.

[0112] Further, the method for increasing the content and / or activity of the above-mentioned PdeAPL protein in the target plant is overexpressing the above-mentioned PdeAPL-1 protein or the above-mentioned PdeAPL-2 protein in the target plant.

[0113] Still further, the xylem cells include xylem vessel cells and xylem fiber cells.

[0114] Still further, the method for overexpression is introducing the coding gene of the above-mentioned PdeAPL-1 protein or the coding gene of the above-mentioned PdeAPL-2 protein into the target plant.

[0115] In the method for breeding a transgenic plant with increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves, the gene encoding the PdeAPL-1 protein or the gene encoding the PdeAPL-2 protein is a cDNA gene of the PdeAPL-1 protein or the PdeAPL-2 protein. The cDNA gene is a cDNA molecule comprising a coding sequence (CDS) of the PdeAPL-1 protein or the PdeAPL-2 protein. The coding sequence can be SEQ ID No. 1 or SEQ ID No. 2. The transgenic plant contains the cDNA gene.

[0116] To solve the above technical problems, the present application finally provides a method for breeding a transgenic plant with decreased plant height and / or decreased number of stem nodes and / or decreased length of stem nodes and / or decreased width of xylem and / or larger xylem cells and / or larger phloem cells and / or larger leaves.

[0117] The method for breeding a transgenic plant with increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves can also be a method for producing a transgenic plant with increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves.

[0118] The method for breeding a transgenic plant with decreased plant height and / or decreased number of stem nodes and / or decreased length of stem nodes and / or decreased width of xylem and / or larger xylem cells and / or larger phloem cells and / or larger leaves provided by the present application comprises the following steps: reducing the content and / or activity of the PdeAPL protein in the target plant to obtain a transgenic plant with decreased plant height and / or decreased number of stem nodes and / or decreased length of stem nodes and / or decreased width of xylem and / or larger xylem cells and / or larger phloem cells and / or larger leaves.

[0119] Further, 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 gene encoding the PdeAPL protein into the target plant.

[0120] Still further, the xylem cells include xylem vessel cells and xylem fiber cells.

[0121] Further, the substance that inhibits or interferes with the expression of the PdeAPL protein-encoding gene is a substance that inhibits or interferes with the expression of the PdeAPL-1 protein-encoding gene and the PdeAPL-2 protein-encoding gene.

[0122] In some embodiments, the substance that inhibits or interferes with the expression of the PdeAPL-1 protein-encoding gene and the PdeAPL-2 protein-encoding gene can be an RNA molecule having the nucleotide sequence of SEQ ID No. 6 or a DNA molecule that produces an RNA molecule having the nucleotide sequence of SEQ ID No. 6. The DNA molecule can specifically be a double-stranded nucleic acid molecule having the nucleotide sequence of SEQ ID No. 5.

[0123] In the method for breeding a transgenic plant with reduced plant height and / or reduced number of stem nodes and / or reduced length of stem nodes and / or reduced width of xylem and / or enlarged xylem cells and / or enlarged phloem cells and / or enlarged leaves, the plant of interest contains a PdeAPL protein or a gene (genomic gene and / or cDNA gene) encoding the PdeAPL protein.

[0124] In the method for breeding a transgenic plant with reduced plant height and / or reduced number of stem nodes and / or reduced length of stem nodes and / or reduced width of xylem and / or enlarged xylem cells and / or enlarged phloem cells and / or enlarged leaves, the transgenic plant can contain a DNA molecule having the nucleotide sequence of SEQ ID No. 5 or an expression vector containing the DNA molecule. The expression vector can be a PGWB2 vector.

[0125] In any of the above-mentioned uses or methods, the transgenic plant not only includes the first generation transgenic plant obtained by transforming a plant of interest with a substance that overexpresses or interferes with a PdeAPL gene, but also includes its progeny. For a transgenic plant, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species using conventional breeding techniques, particularly including commercial varieties. The transgenic plant includes seeds, calli, whole plants, and cells.

[0126] In any of the above-mentioned applications or methods, the plant can be a dicotyledon or a monocotyledon, including food crops such as rice, wheat, barley, corn, soybean, potato, legume, oat, and millet; vegetable crops such as Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, zucchini, leek, onion, and radish; economic crops such as ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, wild sesame, peanut, and rapeseed; fruits such as apple, pear, jujube, peach, kiwi, grape, orange, persimmon, plum, apricot, and banana; flowers such as rose, sword lily, gerbera, carnation, chrysanthemum, lily, and tulip; forage crops such as ryegrass, red clover, orchardgrass, alfalfa, tall fescue, and perennial ryegrass; and wood plants such as poplar, willow, birch, locust, elm, metasequoia, spruce, beech, maple, oak, mahogany, Fraxinus mandshurica, Sophora japonica, Gordonia longicarpa, Dalbergia hupeana, Dalbergia odorifera, Dalbergia variabilis, white poplar, and maple. Further, the dicotyledon is a Salicaceae plant. Further, the Salicaceae plant is a Populus plant. Further, the Populus plant is a Populus sp. In some embodiments, the Populus sp. is a poplar (e.g., wild-type poplar "nanlin895").

[0127] The transgenic plant produced according to the above method also falls within the protection scope of the present application.

[0128] The present application constructs a transgenic poplar overexpressing PdeAPL and a transgenic poplar with reduced expression of PdeAPL gene. Experiments show that, compared with the control, the transgenic poplar overexpressing PdeAPL has significantly increased plant height, stem node number, and stem node length, and the leaf becomes smaller; the transgenic poplar with reduced expression of PdeAPL gene has reduced plant height and stem node number, increased stem node length, and larger leaf. In addition, the transgenic poplar with reduced expression of PdeAPL gene has reduced xylem width, and larger xylem cell and phloem cell areas. The present application is of great significance for revealing the molecular mechanism of regulation of stem vascular development of forest trees, improving tree shape, growth rate, and wood quality, and directionally cultivating forest trees with tree shape or wood quality meeting the actual production needs. BRIEF DESCRIPTION OF DRAWINGS

[0129] Figure 1 shows the detection results of PdeAPL gene expression in PdeAPL interference transgenic poplar lines. WT represents wild-type poplar, mi-L6 and mi-L21 represent two different lines of transgenic poplar into which the recombinant interference vector PGWB2-R is introduced.

[0130] Figure 2 shows the detection results of expression in PdeAPL-1 overexpression transgenic poplar seedlings.

[0131] Figure 3 is the result of expression level detection of PdeAPL-2 overexpression transgenic poplar seedlings.

[0132] Figure 4 is the phenotype observation and quantitative trait statistics chart of wild type poplar seedlings and PdeAPL interference transgenic poplar seedlings. A is the phenotype chart of wild type poplar seedlings and PdeAPL interference transgenic poplar seedlings mi-L229, mi-L6 and mi-L21 grown for one month. B is the plant height statistics chart of wild type poplar seedlings and PdeAPL interference transgenic poplar seedlings. C is the number of stem nodes statistics chart of wild type poplar seedlings and PdeAPL interference transgenic poplar seedlings. D is the stem node length statistics chart of wild type poplar seedlings and PdeAPL interference transgenic poplar seedlings, wherein the stem node refers to the stem section between two consecutive leaves, and the first stem node from the top to the base is defined as 1st, the second stem node is defined as 2nd, the third stem node is defined as 3rd, the fourth stem node is defined as 4th, and the fifth stem node is defined as 5th. The stem node length here is the total stem node length from the first stem node to the fifth stem node. Each plant system is counted for 5 repetitions, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, ns: no significant difference.

[0133] Figure 5 is the phenotype observation and quantitative trait statistics chart of wild type poplar seedlings and PdeAPL overexpression transgenic poplar seedlings. A is the phenotype chart of wild type poplar seedlings and PdeAPL overexpression transgenic poplar seedlings OE1-30 and OE2-24 grown for three months. B is the plant height statistics chart of wild type poplar seedlings and PdeAPL overexpression transgenic poplar seedlings OE1-30 and OE2-24. C is the number of stem nodes statistics chart of wild type poplar seedlings and PdeAPL overexpression transgenic poplar seedlings OE1-30 and OE2-24. D is the stem node length statistics chart of wild type poplar seedlings and PdeAPL overexpression transgenic poplar seedlings OE1-30 and OE2-24. The stem node length here is the total stem node length from the fifth stem node to the eighth stem node. Each plant system is counted for 5 repetitions, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, ns: no significant difference.

[0134] Figure 6 is a wild-type poplar seedling and PdeAPL interference transgenic poplar seedling same time stem node cross-section diagram and xylem width, xylem vessel cell area, xylem fiber cell area, phloem cell area statistical chart. A-C is a wild-type poplar seedling cross-section diagram, B and C are partial enlarged views of the phloem and xylem of the wild-type poplar seedling, respectively, D-F is a cross-section diagram of the PdeAPL interference transgenic poplar seedling, E and F are partial enlarged views of the phloem and xylem of the PdeAPL interference transgenic poplar seedling, respectively, G, H, I and J are statistical charts of the xylem width, xylem vessel cell area, xylem fiber cell area and phloem cell area of the wild-type poplar seedling and the PdeAPL interference transgenic poplar seedling, respectively.

[0135] Figure 7 is a wild-type poplar seedling and transgenic poplar seedling leaf phenotype and trait statistical chart. A is a wild-type poplar seedling and transgenic poplar seedling leaf phenotype chart. B is a wild-type poplar seedling and transgenic poplar seedling leaf area statistical chart. C is a wild-type poplar seedling and transgenic poplar seedling leaf length statistical chart. D is a wild-type poplar seedling and transgenic poplar seedling leaf width statistical chart. Each plant system is counted 5 times, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, ns: no significant difference. Embodiments of the present application

[0136] The present application will be further described in conjunction with the specific embodiments, and 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 of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0137] 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. In the following examples, unless otherwise specified, quantitative tests are set up for three or more repeated experiments.

[0138] The PGWB2 vector in the following examples is described in the literature "Figueiredo J, P Lahaye T, et al. (2011) Agrobacterium-mediated transient expression in citrus leaves: a rapid tool for gene expression and functional gene assay. [J]. Plant Cell Reports, 30(7): 1339-1345.

[0139] The PGWB2-5xMYC vector in the following examples is a vector obtained by linking a 5xMYC tag into the PGWB2 vector.

[0140] 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.”

[0141] The wild-type poplar “nanlin895” in the following examples is described in the literature “Zhu, Y., et al. (2018). A HD-ZIP III gene, PtrHB4, is required for interfascicular cambium development in Populus. Plant Biotechnol J 16(3): 808-817.”

[0142] 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.”

[0143] Example 1, Obtaining of PdeAPL protein and its encoding gene

[0144] ​1. The total RNA was extracted from the whole plant of Populus "nanlin 895" which was frozen in liquid nitrogen and grinded after growing in a tissue culture bottle for one month, and the cDNA of Populus was obtained by reverse transcription of the total RNA.

[0145] 2. The cDNA obtained was used as a template, 5'-ATGTTCCATACCAAGAAACC-3' (SEQ ID No. 7) was used as a forward primer, and 5'-TCAACCAAATGGTGAGTTTC-3' (SEQ ID No. 8) was used as a reverse primer to perform PCR amplification, and the amplification product was obtained.

[0146] 3. The amplification product was detected and separated and purified by agarose gel electrophoresis to obtain a DNA fragment of about 1000 bp, and then the PCR product was connected to the cloning vector peasy-Blunt simple, and the connected vector was sequenced.

[0147] The sequencing results show that two PdeAPL coding region sequences exist in Populus "nanlin 895", which are shown in SEQ ID No. 1 and SEQ ID No. 2 in the sequence listing, the gene shown in SEQ ID No. 1 is named PdeAPL-1, and the amino acid sequence of the PdeAPL-1 protein encoded by the gene is shown in SEQ ID No. 3 in the sequence listing, and the gene shown in SEQ ID No. 2 is named PdeAPL-2 gene, and the amino acid sequence of the PdeAPL-2 protein encoded by the gene is shown in SEQ ID No. 4 in the sequence listing.

[0148] Example 2, Construction of Recombinant Vectors and Recombinant Agrobacterium

[0149] I. Construction of Recombinant Interference Vectors and Recombinant Interference Agrobacterium

[0150] 1. Construction of Recombinant Interference Vector PGWB2-R

[0151] (1) The common coding sequence 5'-TAACGCAGATCGTCGGGCCAA-3' (SEQ ID No. 9) of the PdeAPL gene coding region shown in SEQ ID No. 1 and SEQ ID No. 2 was used as a target sequence, and the primer was designed on the primer design website http: / / wmd3.weigelworld.org / cgi-bin / webapp.cgi?page=Home;project=stdwmd. The primer sequence is as follows:

[0152] I miR-s: gaTAACGCAGATCGTCGGGCCAAtctctcttttgtattcc (SEQ ID No. 10);

[0153] II miR-a: gaTTGGCCCGACGATCTGCGTTAtcaaagagaatcaatga (SEQ ID No. 11);

[0154] III miR*s: gaTTAGCCCGACGATGTGCGTTTtcacaggtcgtgatatg (SEQ ID No. 12);

[0155] IV miR*a: gaAAACGCACATCGTCGGGCTAAtctacatatatattcct (SEQ ID No. 13).

[0156] (2) Using the pRS300 (MIR319a Arabidopsis thaliana) vector as a template, two rounds of nested PCR (for details, refer to Rebecca Schwab, MPI for Developmental Biology, Tuebingen, 2005) were performed using the four primer sequences in step (1) to synthesize the artificial microRNA gene. The nucleotide sequence of the artificial microRNA gene is shown in SEQ ID No. 5 in the sequence listing.

[0157] (3) The artificial microRNA synthesized in step (2) was connected to the pENTR / D-TOPO® vector to obtain the pENTR / D-TOPO®-artificial microRNA vector; then, through LR homologous recombination, the artificial microRNA gene in the pENTR / D-TOPO®-artificial microRNA vector was connected to the PGWB2 vector to obtain the recombinant interference vector PGWB2-R. The recombinant interference vector PGWB2-R expresses an RNA molecule with the nucleotide sequence of SEQ ID No. 6.

[0158] 2. Construction of recombinant interference Agrobacterium PGWB2-R / GV3101

[0159] The recombinant interference vector PGWB2-R was transformed into Agrobacterium tumefaciens GV3101 by Agrobacterium transformation method, and the recombinant interference Agrobacterium PGWB2-R / GV3101 was obtained through PCR detection.

[0160] II. Construction of recombinant overexpression vector and recombinant overexpression Agrobacterium

[0161] ​​​1. Construction of recombinant overexpression vector p35S::pdeAPL-1-5xMYC

[0162] The DNA fragment (PdeAPL-1 CDS sequence) shown in SEQ ID No. 1 in the sequence listing is connected to the pENTR / D-TOPO vector by homologous recombination, to obtain the pENTR / D-TOPO-APL-1 vector; then the PdeAPL-1 CDS sequence in the pENTR / D-TOPO-APL-1 vector is connected to the PGWB2-5xMYC vector by LR homologous recombination, to obtain the recombinant overexpression vector p35S::pdeAPL-1-5xMYC. The DNA fragment (PdeAPL-1 CDS sequence) shown in SEQ ID No. 1 in the sequence listing is connected to the pENTR / D-TOPO vector by homologous recombination, to obtain the pENTR / D-TOPO-APL-1 vector; then the PdeAPL-1 CDS sequence in the pENTR / D-TOPO-APL-1 vector is connected to the PGWB2-5xMYC vector by LR homologous recombination, to obtain the recombinant overexpression vector p35S::pdeAPL-1-5xMYC. The DNA fragment (PdeAPL-1 CDS sequence) shown in SEQ ID No. 1 in the sequence listing is connected to the pENTR / D-TOPO vector by homologous recombination, to obtain the pENTR / D-TOPO-APL-1 vector; then the PdeAPL-1 CDS sequence in the pENTR / D-TOPO-APL-1 vector is connected to the PGWB2-5xMYC vector by LR homologous recombination, to obtain the recombinant overexpression vector p35S::pdeAPL-1-5xMYC.

[0163] 2. Construction of recombinant overexpression agrobacterium p35S::pdeAPL-1-5xMYC / GV3101

[0164] The recombinant overexpression vector p35S::pdeAPL-1-5xMYC is transformed into Agrobacterium tumefaciens GV3101 by Agrobacterium transformation method, and after PCR detection, the recombinant overexpression agrobacterium p35S::pdeAPL-1-5xMYC / GV3101 containing the recombinant overexpression vector p35S::pdeAPL-1-5xMYC is obtained.

[0165] 3. Construction of recombinant overexpression vector p35S::pdeAPL-2-5xMYC

[0166] The DNA fragment (PdeAPL-2 CDS sequence) shown in SEQ ID No. 2 in the sequence listing is connected to the pENTR / D-TOPO vector by homologous recombination, to obtain the pENTR / D-TOPO-APL-2 vector; then the PdeAPL-2 CDS sequence in the pENTR / D-TOPO-APL-2 vector is connected to the PGWB2-5xMYC vector by LR homologous recombination, to obtain the recombinant overexpression vector p35S::pdeAPL-2-5xMYC. The DNA fragment (PdeAPL-2 CDS sequence) shown in SEQ ID No. 2 in the sequence listing is connected to the pENTR / D-TOPO vector by homologous recombination, to obtain the pENTR / D-TOPO-APL-2 vector; then the PdeAPL-2 CDS sequence in the pENTR / D-TOPO-APL-2 vector is connected to the PGWB2-5xMYC vector by LR homologous recombination, to obtain the recombinant overexpression vector p35S::pdeAPL-2-5xMYC. The DNA fragment (PdeAPL-2 CDS sequence) shown in SEQ ID No. 2 in the sequence listing is connected to the pENTR / D-TOPO vector by homologous recombination, to obtain the pENTR / D-TOPO-APL-2 vector; then the PdeAPL-2 CDS sequence in the pENTR / D-TOPO-APL-2 vector is connected to the PGWB2-5xMYC vector by LR homologous recombination, to obtain the recombinant overexpression vector p35S::pdeAPL-2-5xMYC.

[0167] 4. Construction of recombinant overexpression agrobacterium p35S::pdeAPL-2-5xMYC / GV3101

[0168] ​​The recombinant overexpression vector p35S::pdeAPL-2-5xMYC was transformed into Agrobacterium tumefaciens GV3101 by Agrobacterium transformation method, and after PCR detection, the recombinant overexpression Agrobacterium p35S::pdeAPL-2-5xMYC / GV3101 containing the recombinant overexpression vector p35S::pdeAPL-2-5xMYC was obtained.

[0169] Example 3, obtaining and identifying of transgenic poplar

[0170] I. Obtaining of transgenic poplar

[0171] All the recombinant Agrobacterium constructed in Example 2 were used to infect the tender leaves of tissue culture poplar seedlings "nanlin895" by leaf disc method, and after a series of callus induction, bud induction and rooting induction, transgenic poplar seedlings were obtained. The specific steps are as follows:

[0172] 1. The Agrobacterium monoclonal obtained in Example 2 was first cultured in 5 mL 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 cultured bacterial solution was taken into 100 mL liquid YEB medium, and shaken at 28°C to OD 600nm = 0.8, then 100 μm acetyl-syringone was added.

[0173] 2. Take the tender leaves of the poplar seedlings grown in the tissue culture bottle for 1 month, cut off the four sides of the leaves with a scalpel, leaving the leaves around the main vein about 1 cm 2 in size, and gently cut 3-4 small wounds on the main vein, and place them in the shaken Agrobacterium, and gently shake for 30 min.

[0174] 3. Take out the leaves, place them on the co-culture medium (co-culture medium is a basic medium containing 0.5 mg / L kinetin, 0.75 mg / L 2-4-D, 100 μm acetyl-syringone; the solvent of the basic medium is water, and the solutes and their concentrations are shown in Table 1) with the back down, and co-culture at 28°C for two days.

[0175] 4. Transfer the leaves to the callus induction screening medium (screening medium is a basic medium containing 0.5 mg / L kinetin, 0.75 mg / L 2-4-D, 50 mg / L kan, 250 mg / L carb, 300 mg / L Timentin; the solvent of the basic medium is water, and the solutes and their concentrations are shown in Table 1), and culture in the dark, subculture every 14 days, until round callus tissue grows.

[0176] 5. The callus is cut and placed on differentiation medium (the differentiation medium is the basic medium containing 0.2 mg / L thidiazuron, 50 mg / L kan, 250 mg / L carb, 300 mg / L Timentin; the solvent of the basic medium is water, and the solutes and their concentrations are shown in Table 1) under light, and subcultured every 15 days until sprouting.

[0177] 6. The small buds are cut and independently cultured on rooting medium (the rooting medium is the basic medium containing 50 mg / L kan, 250 mg / L carb, 300 mg / L Timentin; the solvent of the basic medium is water, and the solutes and their concentrations are shown in Table 1) until rooting, and transgenic poplar seedlings are obtained. The transgenic poplar seedlings after rooting can be continuously propagated.

[0178] Table 1. Solutes and their concentrations in the basic medium of different culture media

[0179] II. Identification of transgenic poplar

[0180] 1. PCR identification

[0181] The DNA of the transgenic poplar seedlings is extracted for PCR identification, which specifically includes the following steps: the DNA of all transgenic poplar seedlings is extracted by SLS method, and the DNA of each plant is subjected to PCR amplification by designing primers at both ends of the above-mentioned recombinant vector, and wild-type poplar DNA is used as a control.

[0182] The primer sequences designed for the PGWB2-R vector are as follows:

[0183] 5'-GGGGACTCTAGAGTTATCAAC-3' (SEQ ID No. 14);

[0184] 5'-CTAAGCGCTGTTATCAACCAC-3' (SEQ ID No. 15).

[0185] The primer sequences designed for the PGWB2-5xMYC vector are as follows:

[0186] 5'-GGGGACTCTAGAGTTATCAAC-3' (SEQ ID No. 16);

[0187] 5'-GCATGTCTTGCGTTGATGAAGC-3' (SEQ ID No. 17).

[0188] PCR products were detected by electrophoresis, and positive transgenic plants were obtained. For the pGWB2-R vector, a 822 bp PCR product was amplified as a positive transgenic plant. For the PGWB2-5xMYC vector, a 1639 bp PCR product was amplified as a positive transgenic plant.

[0189] 2. Interference transgenic poplar and overexpression transgenic poplar

[0190] 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 1 month under the same growth conditions were taken, the total RNA of the leaves was extracted by the plant RNA small amount extraction kit of Megan company, then the cDNA was synthesized by the reverse transcription kit of Invitrogen company with Oligod(T) as a primer, and real-time quantitative PCR detection was performed with pdeAPL gene specific primers, with Actin as an internal reference gene. The primers are as follows:

[0191] QpdeAPL-1-F: 5'-TTAAGGACCGAGGCTCAAGG-3'(SEQ ID No. 18);

[0192] QpdeAPL-1-R: 5'-TCCTGGTATGGGTGCAATGAA-3'(SEQ ID No. 19);

[0193] QpdeAPL-2-F: 5'-AGATCCAGATTGCACCACCA-3'(SEQ ID No. 20);

[0194] QpdeAPL-2-R: 5'-GGCCAGCTTGACCTTCTACA-3'(SEQ ID No. 21);

[0195] QpdeActinF: 5'-AAACTGTAATGGTCCTCCCTCCG-3'(SEQ ID No. 22);

[0196] QpdeActinR: 5'-GCATCATCACAATCACTCTCCGA-3'(SEQ ID No. 23).

[0197] The expression amount detection results of the pdeAPL-1 and pdeAPL-2 genes of different interference transgenic poplars are shown in Figure 1. The pdeAPL interference transgenic poplar mi-L229, mi-L6 and mi-L21 strains with the most significant down-regulation of the pdeAPL-1 and pdeAPL-2 gene expression amounts compared with the wild type poplar were selected for subsequent research experiments.

[0198] The expression detection results of pdeAPL-1 genes of different overexpression transgenic poplars are shown in Figure 2. The pdeAPL-1 overexpression transgenic poplar OE1-30 strain with the most significant up-regulation of pdeAPL-1 gene expression compared with the wild type poplar was selected for subsequent research experiments.

[0199] The expression detection results of pdeAPL-2 genes of different overexpression transgenic poplars are shown in Figure 3. The pdeAPL-2 overexpression transgenic poplar OE2-24 strain with the most significant up-regulation of pdeAPL-2 gene expression compared with the wild type poplar was selected for subsequent research experiments.

[0200] Example 4, pdeAPL genes significantly affect the height, number of stem nodes and stem node length of poplar

[0201] Test materials: wild type poplar "nanlin895" (WT), pdeAPL interference transgenic poplar mi-L229, mi-L6 and mi-L21 strains, pdeAPL-1 overexpression transgenic poplar OE1-30 strain, pdeAPL-2 overexpression transgenic poplar OE2-24 strain.

[0202] Experimental method: The wild type tissue culture seedlings and pdeAPL interference transgenic poplar tissue culture seedlings grown in tissue culture bottles for 25 days were transferred to soil culture pots, and after growing in the soil culture pots for 1 month, the tree shape of the wild type poplar seedlings and the pdeAPL interference transgenic poplar seedlings, including the height, number of stem nodes and stem node length of the poplar, were observed and the quantitative traits were counted. The wild type tissue culture seedlings and pdeAPL overexpression transgenic poplar tissue culture seedlings grown in tissue culture bottles for 25 days were transferred to soil culture pots, and after growing in the soil culture pots for 3 months, the tree shape of the wild type poplar seedlings and the pdeAPL overexpression transgenic poplar seedlings, including the height, number of stem nodes and stem node length of the poplar, were observed and the quantitative traits were counted.

[0203] The experimental results of the pdeAPL interference transgenic poplar seedlings are shown in Figure 4. The results show that: compared with the wild type poplar seedlings, the height of the pdeAPL interference transgenic poplar seedlings is significantly reduced, among which the average height of WT, mi-L6 strain and mi-L21 strain is 26.5 cm, 16.37 cm and 13.18 cm respectively. Compared with the wild type poplar seedlings, the number of stem nodes of the pdeAPL interference transgenic poplar seedlings is significantly reduced, among which the average number of stem nodes of WT, mi-L6 strain and mi-L21 strain is 21 nodes, 11 nodes and 10 nodes respectively. Compared with the wild type poplar seedlings, the stem node length of the pdeAPL interference transgenic poplar seedlings is significantly increased, among which the average stem node length of WT, mi-L6 strain and mi-L21 strain is 5.25 mm, 11.78 mm and 9.49 mm respectively.

[0204] The experimental results of pdeAPL overexpression transgenic poplar seedlings are shown in Figure 5. It was found that the plant height of pdeAPL overexpression transgenic poplar seedlings was significantly increased compared with wild type poplar seedlings, wherein the average plant height of WT, OE1-30 strain and OE2-24 strain was 83.55 cm, 89.93 cm and 90.86 cm, respectively. The number of stem nodes of pdeAPL overexpression transgenic poplar seedlings was significantly increased compared with wild type poplar seedlings, wherein the average number of stem nodes of WT, OE1-30 strain and OE2-24 strain was 51 nodes, 53 nodes and 55 nodes, respectively. The stem node length of pdeAPL overexpression transgenic poplar seedlings was significantly increased compared with wild type poplar seedlings, wherein the average stem node length of WT, OE1-30 strain and OE2-24 strain was 19.72 mm, 21.44 mm and 22.23 mm, respectively.

[0205] Example 5, pdeAPL gene significantly affects poplar vascular cell area

[0206] Test materials: wild type poplar "nanlin895" (WT), pdeAPL interference transgenic poplar mi-L21 strain.

[0207] Experimental method: Wild type tissue culture seedlings and pdeAPL interference transgenic tissue culture seedlings grown in tissue culture bottles for 25 days were transferred to soil culture pots. The same time stem nodes (i.e. after growing in soil culture pots for 1 month, the first stem node was marked, and after growing for another month, the marked stem node was the same time stem node) of wild type poplar seedlings and pdeAPL interference transgenic poplar seedlings grown in soil culture pots for 2 months were hand sectioned and stained with safranin blue dye. The xylem width, xylem vessel cell area, xylem fiber cell area, and phloem cell area were observed and counted.

[0208] The results are shown in Figure 6. It was found that the xylem width of pdeAPL interference transgenic poplar seedlings was significantly reduced, and the xylem vessel cell area, xylem fiber cell area, and phloem cell area were significantly increased compared with wild type poplar seedlings. Specifically, the xylem width of pdeAPL interference transgenic poplar seedlings was reduced by 17.79% compared with wild type poplar seedlings, the xylem vessel cell area was increased by 43.35% compared with wild type poplar seedlings, the xylem fiber cell area was increased by 85.05% compared with wild type poplar seedlings, and the phloem cell area was increased by 45.55% compared with wild type poplar seedlings.

[0209] Example 6, pdeAPL gene significantly affects poplar leaf development

[0210] Test materials: wild type poplar "nanlin895" (WT), pdeAPL interference transgenic poplar mi-L6 and mi-L21 lines, pdeAPL-1 overexpression transgenic poplar OE1-30 line, and pdeAPL-2 overexpression transgenic poplar OE2-24 line.

[0211] Experimental method: Wild type and transgenic poplar seedlings grown in tissue culture bottles for 25 days were transferred to soil culture pots, and after growing in the soil culture pots for 3 months, the leaves of wild type and transgenic poplar seedlings were observed, and the leaf area, leaf length and leaf width were counted.

[0212] The results are shown in Figure 7. It was found that the leaf area of pdeAPL interference transgenic poplar seedlings was significantly increased, and the leaf area of pdeAPL overexpression transgenic poplar seedlings was significantly reduced, compared with wild type poplar seedlings, wherein the average leaf area of WT, mi-L21 line, OE1-30 line and OE2-24 line was 71.80 cm 2 , 85.41 cm 2 , 40.09 cm 2 and 47.43 cm 2 , respectively. Compared with wild type poplar seedlings, the leaf length of pdeAPL interference transgenic poplar seedlings had no significant change, and the leaf length of pdeAPL overexpression transgenic poplar seedlings was significantly reduced, wherein the average leaf length of WT, mi-L21 line, OE1-30 line and OE2-24 line was 11.56 cm, 12.07 cm, 9.89 cm and 9.65 cm, respectively. Compared with wild type poplar seedlings, the leaf width of pdeAPL interference transgenic poplar seedlings was significantly increased, and the leaf width of pdeAPL overexpression transgenic poplar seedlings was significantly reduced, wherein the average leaf width of WT, mi-L21 line, OE1-30 line and OE2-24 line was 9.37 cm, 11.54 cm, 7.76 cm and 7.92 cm, respectively.

[0213] In summary, pdeAPL gene significantly affects the plant height, number of stem nodes, stem node length, xylem width, xylem cell area, phloem cell area and leaf size of poplar, which provides a new idea for cultivating high-quality, fast-growing and good-shaped poplar.

[0214] The above has been described in detail. For those skilled in the art, the present application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the present application and without unnecessary experiments. Although the present application gives a special embodiment, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art out of the range disclosed in the present application. Some basic features can be applied according to the scope of the following attached claims. Industrial applicability

[0215] The present application provides the application of PdeAPL protein and its encoding gene in regulating the growth and development of poplar and the wood quality. The experiment proves that the plant height, stem node quantity and stem node length of the transgenic poplar with overexpressed PdeAPL are significantly increased, and the leaf becomes smaller; the plant height and stem node quantity of the transgenic poplar with reduced PdeAPL gene expression are reduced, the stem node length is increased, the leaf becomes larger, the xylem width is reduced, and the xylem cell and phloem cell area are larger. The present application has important significance for revealing the regulation molecular mechanism of the stem vascular development of forest trees, improving the tree shape, growth speed and wood quality of forest trees, and directionally cultivating the tree shape or wood quality of forest trees in accordance with the actual production demand.

Claims

1. Use of a PdeAPL protein or a substance that regulates the content and / or activity of the PdeAPL protein in any one of A1) to A10) below: A1) regulating plant stem height; A2) regulating plant stem node number; A3) regulating plant stem node length; A4) regulating plant xylem width; A5) regulating plant xylem cell size; A6) regulating plant phloem cell size; A7) regulating plant leaf size; A8) breeding a transgenic plant with altered stem height and / or stem node number and / or stem node length and / or xylem width and / or xylem cell size and / or phloem cell size and / or leaf size; A9) improving plant tree shape or wood quality or wood properties; A10) plant breeding; the PdeAPL protein is a PdeAPL-1 protein and / or a PdeAPL-2 protein; the PdeAPL-1 protein is any one of B1) to B4) below: B1) a protein with an amino acid sequence of SEQ ID No. 3; B2) a fusion protein with the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid sequence in B1); B3) a protein with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence in B1); B4) a protein with 80% or more identity to the amino acid sequence in B1) and with the same function; the PdeAPL-2 protein is any one of C1) to C4) below: C1) a protein with an amino acid sequence of SEQ ID No. 4; C2) a fusion protein with the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid sequence in C1); C3) a protein with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence in C1); C4) a protein with 80% or more identity to the amino acid sequence in C1) and with the same function.

2. Use of a biological material related to a PdeAPL protein in any one of A1) to A10) below: A1) regulating plant stem height; A2) regulating plant stem node number; A3) regulating plant stem node length; A4) regulating plant xylem width; A5) regulating plant xylem cell size; A6) regulating plant phloem cell size; A7) regulating plant leaf size; A8) breeding a transgenic plant with altered stem height and / or stem node number and / or stem node length and / or xylem width and / or xylem cell size and / or phloem cell size and / or leaf size; A9) improving plant tree shape or wood quality or wood properties; A10) plant breeding; the biological material is any one of E1) to E5) below: E1) a nucleic acid molecule encoding a PdeAPL protein; E2) a nucleic acid molecule for knocking down or knocking out a PdeAPL protein-encoding gene; E3) an expression cassette containing the nucleic acid molecule in E1) or E2); E4) a recombinant vector containing the nucleic acid molecule in E1) or E2); E5) a recombinant microorganism comprising the nucleic acid molecule of E1 ) or E2); the PdeAPL protein is a PdeAPL-1 protein and / or a PdeAPL-2 protein; the PdeAPL-1 protein is any one of the following B1 )-B4): B1 ) a protein having an amino acid sequence of SEQ ID No. 3; 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 of B1 ); 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 of B1 ); B4) a protein having 80% or more identity to the amino acid sequence of B1 ) and having the same function; the PdeAPL-2 protein is any one of the following C1 )-C4): C1 ) a protein having an amino acid sequence of SEQ ID No. 4; C2) 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 of C1 ); C3) 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 of C1 ); C4) a protein having 80% or more identity to the amino acid sequence of C1 ) and having the same function.

3. Use according to claim 2, characterized in that: E1 ) the nucleic acid molecule is any one of the following: F1 ) a DNA molecule having a nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2; F2) a DNA molecule having 75% or more identity to the nucleotide sequence defined in F1 ) and encoding the PdeAPL protein.

4. Use according to claim 2, characterized in that, E2) the nucleic acid molecule is any one of the following: G1 ) an RNA molecule having a nucleotide sequence of SEQ ID No. 6; G2) a nucleic acid molecule having 75% or more identity to the nucleotide sequence defined in G1 ) and inhibiting the expression of the PdeAPL protein-encoding gene.

5. Use according to any one of claims 1 to 4, characterized in that: the plant is any one of the following P1 )-P5): P1 ) a monocotyledonous plant or a dicotyledonous plant; P2) a Salicaceae plant; P3) a Salicaceae plant; P4) a Populus plant; P5) a Populus tree.

6. A method for breeding a transgenic plant having increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves, comprising the step of increasing the content and / or activity of a PdeAPL protein in a plant of interest, to obtain a transgenic plant having increased plant height and / or increased number of stem nodes and / or increased length of stem nodes and / or increased width of xylem and / or smaller xylem cells and / or smaller phloem cells and / or smaller leaves; the PdeAPL protein is a PdeAPL-1 protein and / or a PdeAPL-2 protein; the PdeAPL-1 protein is any one of the following B1 )-B4): B1 ) a protein having an amino acid sequence of SEQ ID No. 3; B2) a fusion protein having the same function as the amino acid sequence of B1) obtained by connecting a tag to the N terminus and / or C terminus of the amino acid sequence of B1); B3) a protein having the same function as the amino acid sequence of B1) obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence of B1); B4) a protein having 80% or more identity to the amino acid sequence of B1) and having the same function; the PdeAPL-2 protein is any one of the following C1) to C4): C1) a protein having an amino acid sequence of SEQ ID No. 4; C2) a fusion protein having the same function as the amino acid sequence of C1) obtained by connecting a tag to the N terminus and / or C terminus of the amino acid sequence of C1); C3) a protein having the same function as the amino acid sequence of C1) obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence of C1); C4) a protein having 80% or more identity to the amino acid sequence of C1) and having the same function.

7. The method of claim 6, wherein: The method for increasing the content and / or activity of the PdeAPL protein in the plant of interest is overexpression of the PdeAPL protein in the plant of interest.

8. The method of claim 7, wherein: The method for overexpression is introduction of the gene encoding the PdeAPL protein into the plant of interest.

9. The method of claim 8, wherein: The nucleotide sequence of the gene encoding the PdeAPL protein is SEQ ID No. 1 or SEQ ID No.

2.

10. The method according to any of claims 6-9, characterized by: The plant is any one of the following P1) to P5): P1) a monocotyledonous plant or a dicotyledonous plant; P2) a plant of the order Salicales; P3) a plant of the family Salicaceae; P4) a plant of the genus Populus; P5) a poplar.

11. A transgenic plant produced according to the method of any one of claims 6 to 10.

12. A method for breeding a transgenic plant having reduced plant height and / or reduced number of stem nodes and / or reduced length of stem nodes and / or reduced width of xylem and / or enlarged xylem cells and / or enlarged phloem cells and / or enlarged leaves, comprising the step of reducing the content and / or activity of the PdeAPL protein in the plant of interest to obtain a transgenic plant having reduced plant height and / or reduced number of stem nodes and / or reduced length of stem nodes and / or reduced width of xylem and / or enlarged xylem cells and / or enlarged phloem cells and / or enlarged leaves; the PdeAPL protein is a PdeAPL-1 protein and / or a PdeAPL-2 protein; the PdeAPL-1 protein is any one of the following B1) to B4): B1) a protein having an amino acid sequence of SEQ ID No. 3; B2) a fusion protein having the same function as the amino acid sequence of B1) obtained by connecting a tag to the N terminus and / or C terminus of the amino acid sequence of B1); B3) a protein having the same function as the amino acid sequence of B1) obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence of B1); B4) a protein having 80% or more identity to the amino acid sequence of B1) and having the same function; the PdeAPL-2 protein is any one of the following C1) to C4): C1) a protein having an amino acid sequence of SEQ ID No. 4; C2) a fusion protein having the same function as the amino acid sequence of C1) obtained by connecting a tag to the N terminus and / or C terminus of the amino acid sequence of C1); C3) a protein having the same function as the amino acid sequence of C1) obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence of C1); C4) a protein having 80% or more identity to the amino acid sequence of C1) and having the same function. C1) the amino acid sequence is the protein of SEQ ID No. 4; C2) a fusion protein having the same function as that of C1) obtained by attaching a tag to the N terminus and / or C terminus of the amino acid sequence of C1); C3) a protein having the same function as that of C1) obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence of C1); C4) a protein having 80% or more identity with the amino acid sequence of C1) and having the same function.

13. The method of claim 12, wherein: The method for reducing the content and / or activity of PdeAPL protein in a plant of interest is introducing into the plant of interest a substance that suppresses the expression of a gene encoding PdeAPL protein.

14. The method of claim 12 or 13, wherein: The substance that suppresses the expression of a gene encoding PdeAPL protein is an RNA molecule whose nucleotide sequence is SEQ ID No.

6.

15. The method of any of claims 12-14, wherein: The plant is any one of P1) to P5) below: P1) a monocotyledonous plant or a dicotyledonous plant; P2) a plant of the order Salicales; P3) a plant of the family Salicaceae; P4) a plant of the genus Populus; P5) a poplar.

16. A transgenic plant produced by the method according to any one of claims 12 to 15.

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