Method for promoting xylem development of poplar by improving expression of PagBEH3b gene in poplar

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for promoting xylem development of a poplar and / or increasing the plant height of the poplar and / or increasing the yield of the poplar by improving expression of a PagBEH3b gene in the poplar. The PagBEH3b gene is transferred into a poplar, and compared with a wild type, the transgenic poplar over-expressing the PagBEH3b has the advantage that the xylem is obviously increased, so that the key regulatory factor PagBEH3b of the brassinolide signal channel is a key regulatory factor for regulating the development of the xylem of the poplar. The invention provides a new choice for xylem development regulation and control means, and has important application value in the field of forest tree genetic engineering.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology by increasing the PagBEH3b Methods for promoting xylem development in poplars by gene expression. Background Art

[0002] With the rapid development of the economy, wood consumption continues to grow, and the demand for wood has increased dramatically. The issue of wood safety has become increasingly prominent. It is urgent to create new germplasms of fast-growing and high-yield plantations through modern biological breeding technology to alleviate the contradiction between wood supply and demand and ensure national wood safety. Molecular design breeding technology has developed rapidly, but it has very high requirements for the selection of target genes. Selecting genes that play a key role in the regulation of wood formation and whose mechanism of action is thoroughly studied will help improve the success rate of molecular design breeding of fast-growing and high-yield plantations.

[0003] The formation of wood mainly comes from the accumulation of secondary xylem year by year, and the development of secondary xylem is affected by the synergistic effects of multiple external environmental factors and endogenous hormones, among which brassinolide has an important influence on the development of secondary xylem. Studies have shown that brassinosteroids can promote the thickening of secondary xylem. BEH is a key factor in the brassinosteroid signal transduction pathway and regulates many downstream genes. Using the model woody plant poplar, the molecular mechanism of BEH regulating secondary xylem is studied, and the target gene regulating wood yield is locked, which is of great significance for the molecular design breeding of fast-growing and high-yield poplars. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to promote the development of poplar xylem and thus increase the yield of poplar.

[0005] In order to solve the above technical problems, the present invention provides a method for improving the quality of poplar trees. PagBEH3b A method for promoting the development of poplar xylem and / or increasing poplar plant height and / or increasing poplar yield by expressing a gene, the method comprising: PagBEH3b The gene is a gene encoding a PagBEH3b protein, and the PagBEH3b protein is a protein of A1, A2 or A3 as follows: A1, the amino acid sequence is the protein of SEQ ID No. 2 in the sequence list; A2, a protein having more than 80% identity with the protein shown in A1 and having similar functions obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2 in the sequence list; A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1 or A2.

[0006] In the above method, SEQ ID No. 2 in the sequence listing consists of 333 amino acid residues.

[0007] In the above method, identity 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 on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0008] In the above method, the above 80% identity can be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0009] In the above method, the PagBEH3b protein may be derived from poplar.

[0010] In the above method, the PagBEH3b The gene may specifically be a nucleic acid molecule whose coding sequence of the coding chain is SEQ ID No. 1 in the sequence list.

[0011] The above method comprises the following steps: PagBEH3b The invention relates to a step of introducing genes into a recipient poplar to obtain a poplar with accelerated xylem development and / or a poplar with increased plant height and / or a poplar with increased yield; the xylem development rate of the poplar with accelerated xylem development is higher than the xylem development rate of the recipient poplar; the plant height of the poplar with increased plant height is higher than the plant height of the recipient poplar; the yield of the poplar with increased yield is higher than the yield of the recipient poplar.

[0012] In the above method, wherein said PagBEH3b The gene can be modified as follows before being introduced into the recipient poplar to achieve better expression: 1) Modifying the gene sequence adjacent to the initiation methionine to allow efficient initiation of translation; for example, using sequences known to be effective in plants; 2) Connected to various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters; the choice of promoter will vary with the temporal and spatial requirements of expression, and also depends on the target species; for example, a tissue or organ-specific expression promoter, depending on the stage of development at which the receptor is required; although it has been demonstrated that many promoters derived from dicotyledons can function in monocotyledons and vice versa, ideally, dicotyledon promoters are selected for expression in dicotyledons, and monocotyledon promoters are selected for expression in monocotyledons; in one embodiment of the present invention, the promoter 35S is used to drive PagBEH3b Gene; 3) Connecting with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminator known to work in plants can be connected with the gene of the present invention; 4) Introducing enhancer sequences, such as intron sequences (e.g. from Adhl and bronze) and viral leader sequences (e.g. from TMV, MCMV and AMV).

[0013] Said PagBEH3b Genes can be introduced into plant cells using conventional biotechnology methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).

[0014] In the above method, the poplars with enhanced xylem development and / or increased plant height and / or increased yield can be transgenic poplars or poplars obtained through conventional breeding techniques such as hybridization.

[0015] The present invention also provides a protein, which is the PagBEH3b protein.

[0016] The present invention also provides biological materials related to the PagBEH3b protein, which also fall within the protection scope of the present invention.

[0017] The biological material related to the PagBEH3b protein provided by the present invention is any one of the following B1 to B5: B1, nucleic acid molecule encoding PagBEH3b protein; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4, a recombinant microorganism containing the nucleic acid molecule described in B1, or a recombinant microorganism containing the expression cassette described in B2, or a recombinant microorganism containing the recombinant vector described in B3; B5. A transgenic plant cell line containing the nucleic acid molecule described in B1, or a transgenic plant cell line containing the expression cassette described in B2, or a transgenic plant cell line containing the recombinant vector described in B3.

[0018] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0019] In the above biological material, the nucleic acid molecule in B1 is PagBEH3b The gene may specifically be a nucleic acid molecule whose coding sequence of the coding chain is SEQ ID No. 1 in the sequence list.

[0020] In the above biological material, the expression cassette described in B2 ( PagBEH3b Gene expression cassette) refers to a gene expression cassette capable of expressing the PagBEH3b The DNA of the gene may include not only PagBEH3b The promoter of gene transcription may also include the PagBEH3bThe terminator of gene transcription. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or a LAP promoter (both inducible by methyl jasmonate); a heat shock promoter (U.S. Pat. No. 5,187,267); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); a seed-specific promoter, such as millet seed-specific promoter pF128 (CN101063139B (China Patent No. 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example, Odell et al. (1991). 985 ) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0021] Existing plant expression vectors can be used to construct PagBEH3b Recombinant expression vector of gene expression cassette. The plant expression vector includes binary Agrobacterium vector and vector that can be used for plant microprojectile bombardment, etc. Such as pMDC32, pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos ), the non-translated region transcribed at the 3' end of plant genes (such as soybean storage protein gene) all have similar functions. When using the gene of the present invention 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 region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging and can be natural or synthetic. The translation initiation region can come 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 and encode enzymes or luminescent compounds that can produce color changes ( GUS genes, luciferase genes, etc.), antibiotic marker genes (such as those that confer resistance to kanamycin and related antibiotics nptII Gene that confers resistance to the herbicide phosphinothricin bar gene, the HPT gene that confers resistance to the antibiotic hygromycin, hph genes, and conferring resistance to methatrexate dhfr Genes, such as EPSPS genes that confer resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0022] In the above biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

[0023] In order to solve the above technical problems, the present invention also provides a plant agent that promotes the development of poplar xylem and / or increases poplar yield and / or increases poplar plant height, wherein the active ingredient of the plant agent is to promote or increase the PagBEH3b gene, a substance that increases the abundance of the PagBEH3b protein. The substance may contain the PagBEH3b protein and / or the biological material.

[0024] The active ingredients of the above-mentioned plant agents may also contain other biological components and / or non-biological components. Those skilled in the art can determine the other active ingredients of the above-mentioned plant agents based on the effects of promoting the development of poplar xylem and / or increasing poplar yield and / or increasing poplar plant height.

[0025] The present invention also protects the use of the method, the PagBEH3b protein, or the biological material in poplar breeding or poplar wood production.

[0026] The invention also protects the use of the plant reagent in the production of poplar wood.

[0027] The present invention uses silver gland poplar variety 84K as material, screens and identifies PagBEH3b The phenotypic identification of its overexpression plants showed that it could regulate the development of poplar xylem, indicating that PagBEH3b The gene is a key regulatory factor in regulating the development of poplar xylem, providing a new option for regulating xylem development and has important application value in the field of forest genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The plant expression vector pMDC32- PagBEH3b Schematic diagram of the structure.

[0029] Figure 2 The present invention overexpresses PagBEH3b Real-time quantitative detection of transcription levels in transgenic poplars. ** in the figure represents the significance analysis result of P<0.01.

[0030] Figure 3 The non-transgenic poplar 84K (WT) of the present invention and PagBEH3b Growth chart of transgenic poplars (OE5, OE11, OE17). In the figure, * represents the significance analysis result of P < 0.05, and ** represents the significance analysis result of P < 0.01.

[0031] Figure 4 The non-transgenic poplar 84K (CK) of the present invention and PagBEH3b Transverse sections of the stems of transgenic poplars (OE5, OE11, OE17). DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0033] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0035] The poplar variety 84K in the following examples is recorded in the non-patent document "KNAT2 / 6b, a class I KNOXgene, impedes xylem differentiation by regulating NAC domain transcription factors in poplar. Zhao et al. (2020) New Phytol. 225: 1531-1544.", which is available to the public from the applicant to repeat the experiments of the present invention.

[0036] Example 1 1. Cloning PagBEH3b Gene At 84K ( P. alba X P. glandulosa ) Populus argentatus was used as the material. Total RNA was extracted from one-month soil-cultured seedlings using the RNeasy Plant Mini kit and the RNase-free DNase I kit (Qiagen, Hilden, Germany). 1.0 μg of RNA was taken from each sample, and the first-strand cDNA was synthesized using the SuperScript III first-strand synthesis system (LifeTechnologies, Carlsbad, CA, USA). With reference to the published genome sequence of Populus trichocarpa, primers were designed using Primer 5 software (amplicon included the start codon and stop codon), and full-length gene amplification was performed (GATEWAY adapters were introduced into the primers); in, PagBEH3b The forward primer for ORF is PagBEH3b ORF-F, reverse primer is PagBEH3b ORF-R, primer sequences are as follows: PagBEH3bORF-F: 5'- GGGGACAACTTTGTACAAAAAAGTTGGAATGACGTCGGGGACGAGAAT -3' (as shown in SEQ ID No. 3 in the sequence list, wherein positions 1-28 are GATEWAY linkers, and positions 29-48 are identical to positions 1-20 of SEQ ID No. 1); PagBEH3b ORF-R: 5'-GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTACTATCTGGTACTAGAGTTTC -3' (as shown in SEQ ID No. 4 in the sequence listing, wherein positions 1-35 are GATEWAY linkers, wherein positions 36-55 are reverse complementary to the sequence of positions 983-1002 of SEQ ID No. 1).

[0037] The PCR reaction system was as follows: 12.5 μl of TaKaRa high-fidelity amplification enzyme mixture PrimeSTAR, 1 μl of forward primer (10 μM), 1 μl of reverse primer (10 μM), 1 μl of template (84K poplar cDNA), and sterile ddH2O to 25 μl. Reaction procedure: pre-denaturation 98°C, 5 min; 98°C, 30 s; 56°C, 30 s; 72°C, 1 min, 10 cycles; 98°C, 30 s; 60°C, 30 s; 72°C, 1 min, 25 cycles; 72°C 10 min.

[0038] The final obtained gene full-length cDNA sequence was 1002bp and was named PagBEH3b The gene sequence is shown as SEQ ID No. 1 in the sequence list, and the PagBEH3b protein sequence compiled and expressed by the gene is shown as SEQ ID No. 2 in the sequence list.

[0039] SEQ ID No.1 SEQ ID No.2 MTSGTRMPTWKERENNKRRERRRRAIAAKIYAGLRMYGSYKLPKHCDNNEVLKALCNEAGWTVEEDGTTYRKGCKPVERMDIGGSASASPCSSYHQSPCASYNPSPASSSFPSPVSSRYAANGNGNVDADANSLIPWLRNLSSGSSSASPKHPNHLFIHTGSISA PVTPPLSSPTARTPRTRNDWDDPAAGQSWMGQNYSFLPSSMPSSTPPSPGRQVLPDSGWLAGIQIPQSGPSSPTFSLVSRNPFGFKEEALSGAGSRMWTPGQSGTCSPAVPAGIDQTADVPMADSMAAEFAFGSNTAGLVKPWEGERIHEECVSDDLELTLGNSSTR two, PagBEH3b Construction of gene plant expression vector The entry vector PDNOR207 is recorded in the non-patent document "Research on the mechanism of PagKNAT2 / 6b gene regulating poplar growth and development [D]; Zhao Yanqiu; Chinese Academy of Forestry; 2020", which can be obtained by the public from the applicant to repeat the experiments of the present invention.

[0040] The plant expression vector pMDC32 is described in the non-patent document “KNAT2 / 6b, a class I KNOX gene, impedes xylem differentiation by regulating NAC domain transcription factors in poplar. Zhao et al. (2020) New Phytol. 225: 1531-1544.”, which can be obtained by the public from the applicant to repeat the experiments of the present invention.

[0041] Using GATEWAY technology, build PagBEH3b Gene overexpression vector: Use specific PCR primer pairs (composed of PagBEH3b ORF-F and PagBEH3b ORF-R), using 84K cDNA as template, was obtained by PCR amplification. PagBEH3b Gene ORF (as shown in SEQ ID No.1 in the sequence list), PagBEH3bThe gene ORF was constructed into the entry vector PDNOR207. The reaction system was 80 ng of PCR product; 0.4 μl of pDNOR207 vector; 0.6 μl of BP enzyme (Cat. No.: Invitrogen 11789020); and sterile ddH2O was added to 5 μl. The reaction procedure was: 25°C for 5 h.

[0042] Positive clones were selected from the screening culture plate for PCR detection and sequencing verification. PagBEH3b The entry vector of the gene was linearized by Mlu I restriction endonuclease and then constructed into the plant expression vector pMDC32 using the Gateway system.

[0043] The reaction system for LR reaction is: linearized and connected PagBEH3b pDNOR207 vector of the gene 50ng; pMDC32 vector 75ng; LR enzyme (Cat. No.: Invitrogen 11791020) 0.6μl; water to 5μl, reaction conditions: 25℃ reaction for 5h; after LR reaction, PagBEH3b The gene was introduced into the plant expression vector pMDC32 to obtain an overexpression vector (pMDC32- PagBEH3b ),exist PagBEH3b The 5' end of the gene is equipped with a strong expression promoter CaMV35S, which can PagBEH3b The gene is highly expressed in poplar; PagBEH3b The 3' end of the gene is equipped with a strong terminator NOS, which can effectively terminate PagBEH3b Transcription of genes.

[0044] like Figure 1 As shown, the plant expression vector pMDC32- PagBEH3b The structure of the vector is as follows: the strong terminator NOS, the hygromycin selection marker sequence, the kanamycin selection marker sequence, the replication region sequence of pBR322, and the 2×35S strong expression promoter are respectively arranged from the top left-handed direction; the hygromycin phosphotransferase HPT is assembled on the vector as a selection marker for transgenic poplars, and hygromycin can be used to select transgenic poplars; the LB and RB sequences are assembled on the vector to promote the assembly between them PagBEH3b The gene expression framework and the selection marker gene HPT were integrated into the poplar receptor chromosome. The overexpression vector was successfully constructed through PCR detection and sequencing verification, and was named pMDC32- PagBEH3b , this gene is located behind the promoter 35S, and driven by the promoter 35S, PagBEH3b The gene can be efficiently expressed in poplar.

[0045] three, PagBEH3b Genetic transformation By electroporation, the constructed pMDC32- PagBEH3b The overexpression vector was transferred into Agrobacterium GV3101 to obtain Agrobacterium GV3101-pMDC32- PagBEH3b mediated by Agrobacterium tumefaciens PagBEH3b The gene was transferred into poplar (silver gland poplar, 84K poplar, the same below), and the specific transformation steps are as follows: The hybrid poplar clone 84K tissue culture seedlings used for genetic transformation were cultured at a temperature of 23-25°C, a light intensity of 16 / 8h (day / night), and a light intensity of 50μM / m 2 / s.

[0046] Contains the expression vector pMDC32- PagBEH3b Agrobacterium GV3101-pMDC32- PagBEH3b 84K leaves were infected at OD600 = 0.6-0.8, and the infected leaves were cultured in adventitious bud induction medium (SIM, 6-benzylaminopurine (6-BA) and naphthylacetic acid (NAA) were added to MS (Murashige-Skoog) basic medium to obtain adventitious bud induction medium, so that the concentration of 6-BA in the adventitious bud induction medium was 0.5 mg / l, and the concentration of NAA was 0.05 mg / l) at a temperature of 23±2℃ for 3 days in the dark; the leaves after co-culture were transferred to SIM containing 3 mg / L hygromycin B and 200 mg / L Timentin, and the culture temperature was 23-25℃, the light intensity was 16 / 8 h (day / night), and the light intensity was 50μM / m 2 / s to induce and screen resistant adventitious buds; after about 30 days of induction culture, the resistant adventitious buds were transferred to a rooting medium (RIM, indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) were added to 1 / 2MS basic medium to make the IBA concentration in the rooting medium to be 0.05 mg / L and the NAA concentration to be 0.02 mg / L) containing 3 mg / L hygromycin B and 200 mg / L Timentin, until adventitious roots were induced; DNA from leaves of rooted plants was extracted for PCR verification.

[0047] Acquired resistance PagBEH3b Gene overexpression 84K poplar and wild-type plants, extract genomic DNA, use PCR to amplify the resistance gene on the expression vector, and if a clear band can be amplified, it is a transgenic plant; select the stem segments of the transgenic plants, use the wild-type stem segments as the control, extract total RNA and reverse transcribe it, PagBEH3b Gene quantitative analysis was used to determine the expression level of the target gene in transgenic plants. The quantitative primers were PagBEH3b-RT-F(ACCATCAGAGTCCATGTGCA) and PagBEH3b- RT- R(GTTTGGATGCTTGGGTGAGG).

[0048] like Figure 2 As shown, the wild type 84K poplar and the overexpression PagBEH3b Quantitative detection of transcription levels of transgenic poplars, where PagBEH3b The expression level of the gene in the overexpression transgenic plant OE5, the overexpression transgenic plant OE11, and the overexpression transgenic plant OE17 was significantly increased compared with the wild type 84K poplar. The overexpression transgenic plant OE5, the overexpression transgenic plant OE11, and the overexpression transgenic plant OE17 were asexually propagated by tissue culture to obtain the overexpression transgenic line OE5, the overexpression transgenic line OE11, and the overexpression transgenic line OE17.

[0049] Four, PagBEH3b Phenotypic observation of transgenic plants Will PagBEH3b Overexpression transgenic lines OE5, OE11, OE17 and wild-type 84K poplar were planted in a greenhouse in three batches, with 6 plants per line in each batch, and the phenotypes of plants in different batches were measured and photographed, e.g. Figure 3 The wild type 84K poplar and the overexpression PagBEH3b Comparison of plant height phenotypes of transgenic poplars. Figure 3 The phenotype photographs are shown two months after transplanting, and the overexpressing plants are taller compared to the non-transgenic 84K poplar plants.

[0050] five, PagBEH3b Observation on xylem sections of transgenic poplars 1. Take a 0.5 cm long stem segment 2 cm above the ground of a soil-grown poplar seedling, fix it in the slice slot of an oscillating slicer Leica VT1200S with LOCTITE 495 glue, cut it into cross-sections with a thickness of 50 μm, and store the slices in 70% alcohol.

[0051] 2. Histochemical staining and photographic observation and analysis: Fresh sections were stained with 0.05% TBO for 1 min, washed three times with water to remove floating color and excess staining solution, covered with coverslips, and the TBO-stained sections were observed and photographed using an Olympus BX51 ordinary optical microscope to analyze the morphological changes of the stem section cross section. The results are shown in Figure 4 As shown, the wild type 84K poplar and the overexpression PagBEH3b Observation of xylem tissue sections of transgenic poplars; compared with the 84K poplar control, PagBEH3bThe xylem area of ​​the overexpressing plants became wider. PagBEH3b The gene affects the development of secondary xylem, which is radial growth.

[0052] Phenotypic and microscopic morphological observations of the different transgenic strains obtained above revealed that PagBEH3b The height of the overexpression strain increased significantly, and the xylem became significantly wider. The above research results prove that PagBEH3b It plays a key regulatory role in the plant height and xylem development of poplars, and has important application value in forest molecular breeding and the selection of superior varieties.

[0053] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. By increasing the PgBEH3b A method for promoting the development of poplar xylem and / or increasing poplar plant height and / or increasing poplar yield through gene expression, characterized in that: Said PgBEH3b The gene is a gene encoding a PagBEH3b protein, and the PagBEH3b protein is a protein of A1, A2 or A3 as follows: A1, the amino acid sequence is the protein of SEQ ID No. 2 in the sequence list; A2, a protein having more than 80% identity with the protein shown in A1 and having similar functions obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2 in the sequence list; A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1 or A2.

2. The method according to claim 1, characterized in that: Said PgBEH3b The coding sequence of the gene as the coding strand is the DNA molecule of SEQ ID No. 1 in the sequence list.

3. The method according to claim 1 or 2, characterized in that: The method comprises the PgBEH3b The invention relates to a step of introducing genes into a recipient poplar to obtain a poplar with accelerated xylem development and / or a poplar with increased plant height and / or a poplar with increased yield; the xylem development rate of the poplar with accelerated xylem development is higher than the xylem development rate of the recipient poplar; the plant height of the poplar with increased plant height is higher than the plant height of the recipient poplar; the yield of the poplar with increased yield is higher than the yield of the recipient poplar.

4. The PagBEH3b protein of claim 1.

5. The PagBEH3b protein-related biomaterial according to claim 4, characterized in that: Any one of B1 to B5 below: B1, nucleic acid molecule encoding PagBEH3b protein; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4, a recombinant microorganism containing the nucleic acid molecule described in B1, or a recombinant microorganism containing the expression cassette described in B2, or a recombinant microorganism containing the recombinant vector described in B3; B5. A transgenic plant cell line containing the nucleic acid molecule described in B1, or a transgenic plant cell line containing the expression cassette described in B2, or a transgenic plant cell line containing the recombinant vector described in B3.

6. The biomaterial according to claim 5, characterized in that: The nucleic acid molecule B1 is a nucleic acid molecule whose coding sequence is the coding chain and is SEQ ID No. 1 in the sequence list.

7. A plant agent, characterized in that: The active ingredient of the reagent is to promote or enhance the PgBEH3b A gene, or a substance that increases the abundance of the PagBEH3b protein described in claim 1 or 2.

8. The plant agent according to claim 7, characterized in that: The substance comprises the PagBEH3b protein according to claim 4 and / or the biological material according to claim 5 or 6.

9. Use of the method according to any one of claims 1 to 3, or the PagBEH3b protein according to claim 4, or the biomaterial according to claim 5 or 6 in poplar breeding or poplar wood production.

10. Use of the plant agent according to claim 7 or 8 in poplar wood production.

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