PagFIP37 protein, gene encoding PagFIP37 protein and use thereof

Abstract

The present invention provides a PagFIP37 protein, a gene encoding the PagFIP37 protein, and applications thereof, belonging to the field of forestry molecular bioscience technology. The PagFIP37 protein is a homologous protein of AtFIP37 discovered from the 84K poplar species. High expression of the PagFIP37 protein in forest trees can promote tree growth, increase plant height, and enhance their ecological value. It has been verified that transgenic poplar plants that highly express the PagFIP37 protein have significantly more stem nodes than wild-type (WT) plants, and the fifth stem node is significantly wider than that of WT plants. The secondary xylem of transgenic plants that highly express the PagFIP37 protein is significantly wider than that of WT plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of forestry molecular bioscience, and specifically relates to a PagFIP37 protein, a gene encoding the PagFIP37 protein, and applications thereof. Background Art

[0002] Forests are an important renewable resource that people rely on for survival and economic development. They not only conserve water and soil and prevent pollution, but also serve as an important energy source.

[0003] The growth and development of trees requires a continuous flow of new cells, primarily derived from their meristems. The meristems at the apex and stem tips primarily promote the elongation of the tree's rhizomes. The thickening of roots, stems, and branches is achieved through the activity of the secondary meristem—the vascular cambium. The vascular cambium continuously proliferates and differentiates, forming secondary phloem outward and secondary xylem (or wood) inward. Wood is the secondary xylem of trees. Wood plays an important role in papermaking, furniture, and construction.

[0004] However, the long growth cycle and low wood density of trees limit their full utilization. Furthermore, due to the long growth cycle, high genetic heterozygosity, and difficulties in genetic analysis, it is difficult to modify the characteristics of trees to meet current development requirements using existing technology. Summary of the Invention

[0005] The purpose of the present invention is to provide a PagFIP37 protein, a gene encoding the PagFIP37 protein and its application. High expression of the PagFIP37 protein can promote the growth of trees.

[0006] The present invention provides a PagFIP37 protein, the amino acid sequence of which is selected from one or more of 1) to 3):

[0007] 1) the amino acid sequence shown in SEQ ID NO. 1;

[0008] 2) a sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO. 1;

[0009] 3) The amino acid sequence shown in SEQ ID NO. 1 includes an amino acid sequence in which one or more amino acid residues are substituted, deleted, and / or inserted;

[0010] The positions not identical in 1) and 2) are located in a fragment other than positions 131 to 285 of the amino acid sequence shown in SEQ ID NO. 1;

[0011] 3) wherein the substitution, deletion and / or insertion of one or more amino acid residues is located outside positions 131 to 285 of the amino acid sequence shown in SEQ ID NO.1.

[0012] The present invention also provides a PagFIP37 gene, which encodes the PagFIP37 protein described in the above scheme.

[0013] Preferably, the nucleotide sequence of the PagFIP37 gene is selected from one or more of (1) to (3):

[0014] (1) the nucleotide sequence shown in SEQ ID NO. 2;

[0015] (2) a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO. 2;

[0016] (3) The nucleotide sequence shown in SEQ ID NO. 2 includes a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0017] The present invention also provides a recombinant vector into which the PagFIP37 gene described in the above scheme is inserted.

[0018] The present invention also provides a recombinant cell comprising the PagFIP37 gene described in the above scheme or the recombinant vector described in the above scheme.

[0019] The present invention also provides the use of the PagFIP37 protein or the PagFIP37 gene or the recombinant vector or the recombinant cell described in the above scheme in promoting tree growth; the tree growth includes secondary growth and / or elongation growth.

[0020] Preferably, the secondary growth comprises secondary xylem growth.

[0021] Preferably, the trees include broadleaf trees.

[0022] The present invention also provides a method for breeding trees, in which the PagFIP37 gene described in the above scheme is overexpressed.

[0023] The present invention also provides a method for breeding trees, which comprises detecting the content of the PagFIP37 protein described in the above scheme in the trees or detecting the expression level of the PagFIP37 gene described in the above scheme in the trees, and selecting plants with a relatively high content of the PagFIP37 protein and / or a relatively high expression level of the PagFIP37 gene for breeding.

[0024] The present invention provides a PagFIP37 protein, a homologous protein of AtFIP37 discovered in the 84K poplar species. Overexpressing PagFIP37 in forest trees can promote tree growth, increase plant height, and enhance their ecological value. Transgenic poplar plants overexpressing PagFIP37 have been shown to have significantly more nodes than wild-type (WT) plants, with the fifth node being significantly wider than that of WT plants. The secondary xylem of transgenic plants overexpressing PagFIP37 is also significantly wider than that of WT plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 The positive clones were detected by PCR of PagFIP37 bacterial solution;

[0027] Figure 2 Comparison of PagFIP37 sequencing results and the original PagFIP37 sequence;

[0028] Figure 3 This is the positive monoclonal detection result of the PagFIP37 overexpression vector;

[0029] Figure 4 Obtaining 84K poplar plants overexpressing PagFIP37; A: Early differentiation of PagFIP37; B: Adventitious buds differentiated from PagFIP37; C: Resistance screening of transgenic PagFIP37 plants;

[0030] Figure 5 The expression analysis results of OE-PagFIP37 plants;

[0031] Figure 6 This is the growth status of OE-PagFIP37 plants for one month;

[0032] Figure 7 is the number of stem nodes of OE-PagFIP37 plants;

[0033] Figure 8 is the diameter of the fifth stem node of OE-PagFIP37 plants;

[0034] Figure 9 This is a semi-thin section from the 10th stem node of the OE-PagFIP37 plant; Xy: Xylem, the abbreviation of xylem;

[0035] Figure 10 The results of hydrophobicity and hydrophilicity analysis of the OE-PagFIP37 protein structure are shown;

[0036] Figure 11 This is the result of the transmembrane structure analysis of the OE-PagFIP37 protein;

[0037] Figure 12 This is the result of signal peptide analysis of OE-PagFIP37 protein. DETAILED DESCRIPTION

[0038] The present invention provides a PagFIP37 protein, the amino acid sequence of which is selected from one or more of 1) to 3):

[0039] 1) the amino acid sequence shown in SEQ ID NO. 1;

[0040] 2) a sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO. 1;

[0041] 3) The amino acid sequence shown in SEQ ID NO. 1 includes an amino acid sequence in which one or more amino acid residues are substituted, deleted, and / or inserted;

[0042] The positions not identical in 1) and 2) are located in a fragment other than positions 131 to 285 of the amino acid sequence shown in SEQ ID NO. 1;

[0043] 3) wherein the substitution, deletion and / or insertion of one or more amino acid residues is located outside positions 131 to 285 of the amino acid sequence shown in SEQ ID NO.1.

[0044] In the present invention, the PagFIP37 protein is a homologous protein of AtFIP37 found in 84K poplar species. AtFIP37 (FKBP12 Interacting Protein 37KD, FIP37) is an RNAm 6 A methyltransferase. The present invention discovered and isolated the homologous protein PagFIP37 of AtFIP37 protein from poplar for the first time, and its sequence structure is different from the homologous sequence structure from other species.

[0045] The present invention discovered for the first time that the PagFIP37 protein has the function of promoting secondary growth, so that the transgenic trees obtained according to the present invention have wider xylem, more wood can be obtained in their subsequent application process, and their economic value is improved.

[0046] In the present invention, SEQ ID The amino acid sequence shown in NO.1 is specifically: MASHNHLDVDDDDFGGDFPGSHNSRRSGNKRSFGDLEDDEDDIFSSKKGNSKVEETAMILSLRESLETCKSLATCQTELEAAKSEIQKWRSAFENESSIPAGASLEPKLVINYLQTLKSSEELLREQLEKAKKKEAAFIVTFAKREQEIAELKSAVRDLK AQLKPPSMQARRLLLDPAIHEEFTRLKNLVEEKDKKVKELQDNIAAMNFTPQSKMGKMLMAKCRTLQEENEEIGNQAAEGKIHELAMKLALQKSQNAELRSQFEGLYEHMEGLTNDVEKSNETVLLLQEKLEEKDQELKKLKLQLQQKTLVEEKTDPCPNKTVSADELKKEAEVN*.

[0047] In the present invention, the at least 90% in 2) includes any value between 90% and 100%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

[0048] In the present invention, the multiple amino acid residues in 3) are no more than 10 amino acid residues, for example, the multiple amino acid residues that are substituted, deleted and / or inserted are no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid residue.

[0049] In the present invention, the extracellular region of the amino acid sequence set forth in SEQ ID NO. 1 is a non-functional region. The extracellular region is a fragment excluding positions 46 to 297 of the amino acid sequence set forth in SEQ ID NO. 1, and more preferably a fragment excluding positions 131 to 285 of the amino acid sequence set forth in SEQ ID NO. 1. Identity changes or residue mutations (substitutions, deletions, and / or insertions) in the non-functional region do not affect the function of the protein.

[0050] In the present invention, the non-identical sites (i.e., sites with different amino acid residues) described in 2) or the substitutions, deletions, and / or insertions of one or more amino acid residues described in 3) are located in the extracellular region of the amino acid sequence described in SEQ ID NO. 1. The sequences outside the extracellular region in 2) and 3) are the same as in 1).

[0051] The present invention also provides a PagFIP37 gene that encodes the PagFIP37 protein described in the above scheme. When the PagFIP37 gene is introduced into trees and the PagFIP37 protein is highly expressed, the growth of the trees can be promoted, the height of the plants can be increased, and their ecological value can be enhanced.

[0052] In the present invention, the nucleotide sequence of the PagFIP37 gene is preferably selected from one or more of (1) to (3):

[0053] (1) the nucleotide sequence shown in SEQ ID NO. 2;

[0054] (2) a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO. 2;

[0055] (3) The nucleotide sequence shown in SEQ ID NO. 2 includes a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0056] In the present invention, the nucleotide sequence shown in SEQ ID NO.2 is specifically:

[0057]

[0058] In the present invention, based on the codon optimization strategy of different plants, codon optimization can be performed on the basis of the nucleotide sequence shown in SEQ ID NO. 2, so that the nucleotide sequence is suitable for application in different plants.

[0059] In the present invention, the at least 90% in (2) preferably includes any value between 90% and 100%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

[0060] In the present invention, the multiple nucleotides in (3) are preferably no more than 30 nucleotides, and can be any value from 1 to 30, for example, the multiple nucleotides substituted, deleted and / or inserted are no more than 30, 27, 24, 21, 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide.

[0061] In the present invention, the change in identity described in (2) or the base mutation such as substitution, deletion and / or insertion described in (3) does not result in a change in the function of the protein encoded by the gene. In the present invention, the site of non-identity described in (2) corresponds to the site of non-identity described in the above scheme 2); the site of base mutation in (3) corresponds to the site of substitution, deletion and / or insertion of one or more amino acid residues in the above scheme 3).

[0062] The present invention also provides a recombinant vector into which the PagFIP37 gene described in the above scheme is inserted. In the present invention, the original vector of the recombinant vector preferably includes a plasmid, a viral vector or a bacteriophage, such as a 35S plasmid.

[0063] The present invention also provides a recombinant cell comprising the PagFIP37 gene described in the above scheme or the recombinant vector described in the above scheme.

[0064] The recombinant cells of the present invention are exogenously introduced with the PagFIP37 gene described in the above-mentioned scheme; the recombinant cells highly express the PagFIP37 protein. In the present invention, the recombinant cells include host cells or targeted plant cells; the host cells are preferably prokaryotic, eukaryotic, or fungal host cells; the prokaryotic host cells are preferably Escherichia coli or Agrobacterium; the eukaryotic host cells are preferably yeast; and the tree-targeted cells are preferably one or more of the cells from the roots, stems, trunks, leaves, flowers, and callus tissues of trees.

[0065] The present invention also provides the use of the PagFIP37 protein or the PagFIP37 gene or the recombinant vector or the recombinant cell described in the above scheme in promoting tree growth; the tree growth includes secondary growth and / or elongation growth. In the present invention, the secondary growth preferably includes secondary xylem growth. In the present invention, the promotion of secondary growth is manifested in that the transgenic trees after the introduction of the PagFIP37 gene have wider secondary xylem relative to wild-type trees without the introduction of the PagFIP37 gene. In the present invention, the promotion of tree elongation growth is manifested in that the transgenic trees after the introduction of the PagFIP37 gene have increased plant height relative to wild-type trees without the introduction of the PagFIP37 gene.

[0066] In the present invention, the trees preferably include broad-leaved trees; the broad-leaved trees preferably include deciduous broad-leaved trees or evergreen broad-leaved trees, more preferably broad-leaved trees of the Salicaceae family; the broad-leaved trees of the Salicaceae family are preferably trees of the genus Salix, Populus or Salix in the Salicaceae family; the trees of the genus Populus preferably include poplars of the Tacamahaca, Leuce, Aigeiros, Turanga or Leucoides families, and further preferably, the trees are Populus tomentosa, Populus alba, Populus montana, Populus hebeiensis, Populus rapa, Populus tomentosa, Triploid Populus tomentosa or 84k Populus in the Populus family.

[0067] The present invention also provides a method for breeding trees, in which the PagFIP37 gene described in the above scheme is overexpressed.

[0068] In the present invention, overexpressing the PagFIP37 gene described in the above scheme in the tree comprises introducing the PagFIP37 gene into the tree. In the present invention, the introduction of the PagFIP37 gene into the tree is preferably carried out by the following method:

[0069] S1: Obtain the coding region sequence of the PagFIP37 gene;

[0070] S2: The coding region sequence of the PagFIP37 gene was connected to the expression vector by double enzyme digestion to construct a PagFIP37 overexpression vector;

[0071] S3: introducing the PagFIP37 overexpression vector into trees to obtain overexpression transgenic plants;

[0072] S4 extracts the genome of the overexpressed transgenic plant and identifies the positive transgenic plant;

[0073] The present invention first obtains the coding region sequence of the PagFIP37 gene. The present invention does not particularly limit the method for obtaining the coding region sequence of the PagFIP37 gene; conventional methods in the art can be used. In a specific implementation of the present invention, RNA is extracted from 84K poplar leaves using PCR, reverse transcribed, and the resulting cDNA is used as a template for amplification to obtain the coding region sequence of the PagFIP37 gene.

[0074] After obtaining the coding region sequence of the PagFIP37 gene, the present invention connects the coding region sequence of the PagFIP37 gene to an expression vector by double enzyme digestion to construct a PagFIP37 overexpression vector. In the present invention, the PagFIP37 overexpression vector is preferably a 35S::PagFIP37 overexpression vector.

[0075] After constructing the PagFIP37 overexpression vector, the present invention introduces the PagFIP37 overexpression vector into trees to obtain overexpressing transgenic plants. In the present invention, the method for introducing the PagFIP37 overexpression vector into trees is preferably Agrobacterium-mediated genetic transformation.

[0076] After obtaining the overexpressed transgenic plants, the present invention extracts the genome of the overexpressed transgenic plants and identifies the positive transgenic plants.

[0077] In the present invention, the trees fall within the range of trees defined in the above embodiment, and are preferably trees of the poplar family.

[0078] The trees bred by the breeding method of the present invention have wider secondary xylem and / or increased plant height compared to wild-type trees.

[0079] The present invention also provides a method for breeding trees, which comprises detecting the content of the PagFIP37 protein described in the above scheme in the trees or detecting the expression level of the PagFIP37 gene described in the above scheme in the trees, and selecting plants with a relatively high content of the PagFIP37 protein and / or a relatively high expression level of the PagFIP37 gene for breeding.

[0080] The present invention has no particular limitation on the method for detecting the content of the PagFIP37 protein described in the above scheme in trees or the expression level of the PagFIP37 gene described in the above scheme in trees, and conventional methods in the art can be used.

[0081] To further illustrate the present invention, the PagFIP37 protein, the gene encoding the PagFIP37 protein, and its application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0082] Example 1: Cloning of the coding region sequence of the poplar PagFIP37 gene

[0083] The AtFIP37 gene sequence was downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and the full genome sequence of Populus 84K was downloaded from Figshare (https: / / figshare.com / ). The PtrFIP37 gene sequence in Populus 84K was obtained by blast analysis. Based on the base sequence of the coding region, primers for PagFIP37 amplification were designed and amplified from Populus 84K. The PagFIP37 gene was amplified. The gene sequence is shown in SEQ ID NO. 2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 1.

[0084] The present invention utilizes the PCR method to reverse transcribe the extracted 84K poplar leaf RNA, and then uses the obtained cDNA as a template to amplify the coding region sequence of the PagFIP37 gene. The specific method is as follows:

[0085] (1) Total RNA extraction from 84K poplar leaves

[0086] Cut the leaves of 84K tissue culture seedlings and grind them under liquid nitrogen for later use. Specific steps are described in the instructions for the RNeasy Plant mini Kit (Beijing Tiangen Biochemical Technology Co., Ltd.).

[0087] (2) cDNA synthesis

[0088] 1) Prepare the reverse transcription mixture according to the following system and place it in a 200 μL RNase-free PCR tube; the system is as follows:

[0089] Table 1: Reverse transcription system

[0090]

[0091] 2) Mix thoroughly and incubate at 42°C for 30 min;

[0092] 3) Heat at 85°C for 5 seconds to inactivate TransScript RT / RI and gDNA Remover;

[0093] 4) The obtained cDNA was stored at -20°C. All reagents used in the reaction were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0094] (3) Amplification of the target gene PagFIP37

[0095] 1) Based on the 84K poplar PagFIP37 gene sequence obtained by blast, primers for amplification of the PagFIP37 coding region were analyzed and designed:

[0096] PagFIP37-F1:ATGGCATCGCACAACCATC (SEQ ID NO.3);

[0097] PagFIP37-R1: CTAGTTGACCTCAGCTTCCTTCTT (SEQ ID NO. 4).

[0098] 2) Using the 84K poplar cDNA sequence as a template, the 84K poplar PagFIP37 cDNA sequence was amplified using the following PCR reaction system; the following reagents and amounts were referred to the instructions for the TranStart FstPfu Fy DNA polymerase kit from Quanshijin Company, as shown in the table below:

[0099] Table 2: PCR amplification system

[0100]

[0101] 3) The PCR reaction procedure is as follows:

[0102] Pre-denaturation: 98°C for 2 min; (denaturation: 98°C for 30 s, annealing: 58°C for 30 s, extension: 72°C for 30 s) × 36 cycles; extension: 72°C for 5 min; incubation at 4°C. The amplified PagFIP37 gene sequence is shown in SEQ ID NO. 2.

[0103] 4) Agarose gel electrophoresis detection

[0104] Weigh 0.5g agarose, add 50mL 1×TAE, heat in a microwave oven, add 5μL GoldView (purchased from Zhongke Ruitai Biotechnology Co., Ltd.) and shake thoroughly, then pour into the gel plate. After the agarose gel solidifies, add the mixture of PCR product and Loading buffer (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to the gel well for electrophoresis detection and recover the cDNA fragment. Connect the target fragment to the T vector, then transform, and apply ampicillin resistance plate to screen positive monoclones. Identify the recombinant plasmid by monoclonal bacterial liquid PCR. A total of 20 monoclonal bacteria were picked for bacterial liquid PCR. The agarose gel electrophoresis results showed that there were 12 lanes with bands consistent with the size of the target fragment, about 1000bp ( Figure 1 ). Six positive monoclonal bacterial cultures were selected from the monoclonal cultures and sent to Beijing Ruibo Sequencing Co., Ltd. for sequencing.

[0105] 5) Sequence alignment

[0106] The sequencing results of PagFIP37 were compared with the original sequence obtained by blasting using DNAMAN software. It was found that the base similarity between the sequencing results and the original sequence was 99.60%, the number of bases was the same, and the size was 1011 bp ( Figure 2 ).

[0107] Example 2: Construction of PagFIP37 gene overexpression vector

[0108] 1) Analyze the PagFIP37 gene using Primer Premier 5 and design primers with Kpn I and Xba I restriction sites (restriction sites in bold italics):

[0109] PagFIP37-F2:

[0110] PagFIP37-R2:

[0111] 2) Amplify PagFIP37 with restriction enzyme cleavage sites and recover the PCR product (the method is as described in Example 1).

[0112] 3) Construction of pCAMBIA2300-35S::PagFIP37: The PagFIP37 product with restriction enzyme cleavage sites and the expression vector pCAMBIA2300 were double-digested with the endonucleases Kpn I and Xba I. (The endonucleases Kpn I and Xba I were purchased from Bio-Rad Biotechnology Co., Ltd., Beijing. For specific procedures and dosages, refer to the instructions.) The digestion products were recovered and ligated with T4-DNA ligase (purchased from Bio-Rad Biotechnology Co., Ltd., Beijing. For specific procedures and dosages, refer to the instructions).

[0113] 4) The ligation product was transformed into competent E. coli TOP 10 (purchased from Beijing Adlai Biotechnology Co., Ltd.; refer to the instructions for specific operation methods and dosages). Then, the competent E. coli cells were transformed, spread on LB solid plates containing Kan antibiotics, and cultured in a 37°C incubator.

[0114] 5) Pick a single colony from the solid plate and place it in LB medium (containing antibiotics) and culture at 37°C for 4-5 hours. Use the bacterial solution as a template for PCR amplification and 1% agarose gel electrophoresis for detection ( Figure 3Positive clones were selected and sent to Beijing Ruibo Biotechnology for sequencing. Sequencing results were aligned with the original gene sequence using DNAMAN software. Positive clones with correct sequencing results were amplified, supplemented with 60% glycerol, and frozen at -80°C. The strain was preserved and named 35S::PagFIP37.

[0115] Example 3: Poplar genetic transformation

[0116] The overexpression vector 35S::PagFIP37 was introduced into poplar leaves through Agrobacterium-mediated genetic transformation, and after pre-culture, infection, dark culture, induction of adventitious buds, induction of bud rooting, propagation, and hardening, the leaves were transplanted into a greenhouse.

[0117] 1) Pretreatment of wild-type 84K poplar leaves:

[0118] Before using the clean bench, wipe the table top with 75% alcohol in advance, put the scissors, tweezers and scalpel in the high-temperature sterilizer, turn on the high-temperature sterilizer, and finally turn on the ultraviolet light for sterilization for 25 to 30 minutes. Then operate in the sterile clean bench, cut the leaves of the sterile seedlings in good growth condition, generally choose the leaves in the 3rd to 6th leaf order position from the bottom to the top of the plant, use a scalpel to make evenly spaced wounds on the back of the leaves in the direction perpendicular to the main vein, and spread the leaves with the back facing up on WPM co-cultivation medium (containing acetosyringone) and culture in the light for 1 to 2 days.

[0119] 2) Preparation of Agrobacterium infection solution:

[0120] Pipette 80 μL of the frozen positive clone pCAMBIA2300-35S::PagFIP37 Agrobacterium into 80 mL of LB liquid containing Kan antibiotics, mix well, and place in a 28°C constant temperature shaker at 200 rpm overnight. Use a 50 mL sterile centrifuge tube to collect the bacteria by centrifugation at 5000 rpm for 10 minutes at room temperature. Finally, resuspend the bacteria in 1 / 2 MS resuspension buffer and dilute to OD 600 The concentration of 0.8 was used to infect the pretreated leaves.

[0121] 3) Infection process:

[0122] The infection process needs to be carried out in a sterile clean bench. Soak the pre-treated cultured leaves in the prepared Agrobacterium

[0123] Resuspended bacterial solution (OD 600 The leaf is immersed in a 0.8% agarose gel for 10-13 minutes, shaking the leaf several times periodically to ensure adequate contact between the wound and the bacterial solution. Remove the infected leaf and place it on sterile filter paper to remove excess Agrobacterium from the leaf surface. Finally, lay the leaf flat, dorsal surface facing up, on a dark-treated medium without antibiotics.

[0124] 4) Co-cultivation:

[0125] Place the infected leaves on the dark-treated culture medium in a dark box for dark culture for about 3 days until a small amount of Agrobacterium bacteria can be seen around the leaves with the naked eye, then the differentiation stage can begin.

[0126] (5) Screening and culture:

[0127] In a cleanroom, use sterile filter paper to remove excess bacterial flora from the dark-cultured leaves. Transfer the leaves to a differentiation medium containing Kan antibiotics and place them in a culture room at approximately 25°C for resistance differentiation screening. Change the differentiation medium approximately every 20 days until adventitious buds differentiate.

[0128] (6) Subculture resistance screening:

[0129] In a clean bench, use a sterile scalpel to cut off the differentiated adventitious buds, place them on new differentiation medium and continue to culture for about 3 weeks to continue inducing differentiation.

[0130] (7) Rooting culture:

[0131] When the adventitious buds grow to about 1.5 cm, use a sterile scalpel to separate the adventitious buds into single buds in a clean bench, insert them into a rooting screening medium containing Kan antibiotics, and culture for about 7 to 10 days for the base of the adventitious buds to take root. After rooting, first cut the leaves of the sterile rooted plants to extract the genome test to verify whether it is a positive plant. Use a sterile scalpel to cut the top buds and stem segments of the transgenic plants identified as positive for the first propagation and subculture (the subculture medium needs to contain Kan antibiotics) ( Figure 4 ).

[0132] Example 4: Identification of transgenic poplars and observation of growth rate and secondary xylem

[0133] 1) After the PagFIP37 overexpression-resistant plants were grown in culture for approximately one month, RNA levels were detected in the overexpression plants. A highly specific sequence of approximately 150 to 200 bp was selected, and qRT-PCR primers for PagFIP37 were designed online through the NCBI website. The expression level of the PagFIP37 gene was detected by fluorescence quantitative PCR ( Figure 5 ), the results showed that the expression level of the OE-PagFIP37 gene in overexpressed plants was 28 times that of the wild type.

[0134] The primers were designed as follows:

[0135] OE-PagFIP37-F: CTCAACTCAAGCCACCAT, (SEQ ID NO. 7);

[0136] OE-PagFIP37-R:TGTCCTACACTTTGCCATC, (SEQ ID NO. 8).

[0137] 2) Testing the growth rate of transgenic poplars: Cut the first two internodes and about 1-2 cm including the stem tip of the identified transgenic and wild-type poplar tissue culture seedlings and grow them in rooting medium without antibiotics for 5-8 weeks. After hardening, transplant the tissue culture seedlings to the greenhouse for growth. After one month of growth, take pictures. Figure 6 shown.

[0138] Under the same culture conditions, the internode numbers and the diameter of the fifth stem node of WT and OE-PagFIP37 plants grown in the greenhouse for 3 months were counted. The results of prism statistics showed that the number of stem nodes of the overexpression plants was significantly more than that of the WT ( Figure 7 ), the fifth stem node of the overexpressing plants was significantly wider than that of the WT plants ( Figure 8 ).

[0139] 3) Observation of xylem in transgenic plants

[0140] In order to observe the effects of PagFIP37 at the cytological level, one-month-old tissue culture seedlings were transferred to nutrient soil. After growing in the greenhouse for one month, the 10th stem nodes of the transgenic plants and WT plants were selected and fixed in fixative for three days. They were then dehydrated, infiltrated, embedded, and finally cut into pieces and sliced using a semi-thin microtome. The slices were placed on water droplets on a glass slide with water droplets, and then the glass slides were placed on a slide baker and baked at 60°C. When the water droplets on the slides were dried, 1% (v / v) methylamine blue dye was added to the slices attached to the slides and stained for 20 to 30 seconds; excess dye was washed off with ddH2O, and the slices were placed on a slide baker until they were completely dry; 1 drop of xylene transparent solution and a mixture of equal volumes of neutral gum were added, and the coverslips were covered and sealed; the slices were observed and photographed under an optical microscope. The results are shown in the figure below. Figure 9 As shown, the secondary xylem of transgenic plants was significantly wider than that of wild-type plants.

[0141] Example 5: Structural and functional analysis of OE-PagFIP37 protein

[0142] Bioinformatics analysis of the amino acid sequence of PagFIP37 was performed using DNAMAN software, and it was found that the CDS sequence of PagFIP37 encodes 336 amino acids, with a molecular mass of approximately 38.065kDa and an isoelectric potential of 5.22. In order to further study the biological function of the PagFIP37 protein, the amino acid sequence of PagFIP37 was analyzed for protein transmembrane structure prediction, hydrophobicity, hydrophilicity, and signal peptide. The analysis results showed that the lowest hydrophobicity score of PagFIP37 was -2.933, which was the serine at position 27, and the highest score was 1.722, which was the isoleucine at position 140. The total hydrophobicity score was -0.815, and the values of most amino acids were negative, so it was speculated that PagFIP37 was a hydrophilic protein ( Figure 10 DNAMAN software was used to analyze the transmembrane domain, and the results showed that PagFIP37 did not have a transmembrane domain ( Figure 11 ). Predicting protein signal peptides can provide a reference for determining functional domains and subcellular localization. Through software prediction, it was found that PagFIP37 does not have a signal peptide ( Figure 12 ).

[0143] This patent is supported by the Basic Research Business Expenses of the Central Universities (2021ZY57), the National Natural Science Foundation of China (31970182), and the "College Student Innovation and Entrepreneurship Training Program" of Beijing Forestry University (202110022069).

[0144] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. PagFIP37 protein, PagFIP37 Application of genes, recombinant vectors or recombinant cells in promoting the growth of poplars; the growth of the poplars is secondary growth; The amino acid sequence of the PagFIP37 protein is shown in SEQ ID NO.1; described PagFIP37 The nucleotide sequence of the gene is shown in SEQ ID NO. 2; The recombinant vector is inserted with the PagFIP37 Gene; The recombinant cell comprises PagFIP37 gene or the recombinant vector.

2. The use according to claim 1, characterized in that The secondary growth includes secondary xylem growth.

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