CN115927233A - PagFIP37 protein, coding gene of PagFIP37 protein and application thereof - Google Patents

Abstract

The invention provides a PagFIP37 protein, a coding gene of the PagFIP37 protein and application thereof, belonging to the technical field of molecular biological science of forestry; the PagFIP37 protein is a homologous protein to AtFIP37 found in 84K poplar species. The PagFIP37 protein is highly expressed in forest trees, so that the growth of the trees can be promoted, the plants can be increased, and the ecological value of the trees can be increased. Through verification, the stem node number of a transgenic poplar plant with high expression of the PagFIP37 protein is obviously more than that of a Wild Type (WT) plant, and the 5 th stem node is obviously more than that of the WT plant; the secondary xylem of the transgenic plant with high expression of the PagFIP37 protein is obviously wider than that of the WT plant.

Description

PagFIP37 protein, coding gene of PagFIP37 protein and application of coding gene

Technical Field

The invention belongs to the technical field of forestry molecular bioscience, and particularly relates to a PagFIP37 protein, a coding gene of the PagFIP37 protein and application of the coding gene.

Background

Forest trees are important renewable resources which people rely on to live and promote economic development. The forest trees not only can maintain water and soil and prevent pollution, but also are important energy substances.

The growth and development of forest trees require the continuous generation of new cells which are mainly derived from the meristematic tissues of forest trees. The meristems of the tip and shoot tip primarily promote the elongation of the wood rootstock. 3242 the thickening of the stems and branches of the Lin Mugen is achieved by the action of the secondary meristem, the vascular cambium. The vascular formation layer is continuously proliferated and differentiated to be outwardly differentiated into a secondary phloem and inwardly differentiated into a secondary xylem, namely wood. Wood is the secondary xylem of forest trees. The wood plays an important role in papermaking, furniture, buildings and the like.

However, the trees have long growth cycles and low wood density, which limits the full utilization of the trees. In addition, due to the difficulties of long growth period, high genetic heterozygosity, genetic analysis and the like, the properties of the forest trees are difficult to change to adapt to the current development requirements in the prior art.

Disclosure of Invention

The invention aims to provide a PagFIP37 protein, a coding gene of the PagFIP37 protein and application thereof, wherein the high-expression PagFIP37 protein can promote the growth of forest trees.

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

1) An amino acid sequence shown as SEQ ID NO. 1;

2) A sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No. 1;

3) The amino acid sequence shown in SEQ ID NO.1 comprises an amino acid sequence with one or more amino acid residues substituted, deleted and/or inserted;

1) And 2) a fragment in which the site having no identity in SEQ ID NO.1 is located other than at positions 131 to 285 of the amino acid sequence shown in SEQ ID NO. 1;

3) The fragment in (1) in which one or more amino acid residues are located outside positions 131 to 285 of the amino acid sequence shown in SEQ ID NO. 1.

The invention also provides a PagFIP37 gene, and the PagFIP37 gene codes the PagFIP37 protein in the scheme.

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

(1) A nucleotide sequence shown as SEQ ID NO. 2;

(2) A nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID No. 2;

(3) The nucleotide sequence shown in SEQ ID NO.2 comprises a nucleotide sequence with one or more nucleotides substituted, deleted and/or inserted.

The invention also provides a recombinant vector, into which the PagFIP37 gene described in the above scheme is inserted.

The invention also provides a recombinant cell which comprises the PagFIP37 gene or the recombinant vector.

The invention also provides the application of the PagFIP37 protein or the PagFIP37 gene or the recombinant vector or the recombinant cell in promoting the growth of trees; the tree growth includes secondary growth and/or elongation growth.

Preferably, the secondary growth comprises secondary xylem growth.

Preferably, the trees comprise broadleaf trees.

The invention also provides a breeding method of trees, and the PagFIP37 gene in the scheme is over-expressed in the trees.

The invention also provides a tree breeding method, which is used for detecting the content of the PagFIP37 protein in the tree or detecting the expression quantity of the PagFIP37 gene in the tree in the scheme, and selecting plants with high PagFIP37 protein content and/or high PagFIP37 gene expression quantity for breeding.

The invention provides a PagFIP37 protein, wherein the PagFIP37 protein is a homologous protein of AtFIP37 found in 84K poplar species. The PagFIP37 protein is highly expressed in forest trees, so that the growth of the trees can be promoted, the plants can be increased, and the ecological value of the trees can be increased. Through verification, the stem node number of a transgenic poplar plant with high expression of the PagFIP37 protein is obviously more than that of a Wild Type (WT) plant, and the 5 th stem node is obviously more than that of the WT plant; the secondary xylem of the transgenic plant with high expression of the PagFIP37 protein is obviously wider than that of the WT plant.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 shows positive clones detected by PCR of PagFIP37 bacterial liquid;

FIG. 2PagFIP37 sequencing results and PagFIP37 original sequence alignment results;

FIG. 3 shows the positive monoclonal detection results of the PagFIP37 overexpression vector;

FIG. 4 is a depiction of the overexpression of 84K Yang Zhizhu by PagFIP 37; wherein A is the PagFIP37 differentiation initial stage B: differentiating adventitious buds C by PagFIP37, and screening the resistance of PagFIP37 transgenic plants;

FIG. 5 shows the results of analysis of OE-PagFIP37 plant expression levels;

FIG. 6 shows the one month growth status of OE-PagFIP37 plants;

FIG. 7 shows the number of shoot nodes of OE-PagFIP37 plants;

FIG. 8 shows the diameter of the 5 th stem node of OE-PagFIP37 plants;

FIG. 9 is a 10 th stem node half-thin section of an OE-PagFIP37 plant; and Xy: xylem, english abbreviation for Xylem;

FIG. 10 shows the results of the structural hydrophobicity and hydrophilicity analyses of OE-PagFIP37 protein;

FIG. 11 shows the results of analysis of the transmembrane structure of OE-PagFIP37 protein;

FIG. 12 shows the signal peptide analysis results of OE-PagFIP37 protein.

Detailed Description

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

1) An amino acid sequence shown as SEQ ID NO. 1;

2) A sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No. 1;

3) The amino acid sequence shown in SEQ ID NO.1 comprises an amino acid sequence with one or more amino acid residues substituted, deleted and/or inserted;

1) And 2) a fragment in which the site having no identity is located outside positions 131 to 285 of the amino acid sequence represented by SEQ ID NO. 1;

3) The fragment in (1) in which one or more amino acid residues are located outside positions 131 to 285 of the amino acid sequence shown in SEQ ID NO. 1.

In the present invention, the PagFIP37 Protein is a homologous Protein of AtFIP37 found in 84K poplar species, and AtFIP37 (FKBP 12 Interacting Protein 37KD, FIP 37) is an RNAm in Arabidopsis thaliana ⁶ A methyltransferase. The invention discovers and separates the homologous protein PagFIP37 of the AtFIP37 protein from the poplar for the first time, and the sequence structure of the homologous protein PagFIP37 is different from the homologous sequence structure of other species sources.

The invention discovers that the PagFIP37 protein has the function of promoting secondary growth for the first time, so that the transgenic tree obtained according to the invention has wider xylem, more wood can be obtained in the subsequent application process, and the economic value of the transgenic tree is improved.

In the invention, the amino acid sequence shown in SEQ ID NO.1 is specifically: MASHNHLDVDDDDFGGDFPGSHNSRRSGNKRSFGDLEDDEDDIFSSKKGNSKVEETAMILSLRESLETCKSSLATCQTELEAAKSEIQKWRSAFENESSIPAGASLEPKLVINYLQTLKSSEELLREQLEKAKKKEAAFIVTFAKREQEIAELKSAVRDLKAQLKPPSMQARRLLLDPAIHEEFTRLKNLVEEKDKKVKELQDNIAAMNFTPQSKMGKMLMAKCRTLQEENEEIGNQAAEGKIHELAMKLALQKSQNAELRSQFEGLYEHMEGLTNDVEKSNETVLLLQEKLEEKDQELKKLKLQLQQKTLVEEKTDPCPNKTVSADELKKEAEVN.

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%.

In the present invention, the plurality of amino acid residues in 3) is not more than 10 amino acid residues, for example, the plurality of amino acid residues substituted, deleted and/or inserted is not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or not more than 1 amino acid residue.

In the present invention, the extracellular region of the amino acid sequence shown in SEQ ID NO.1 is a non-functional region, and the extracellular region is a fragment other than positions 46 to 297 of the amino acid sequence shown in SEQ ID NO.1, and more preferably, a fragment other than positions 131 to 285 of the amino acid sequence shown in SEQ ID NO. 1. Changes in identity or residue mutations (substitutions, deletions and/or insertions) in non-functional regions do not affect the function of the protein.

In the present invention, the site having no identity described in 2) (i.e., the site differing in amino acid residue) or the substitution, deletion and/or insertion of one or more amino acid residues in the scheme 3) is/are located in the extracellular region of the amino acid sequence described in SEQ ID NO. 1. 2) And 3) the sequence outside the extracellular region is the same as 1).

The invention also provides a PagFIP37 gene, and the PagFIP37 gene codes the PagFIP37 protein in the scheme. The PagFIP37 gene is introduced into the tree, and after the PagFIP37 protein is highly expressed, the growth of the tree can be promoted, the plant height is increased, and the ecological value is increased.

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

(1) A nucleotide sequence shown as SEQ ID NO. 2;

(2) A nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID No. 2;

(3) The nucleotide sequence shown in SEQ ID NO.2 comprises a nucleotide sequence with one or more nucleotides substituted, deleted and/or inserted.

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

atggcatcgcacaaccatctcgacgtcgatgatgatgattttggcggtgattttcctgggagccacaatagcagacgttctggcaacaagagaagctttggagatctcgaggatgacgaagatgatatttttagctccaaaaagggtaattccaaggtagaagaaactgcaatgattttgtcgcttcgtgagagtcttgagacttgtaaaagttcactcgcaacatgccagacagagcttgaagctgcaaaatctgaaattcagaagtggcgttctgcatttgagaatgagtcctccatacctgctggggcatctcttgaacctaaactagtgatcaactatcttcagaccctgaaatcttctgaggagttattgagagagcagttagaaaaggctaagaagaaggaagctgcctttattgtaacttttgcaaaacgggagcaggagatagcagagctaaagtctgcagtccgggatctgaaagctcaactcaagccaccatcaatgcaggcaaggaggttactactggatccagcaattcatgaggaatttacacgtttgaagaatttggttgaggagaaggacaagaaggtgaaggaattgcaggataatattgctgctatgaattttactccacaaagcaagatggggaagatgctgatggcaaagtgtaggacactgcaagaggaaaatgaggagattgggaatcaagctgcagaaggaaagatacatgaattagcaatgaaacttgctttgcagaaatcacagaatgcagaacttagaagtcaatttgaaggactgtacgaacacatggagggcctgacaaatgatgtagagaaatcaaatgaaacggtacttctgttgcaagaaaagctagaggagaaggatcaggagcttaaaaagctgaagcttcaactacagcagaagacattagtggaggaaaaaactgatccgtgtccaaataaaacagtcagcgctgatgaacttaagaaggaagctgaggtcaactag。

in the invention, based on codon optimization strategies of different plants, codon optimization can be carried out on the basis of the nucleotide sequence shown in SEQ ID NO.2, so that the nucleotide sequence is suitable for application of different plants.

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%.

In the present invention, the plurality of nucleotides in (3) is preferably not more than 30 nucleotides, and may be any of 1 to 30, for example, the plurality of nucleotides substituted, deleted and/or inserted is not more than 30, 27, 24, 21, 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 or not more than 1 nucleotide.

In the present invention, the alteration of the identity in (2) or the base mutation such as substitution, deletion and/or insertion in (3) does not result in alteration of the function of the protein encoded by the gene. In the present invention, the site having no identity in (2) corresponds to the site having no identity in the above-mentioned scheme 2); (3) The site of the base mutation(s) corresponds to the site of substitution, deletion and/or insertion of one or more amino acid residues in the above-mentioned scheme 3).

The 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 phage, such as a 35S plasmid.

The invention also provides a recombinant cell which comprises the PagFIP37 gene or the recombinant vector.

The PagFIP37 gene in the scheme is exogenously introduced into the recombinant cell; the recombinant cells highly express the PagFIP37 protein. In the present invention, the recombinant cell includes a host cell or a target cell of a plant; the host cell is preferably a prokaryotic, eukaryotic or fungal host cell; the host cell of the prokaryote is preferably Escherichia coli or Agrobacterium; the host cell of the eukaryote is preferably a yeast; the targeted cells of the tree are preferably one or more of the cells from roots, stems, trunks, leaves, flowers and calli of the tree.

The invention also provides the application of the PagFIP37 protein or the PagFIP37 gene or the recombinant vector or the recombinant cell in promoting the growth of trees; the tree growth includes secondary growth and/or elongation growth. In the present invention, the secondary growth preferably comprises secondary xylem growth. In the present invention, the promotion of secondary growth is manifested in that the transgenic tree into which the PagFIP37 gene has been introduced has a wider secondary xylem relative to the wild-type tree into which the PagFIP37 gene has not been introduced. In the invention, the promotion of elongation growth of trees is shown by the fact that plants in transgenic trees into which the PagFIP37 gene is introduced are increased relative to wild trees into which the PagFIP37 gene is not introduced.

In the present invention, the tree preferably includes a broadleaf tree; the broad-leaved trees preferably comprise deciduous broad-leaved trees or evergreen broad-leaved trees, and more preferably broad-leaved trees in the family of salicaceae; the broadleaf tree of the Salicaceae family is preferably a tree of the genus Salix, populus or Salix in the Salicaceae family; the tree of the genus populus preferably includes a populus tremuloides (Tacamahaca), a populus tremula (Leuce), a populus nigra (Aigeiros), a populus tremula (Turanga), or a populus tremula (Leucoses), and more preferably, the tree is a populus tomentosa, a populus alba, a populus tremuloides, a populus deltoides, a populus sonota, a populus tomentosa Mao Yang, a triploid populus tremula, or a populus 84k in the populus tremula.

The invention also provides a breeding method of trees, and the PagFIP37 gene in the scheme is over-expressed in the trees.

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

s1: obtaining the coding region sequence of the PagFIP37 gene;

s2: connecting the coding region sequence of the PagFIP37 gene to an expression vector by a double enzyme digestion method to construct a PagFIP37 overexpression vector;

s3: introducing the PagFIP37 overexpression vector into a tree to obtain an overexpression transgenic plant;

s4, extracting the genome of the overexpressed transgenic plant, and identifying a positive transgenic plant;

the present invention first obtains the coding region sequence of the PagFIP37 gene. The method for obtaining the coding region sequence of the PagFIP37 gene is not particularly limited, and the conventional method in the field is adopted, and in the specific implementation process of the invention, the RNA of 84K poplar leaves is extracted by a PCR method for reverse transcription, and then the coding region sequence of the PagFIP37 gene is obtained by taking the obtained cDNA as a template for amplification.

After the coding region sequence of the PagFIP37 gene is obtained, the coding region sequence of the PagFIP37 gene is connected to an expression vector by a double enzyme digestion method to construct a PagFIP37 overexpression vector. In the present invention, the PagFIP37 overexpression vector is preferably a 35S:PagFIP37 overexpression vector.

After the PagFIP37 overexpression vector is constructed, the PagFIP37 overexpression vector is introduced into trees to obtain overexpression transgenic plants. In the present invention, the method for introducing the PagFIP37 overexpression vector into trees is preferably an agrobacterium-mediated genetic transformation method.

After obtaining the transgenic plant with over-expression, the invention extracts the genome of the transgenic plant with over-expression and identifies the positive transgenic plant.

In the present invention, the tree is suitable for the range of trees defined in the above scheme, preferably a tree of aspen type.

The trees bred by the breeding method have wider secondary xylem and/or plant height compared with wild trees.

The invention also provides a tree breeding method, which is used for detecting the content of the PagFIP37 protein in the tree or detecting the expression quantity of the PagFIP37 gene in the tree in the scheme, and selecting plants with high PagFIP37 protein content and/or high PagFIP37 gene expression quantity for breeding.

The method for detecting the content of the PagFIP37 protein in the tree or the expression level of the PagFIP37 gene in the tree in the scheme is not particularly limited, and the conventional method in the field can be adopted.

In order to further illustrate the present invention, the following will describe in detail the PagFIP37 protein, the gene encoding the PagFIP37 protein and the applications thereof provided by the present invention with reference to the drawings and examples, which should not be construed as limiting the scope of the present invention.

Example 1: cloning of coding region sequence of poplar PagFIP37 gene

The AtFIP37 gene sequence was downloaded in NCBI (https:// www.ncbi.nlm.nih.gov /) database, the entire genome sequence of 84K Populus was downloaded by logging in figshare (https:// figshare. Com /), and the gene sequence of PtrFIP37 in 84K Populus was obtained by blast. According to the base sequence of the coding region, 84K poplar PagFIP37 amplification primers are analyzed and designed, so that a PagFIP37 gene is amplified, the gene sequence is shown as SEQ ID NO.2, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 1.

The invention utilizes a PCR method to carry out reverse transcription on extracted 84K poplar leaf RNA, and then uses the obtained cDNA as a template to amplify the coding region sequence of the PagFIP37 gene, and the specific method is as follows:

(1) 84K poplar leaf total RNA extraction

Cutting 84K tissue culture seedling leaves, and grinding the leaves in a liquid nitrogen environment for later use. The specific operation steps refer to the instruction of a Plant total RNA extraction Kit (RNeasy Plant mini Kit) of Tiangen Biochemical technology Beijing GmbH.

(2) cDNA Synthesis

1) Preparing reverse transcription mixed solution according to the following system, and placing the reverse transcription mixed solution into a 200 mu L RNase free PCR tube; the system is as follows:

table 1: reverse transcription system

2) Mixing, and incubating at 42 deg.C for 30min;

3) Heating at 85 deg.C for 5s to inactivate TransScript RT/RI and gDNARMOVER;

4) The obtained cDNA was stored at-20 ℃. All reagents used in the reaction were purchased from Beijing Quanjin Biotechnology, inc.

(3) Amplification of the Gene of interest PagFIP37

1) According to the 84K poplar PagFIP37 gene sequence obtained by blast, analyzing and designing PagFIP37 coding region amplification primers:

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

PagFIP37-R1：CTAGTTGACCTCAGCTTCCTTCTT(SEQ ID NO.4)。

2) Amplifying according to the following PCR reaction system by taking the 84K poplar cDNA sequence as a template to obtain an 84K poplar PagFIP37 cDNA sequence; the following reagents and amounts were used in accordance with the instructions of the TransStart Fstpfu Fy DNA polymerase kit of the entire gold company, as shown in the following Table:

table 2: PCR amplification system

3) The PCR reaction procedure was as follows:

pre-denaturation: 2min at 98 ℃; (denaturation: 98 ℃ for 30s, annealing: 58 ℃ for 30s, extension: 72 ℃ for 30 s). Times.36 cycles; extension: 5min at 72 ℃; keeping the temperature at 4 ℃. The sequence of the amplified PagFIP37 gene is shown as SEQ ID NO. 2.

4) Agarose gel electrophoresis detection

0.5g of agarose was weighed, added to 50mL of 1 XTAE, heated in a microwave oven, added to 5. Mu.L of GoldView (from Zhongke Thai Biotech Co., ltd.) and shaken well and poured into a slab rubber. After the agarose gel is solidified, the mixture of the PCR product and Loading buffer (purchased from Beijing Quanyu gold Biotechnology Co., ltd.) is added into the gel hole for electrophoresis detection, and the cDNA fragment is recovered. The target fragment was ligated to the T vector, followed by transformation, and screening for positive monoclonals by plating ampicillin resistant plates. Recombinant plasmids were identified by PCR of monoclonal bacterial solutions. 20 monoclonal bacteria were selected for PCR, and agarose gel electrophoresis showed that 12 lanes showed a band of about 1000bp (FIG. 1) identical to the size of the target fragment. 6 positive monoclonal bacteria solutions were selected from the monoclonals and sent to Beijing Rui Bo sequencing company for sequencing.

5) Sequence alignment

Sequence analysis and comparison are carried out on the PagFIP37 sequencing result and the original sequence obtained by blast through DNAMAN software, and the base similarity rate of the sequencing result and the original sequence is 99.60%, the base numbers are the same, and the size is 1011bp (figure 2).

Example 2: construction of overexpression vector for PagFIP37 Gene

1) The PagFIP37 gene was analyzed using Primer Premier 5 and primers with Kpn I and Xba I cleavage sites were designed (in bold italics):

PagFIP37-F2：

PagFIP37-R2：

/>

2) PagFIP37 with cleavage sites was amplified and the PCR product recovered (as described in example 1).

3) Constructing pCAMBIA2300-35S, wherein the ratio of PagFIP37: the PagFIP37 product with the cleavage sites and the expression vector pCAMBIA2300 were double-digested with the endonucleases Kpn I and Xba I. Respectively recovering products after enzyme digestion, and connecting the target gene fragment with an expression vector by using T4-DNA ligase (purchased from Beijing Limited company of medical science and technology of Baori, and the specific operation method and the dosage refer to the specification).

4) The ligation products were transformed into E.coli competent TOP 10 (purchased from Beijing Ederly Biotech Co., ltd., for details of the procedure and amounts, see the instructions), after which E.coli competent cells were transformed, spread on LB solid plates containing Kan antibiotics, and cultured in an incubator at 37 ℃.

5) Picking single colony on solid plate, putting it into LB culture medium (containing antibiotic), culturing 4-5 h at 37 deg.C. The bacterial suspension was used as a template, amplified by PCR, and detected by 1% agarose gel electrophoresis (FIG. 3). The positive clone was selected and transferred to Beijing Rui Bo Biotech for sequencing. And (3) comparing the sequencing result with the original gene sequence by DNAMAN software, amplifying and shaking the positive clone bacteria liquid with correct sequencing, adding 60% of glycerol, freezing and storing at-80 ℃, reserving the strain, and naming the vector as 35S: pagFIP37.

Example 3: genetic transformation of poplar

Introducing the excessive expression vector 35S, namely PagFIP37, into poplar leaves by an agrobacterium-mediated genetic transformation method, and transplanting in a greenhouse after preculture, infection, dark culture, adventitious bud induction, bud rooting induction, propagation and seedling hardening.

1) Pretreating wild type 84K poplar leaves:

before the super clean bench is used, the tabletop of the super clean bench is wiped clean by using 75% alcohol in advance, scissors, tweezers, a scalpel and the like are placed in a high-temperature sterilizer, the high-temperature sterilizer is opened, and finally an ultraviolet lamp is opened for sterilization for 25-30 min. Then operating in an aseptic super clean bench, shearing leaves with good growth state of aseptic seedlings, generally selecting leaves at the 3 rd to 6 th phyllotaxy positions of plants from bottom to top, drawing wounds with uniform intervals on the back surfaces of the leaves in the direction vertical to the main veins by using a scalpel, enabling the back surfaces of the leaves to face upwards, and flatly paving the leaves on a WPM (acetosyringone) -co-culture medium for visible light culture for 1-2 days.

2) Preparing an agrobacterium infection solution:

absorbing 80 mu L of PagFIP37 agrobacterium into 80mL LB liquid containing Kan antibiotic, mixing evenly, putting into a constant temperature shaking table at 28 ℃, and culturing overnight at 200 rpm. The cells were collected by centrifugation in a 50mL sterile centrifuge tube at 5000rpm for 10min at room temperature. Finally, the cells were resuspended in 1/2MS resuspension and diluted to OD ₆₀₀ 0.8, followed by infection of the pretreated leaves.

3) And (3) infection process:

the infestation process needs to be conducted in a sterile clean bench. Soaking the pretreated and cultured leaves in the prepared agrobacterium

Heavy suspension bacterial liquid (OD) ₆₀₀ 0.8), the soaking time is 10-13 min, and the process needs to shake for a plurality of times at intervals, so that the wound of the blade can be in full contact with the bacterial liquid. And then taking out the infected leaves, and placing the infected leaves on sterile filter paper to suck the redundant agrobacterium on the surfaces of the leaves. And finally, the back of the leaf is upwards laid on a dark treatment culture medium without antibiotics.

4) Co-culturing:

the infected leaves placed on the dark treatment culture medium are placed in a dark box for dark culture for about 3 days, and the differentiation stage can be carried out until a small amount of agrobacterium floras around the leaves can be seen by naked eyes.

(5) Screening and culturing:

in a super clean bench, using sterile filter paper to absorb excess flora around dark cultured leaves, transferring the dark cultured leaves to a differentiation medium containing Kan antibiotics, placing the leaves in a culture room with the temperature of about 25 ℃, and performing resistance differentiation screening culture. The differentiation medium was changed approximately every 20 days until adventitious buds were differentiated.

(6) Secondary resistance screening:

the adventitious buds differentiated in the culture medium were excised using a sterile scalpel in a super clean bench, and cultured on a new differentiation medium for about 3 weeks to induce differentiation.

(7) Rooting culture:

when the adventitious bud grows to about 1.5cm, dividing the adventitious bud into single buds in a super clean bench by using a sterile scalpel, inserting the single buds into a rooting screening culture medium containing Kan antibiotics, and culturing for about 7-10 days to root the adventitious bud substrate. After rooting, firstly, cutting leaves of sterile rooting plants, extracting genomes, and detecting whether the plants are positive plants or not. The transgenes identified as positive plants were first propagated using a sterile scalpel to cut the apical buds and stem segments (the subculture medium required to contain Kan antibiotics) (fig. 4).

Example 4: identification of transgenic poplar, growth rate and observation of secondary xylem

1) When the PagFIP37 overexpressing resistant plants were grown in culture for approximately 1 month, the overexpressed plants were tested for RNA levels. Selecting a sequence with stronger specificity about 150-200 bp, designing a qRT-PCR primer of PagFIP37 on line through an NCBI website, and detecting the expression quantity of the PagFIP37 gene through a fluorescent quantitative PCR experiment (figure 5), wherein the result shows that the expression quantity of the over-expressed plant OE-PagFIP37 gene is 28 times of that of a wild type.

Primers were designed as follows:

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

OE-PagFIP37-R:TGTCCTACACTTTGCCATC，(SEQ ID NO.8)。

2) Detecting the growth rate of the transgenic poplar: the first two internodes and about 1-2cm including the stem tip of the identified transgenic tissue culture seedling and the tissue culture seedling of the wild poplar are cut and placed in a rooting culture medium without antibiotics for growth for 5-8 weeks. The tissue culture seedlings were acclimatized and then transplanted to a greenhouse for growth, and after they grew for 1 month, they were photographed as shown in fig. 6.

Under the same culture conditions, the internode number and the diameter of the 5 th node of WT and OE-PagFIP37 plants grown in the greenhouse for 3 months were counted, and prism statistics showed that the number of the stem nodes of the over-expressed plants was significantly greater than WT (FIG. 7) and that the 5 th node of the over-expressed plants was significantly wider than WT plants (FIG. 8).

3) Observation of xylem of transgenic plants

In order to observe the effect of PagFIP37 on a cytological level, one month of tissue culture seedlings are transferred into nutrient soil, 10 th stem nodes of transgenic plants and WT plants are selected and put into a fixing solution for fixing for three days after one month of greenhouse growth, and then are dehydrated, infiltrated and embedded, and finally are trimmed and sliced under a semi-thin slicer. Placing the slices on water drops of a glass slide on which water drops are dropped, then placing the glass slide on a baking machine for baking the slices at 60 ℃, dropping 1% (v/v) methylamine blue dye solution on the slices stuck on the glass slide when the water drops on the glass slide are baked, and dyeing for 20-30 s; by ddH ₂ Washing off redundant dyeing liquid, and placing on a baking sheet machine until the slices are completely dried; adding 1 drop of mixed solution of xylene and neutral gum with equal volume, covering with a cover glass, and sealing; the images were observed under an optical microscope and photographed. The results are shown in FIG. 9, the secondary xylem of transgenic plants is significantly broader than that of wild type plants.

Example 5: structural and functional analysis of OE-PagFIP37 protein

Bioinformatics analysis of the amino acid sequence of PagFIP37 by DNAMAN software revealed that the CDS sequence of PagFIP37 encodes 336 amino acids, with a molecular mass of about 38.065kDa and an isoelectric potential of 5.22. In order to investigate the biological functions of the PagFIP37 protein in depth, the amino acid sequence of PagFIP37 was analyzed for prediction of the transmembrane structure of the protein, hydrophobicity, hydrophilicity, and signal peptide, respectively. The analysis results showed that PagFIP37 had a minimum hydrophobic score of-2.933, serine at position 27, a maximum score of 1.722, isoleucine at position 140, a total hydrophobic score of-0.815, and a majority of amino acid values were negative, so PagFIP37 was presumed to be a hydrophilic protein (fig. 10). Analysis of the transmembrane domain by DNAMAN software showed that the transmembrane domain was absent from PagFIP37 (fig. 11). Prediction of protein signal peptides can provide a reference for determining functional domains and subcellular localization. Predicted by the software, the absence of signal peptide was found for PagFIP37 (fig. 12).

The patent is composed of a basic scientific research business fee special item (2021 ZY 57) of central colleges and universities, a national natural science fund (31970182), beijing forestry university "university student innovation business creation training plan" (202110022069) project support.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims (10)

1. A PagFIP37 protein, characterized in that the amino acid sequence is selected from one or more of 1) to 3):

1) An amino acid sequence shown as SEQ ID NO. 1;

2) A sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No. 1;

3) The amino acid sequence shown in SEQ ID NO.1 comprises an amino acid sequence with one or more amino acid residues substituted, deleted and/or inserted;

1) And 2) a fragment in which the site having no identity is located outside positions 131 to 285 of the amino acid sequence represented by SEQ ID NO. 1;

3) The fragment in (1) in which one or more amino acid residues are located outside positions 131 to 285 of the amino acid sequence shown in SEQ ID NO. 1.

2. A PagFIP37 gene, wherein the PagFIP37 gene encodes the PagFIP37 protein of claim 1.

3. The PagFIP37 gene according to claim 2, characterized in that the nucleotide sequence of the PagFIP37 gene is selected from one or more of (1) to (3):

(1) A nucleotide sequence shown as SEQ ID NO. 2;

(2) A nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID No. 2;

(3) The nucleotide sequence shown in SEQ ID NO.2 comprises a nucleotide sequence with one or more nucleotides substituted, deleted and/or inserted.

4. A recombinant vector into which the PagFIP37 gene according to claim 2 or 3 has been inserted.

5. A recombinant cell comprising the PagFIP37 gene of claim 2 or 3 or the recombinant vector of claim 4.

6. Use of the PagFIP37 protein of claim 1 or the PagFIP37 gene of claim 2 or 3 or the recombinant vector of claim 4 or the recombinant cell of claim 5 for promoting tree growth; the tree growth includes secondary growth and/or elongation growth.

7. The use of claim 6, wherein the secondary growth comprises secondary xylem growth.

8. The use of claim 6, wherein the tree comprises a hardwood tree.

9. A method of breeding trees, wherein the PagFIP37 gene of claim 2 or 3 is overexpressed in said trees.

10. A method for tree breeding, characterized in that the content of the PagFIP37 protein according to claim 1 or the expression level of the PagFIP37 gene according to claim 2 or 3 in a tree is detected, and plants with a relatively high content of the PagFIP37 protein and/or a relatively high expression level of the PagFIP37 gene are selected for breeding.

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