阅读说明：本技术 一种PagARGOS蛋白、其编码基因及其应用 (PagARGOS protein, coding gene and application thereof ) 是由 李晓娟 姚小敏 林金星 于 2021-11-25 设计创作，主要内容包括：本发明公开了一种PagARGOS蛋白、其编码基因及其应用,尤其是在降低木质素含量和/或加快树木生长中的应用,具体涉及杨树PagARGOS基因的克隆；将PagARGOS基因导入杨树中进行超量表达PagARGOS蛋白；观察转基因植株的表现并对转基因植株的木质素含量、株高进行检测。本发明通过将PagARGOS基因导入杨树,说明PagARGOS蛋白在降低木质素含量、加快植株生长上起着重要作用；获得的新型速生低木质素转基因树种,对解决造纸原料短缺以及生产过程中木质素引起的环境污染等问题具有重要价值。(The invention discloses a PagARGOS protein, a coding gene thereof and application thereof, in particular to application in reducing lignin content and/or accelerating tree growth, and particularly relates to poplar PagARGOS Cloning a gene; will be provided with PagARGOS The gene is introduced into poplar to carry out overexpression of PagARGOS protein; and observing the expression of the transgenic plant and detecting the lignin content and the plant height of the transgenic plant. The invention is prepared by PagARGOS The gene is introduced into poplar, which shows that the PagARGOS protein plays an important role in reducing the lignin content and accelerating the plant growth; the obtained novel fast-growing low-lignin transgenic treeThe method has important value for solving the problems of shortage of papermaking raw materials, environmental pollution caused by lignin in the production process and the like.)

1. A PagARGOS protein, wherein the amino acid sequence of said protein is selected from the group consisting of:

(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; alternatively, the first and second electrodes may be,

(3) and the amino acid sequence shown in SEQ ID NO.1 and including substitution, deletion and/or insertion of one or more amino acid residues,

wherein the site having no identity in (2) or the site of substitution, deletion and/or insertion in (3) is a fragment located other than positions 46 to 95 of SEQ ID NO. 1.

2. A kind ofPagARGOSA gene encoding the PagARGOS protein of claim 1.

3. The method of claim 2PagARGOSA gene, characterized in that the nucleotide sequence of said gene is selected from the group consisting of:

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

(2) a nucleotide sequence having at least 90% identity to the sequence shown in SEQ ID No. 2; alternatively, the first and second electrodes may be,

(3) and SEQ ID NO.2, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

4. An expression vector comprising the gene of any one of claims 2 to 3.

5. A cell comprising the gene of any one of claims 2-3.

6. Use of the protein of claim 1, the gene of any one of claims 2-3, the vector of claim 4, or the cell of claim 5, wherein the use comprises:

(1) reducing the lignin content in the tree; and/or the presence of a gas in the gas,

(2) and the growth of trees is promoted.

7. The use of claim 6, wherein the tree is a hardwood tree.

8. A tree breeding method is characterized in that the tree is highly expressedPagARGOSA gene.

9. The breeding method of claim 8, wherein the breeding method comprises breedingPagARGOSThe gene is introduced into trees.

10. Use of a tree produced by the propagation method of any one of claims 8 to 9, wherein the use comprises:

(1) as an energy source substance;

(2) manufacturing paper pulp;

(3) building decoration; or the like, or, alternatively,

(4) and (5) environmental protection.

Technical Field

The invention belongs to the field of forestry molecular bioscience, and particularly relates to a PagARGOS protein, a coding gene thereof and application thereof, in particular to application in reducing lignin content and/or accelerating tree growth.

Background

Forest is the most abundant renewable resource on the earth, can be used as an energy source substance, and plays an important role in paper pulp manufacturing, building decoration and environmental protection. Along with the development of society, the wood consumption in China is more and more, wherein the consumption of paper pulp manufacturing is particularly obvious. However, the wood growth cycle is long, which results in the production of difficult to meet the papermaking requirements. In addition, in the pulp manufacturing process, lignin, one of the three main chemical components of wood, cannot be fully utilized, becomes one of the main pollution sources, and causes serious pollution to the environment. The tree species with higher growth rate and lower lignin content is urgently needed, the short-rotation directional cultivation is carried out on the tree species, the wood yield of unit area is improved, and the problem of insufficient papermaking wood is solved. In the process of genetic improvement of forest trees, the defects of long breeding improvement period and the like exist by using a conventional genetic breeding method. However, the conventional breeding technology is combined with molecular biology means, so that the breeding period can be greatly shortened, the quality of the wood of the artificial forest can be improved from the source by utilizing the genetic engineering technology, and the growth rate of the wood is accelerated.

Plant growth is dependent on the proliferation, expansion and differentiation of cells. In primordia, the size of an organ depends on the number of cells and the rate and time of cell division. The research finds that the content of the active ingredients in the active ingredients is high,AtARGOSis a gene controlling the size of lateral organs in Arabidopsis thaliana. During plant growth, the signal of ARGOS protein is transduced into the signal of coding transcription factorAINTEGUMENTA (ANT)Further controlling cell division of above ground organ primordia and maintaining the organ primordiaCYCD3;1To prolong the duration of cell division, resulting in increased growth rate of the plants, increased plant height and increased lateral organs.

Poplar (Latin science name:Populusl.) is a plant of the genus Populus, with more than about 100 species of the whole genus, about 62 species (including 6 hybrids) in China, with 57 species of China distributed, about 4 species introduced for cultivation, and many varieties, variations and introduced lines. The poplar (Populus) classification system is further divided into five groups: populus style（Tacamahaca）Populus style（Leuce）Black poplar pie（Aigeiros）Populus diversifolia pie（Turanga）Dayeyang pie（Leucoides）. The poplar has wide application range, is not only used as wood, but also mainly used as material in the processing industry, and the poplar becomes an important processing raw material in the industries of plywood, fiberboard, paper-making matches, sanitary chopsticks and packaging. However, the lignin content in poplar is high, and the poplar can be harvested and utilized within 5-12 years generally, which limits further industrial application of poplar.

Disclosure of Invention

In view of the above, in order to overcome the above drawbacks, the present invention finds a new homologous protein of AtARGOS from 84K poplar species through experimental exploration, and names it as PagARGOS protein, and applies it to a tree cultivation method, specifically:

in a first aspect of the invention, there is provided a PagARGOS protein, the amino acid sequence of which is selected from:

(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; alternatively, the first and second electrodes may be,

(3) and the amino acid sequence shown in SEQ ID NO.1 and comprising substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, sequences of at least 90% identity in the protocol (2) include any number between 90% and 100%, for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

Preferably, in scheme (3), the plurality of amino acid residues is no more than 10 amino acid residues, e.g., the plurality of amino acid residues substituted, deleted and/or inserted is no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid residue.

It will be appreciated by those skilled in the art that changes in identity or residue mutations (substitutions, deletions and/or insertions) in non-functional regions do not affect the function of the protein. Preferably, the site of non-identity in scheme (2) (i.e., the site of amino acid residue difference) or the mutation site in scheme (3) is located in the extracellular region, more preferably, the site of non-identity or the mutation is located in a segment other than positions 69-77 of SEQ ID NO.1, and even more preferably, the mutation site is located in a segment other than positions 46-95 of SEQ ID NO. 1. In other words, the sequences in schemes (2) and (3) outside the extracellular region are preferably identical to SEQ ID NO.1 at positions 69 to 77 of SEQ ID NO.1, more preferably at positions 46 to 95 of SEQ ID NO. 1.

Sites of non-identity in the scheme (2) are also understood as amino acid residues other than those having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 1.

In a second aspect of the invention, there is provided a polypeptide encoding a PagARGOS protein as described abovePagARGOSA gene.

Preferably, the nucleotide sequence of the gene is selected from the group consisting of:

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

(2) a nucleotide sequence having at least 90% identity to the sequence shown in SEQ ID No. 2; alternatively, the first and second electrodes may be,

(3) and SEQ ID NO.2, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Based on the codon optimization strategy of different tree species, the 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 the application of different tree species.

Preferably, the nucleotide sequence of at least 90% identity in the protocol (2) includes any value between 90% and 100%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

Preferably, in the scheme (3), the plurality of nucleotides is 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.

It will be appreciated by those skilled in the art that such identity changes or base mutations (substitutions, deletions and/or insertions) do not result in a change in the function of the protein encoded by the gene. Preferably, the site having no identity or the site of base mutation corresponds to the site of mutation of the above-mentioned protein.

In a third aspect of the invention, an expression vector is provided, wherein the expression vector comprises the gene.

Preferably, the vector comprises a plasmid, a viral vector or a phage, among others. Such as the 35S plasmid.

In a fourth aspect of the invention, there is provided a cell comprising an exogenous nucleic acid introduced as described abovePagARGOSA gene.

Preferably, the cell highly expresses the PagARGOS protein.

Preferably, the cell may be a host cell, e.g., a host cell from a prokaryote, eukaryote, fungus, e.g., E.coli, Agrobacterium, yeast, etc.; the cells may also be targeted cells from any tree, such as cells from the roots, stems, trunks, leaves, flowers, calli, and the like of a tree.

In a fifth aspect of the invention, there is provided a PagARGOS protein as described above,PagARGOSUse of a gene, an expression vector, a cell, the use comprising:

(1) reducing the lignin content in the tree; and/or the presence of a gas in the gas,

(2) and the growth of trees is promoted.

Preferably, the reduction of the lignin content in the tree is expressed relative to the absence of the lignin introducedPagARGOSIntroduction of the Gene into a wild-type TreePagARGOSThe transgenic tree after gene has lower lignin content.

Preferably, the promotion of tree growth is manifested relative to non-introductionPagARGOSIntroduction of the Gene into a wild-type TreePagARGOSThe transgenic trees after the gene promote the leaf enlargement, the tree plant increase and/or the rotation cutting period is shortened.

Preferably, the tree is a broad-leaved tree, including deciduous broad-leaved trees, evergreen broad-leaved trees, such as trees of the genera tamarix, populus and salix in the family salicaceae, and more preferably, the tree is a tree of the genus populus, including populus type（Tacamahaca）Populus style（Leuce）Black poplar pie（Aigeiros）Populus diversifolia pie（Turanga）Dayeyang pie（Leucoides）The poplar of (4), further preferably, the tree is populus tomentosa, populus alba, populus tremuloides, populus deltoids, populus tremuloides, populus tremula, and populus 84k in a populus tremula.

In a sixth aspect of the present invention, a method for breeding trees with high expression is providedPagARGOSA gene.

Preferably, the breeding method comprises the following steps ofPagARGOSThe gene is introduced into trees.

More preferably, the mixture isPagARGOSThe gene is introduced into trees by the following steps:

cloning of the S1 Gene: to obtainPagARGOSThe coding region sequence of the gene is, for example, extracted from 84K poplar leaf by PCR method, reverse transcribed, and amplified using the cDNA as templatePagARGOSThe coding region sequence of the gene;

s2 vector construction: will be provided withPagARGOSThe coding region sequence of the gene is connected to an expression vector by a double enzyme digestion method to constructPagARGOS(ii) a overexpression vector; preferably, the overexpression vector is 35S:PagARGOSoverexpression vectors

S3 genetic transformation: introducing the expression vector into a tree by utilizing an agrobacterium-mediated genetic transformation method to obtain a transgenic plant with over-expression;

and (3) identifying S4 positive seedlings: extracting the genome of the plant, and identifying a positive transgenic plant;

the trees have low lignin content and/or faster growth, as indicated by lower lignin concentration and/or increased leaf area, higher plant height, and/or reduced rotation relative to wild-type trees.

Preferably, the tree is adapted to the tree range defined above. More preferably, the tree is a aspen tree.

The seventh aspect of the invention provides a tree prepared by the breeding method, wherein the tree is high in expressionPagARGOSA gene.

Preferably, the tree has a lower lignin concentration and/or increased leaf area, increased plant length, and/or reduced rolling period after the transgene compared to a wild-type tree.

Preferably, the tree is adapted to the tree range defined above. More preferably, the tree is a aspen tree.

In an eighth aspect of the present invention, there is provided an application of the tree, the application includes:

(1) as an energy source substance;

(2) manufacturing paper pulp;

(3) building decoration; or the like, or, alternatively,

(4) and (5) environmental protection.

Compared with the prior art, the invention has the following beneficial technical effects:

the invention discovers and separates the homologous protein PagARGOS of the AtARGOS protein from the poplar for the first time, and the sequence structure of the homologous protein PagARGOS is different from the homologous sequence structure of other species sources.

The invention discovers for the first time that the PagARGOS protein has the function of reducing lignin, so that the transgenic tree obtained according to the invention has low content of lignin, and the adverse effect caused by the lignin can be reduced in the subsequent application process, especially in the pulp manufacturing process, the pollution of the lignin to the environment is reduced.

The invention finds introduction into treesPagARGOSAfter the gene and the PagARGOS protein are highly expressed, the growth of trees can be promoted, the perimeter of leaves is increased, the tree plants are increased, the cycle time can be effectively shortened, and more economic values can be generated for the trees.

Drawings

FIG. 1 is a map of pCAMBIA2300 vector;

FIG. 2 is a phenotype diagram of leaves of transgenic poplar and wild-type poplar, wherein WT is wild-type poplar, 35S:PagARGOSis a transgenic poplar;

FIG. 3 shows the height phenotype of transgenic poplar and wild-type poplar plants, wherein WT is wild-type poplar, 35S:PagARGOSis a transgenic poplar;

FIG. 4 is a statistical plot of the plant heights of transgenic poplar and wild-type poplar, wherein WT is wild-type poplar and OE is transgenic poplar;

FIG. 5 is a graph comparing lignin content of transgenic poplar and wild-type poplar, wherein WT is wild-type poplar and OE is transgenic poplar;

FIG. 6 is a graph of the hydrophobicity analysis of the PagARGOS protein.

FIG. 7 is a diagram of prediction of transmembrane structure of PagARGOS protein.

FIG. 8 shows the results of prediction of transmembrane structure of PagARGOS protein.

Detailed Description

The following materials or reagents, unless otherwise specified, were all purchased.

The present invention is further illustrated by the following specific examples.

Example 1: poplar treePagARGOSCloning of coding region sequence of Gene

Downloading in NCBI (https:// www.ncbi.nlm.nih.gov /) databaseAtARGOSGene sequence, placing the sequence in a Phytozome (https:// Phytozome. jgi. doe. gov/pz/portal. html) database, selecting Populus Trichocarpa V3.0 to obtain Populus tomentosaPtrARGOSGene sequence, based on the base sequence of its coding region, 84K poplar was designed by analysisPagARGOSHomologous amplification primers, thereby amplifyingPagARGOSThe gene and the gene sequence are shown in SEQ ID NO.2, and the amino acid sequence of the coded protein is shown in SEQ ID NO. 1.

The invention uses PCR method to make reverse transcription of 84K poplar leaf RNA and then uses the obtained cDNA as template to make amplificationPagARGOSThe coding region sequence of the gene is specifically prepared by the following steps:

(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 liquor according to the following system, and placing the reverse transcription mixed liquor into a 200 muL RNase free PCR tube; the system is as follows:

table 1: reverse transcription system

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

3) heating at 85 deg.C for 5s to inactivate TransScript RT/RI and gDNA Remover;

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

(3) Target genePagARGOSAmplification of

1) Obtaining Chinese white poplar according to PhytozomePtrARGOSGene sequence, analysis designPagARGOSCoding region homologous amplification primers:

PagARGOS-F1：ATGGATGTGAGAGCAAG（SEQ ID NO.3）

PagARGOS-R1：TTACACATATGCAAAAGT（SEQ ID NO.4）

2) 84K poplar cDNA sequence is used as a template, and the 84K poplar is obtained by amplification according to the following PCR reaction systemPagARGOSA 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: 2 min at 98 ℃; (denaturation: 98 ℃ for 30 s, annealing: 58 ℃ for 30 s, extension: 72 ℃ for 30 s) x 36 cycles; extension: 5 min at 72 ℃; keeping the temperature at 4 ℃. Amplified outPagARGOSThe gene sequence is shown in SEQ ID NO. 2.

4) Agarose gel electrophoresis detection

0.5 g of agarose was weighed, 50 mL of 1 XTAE was added, and after heating in a microwave oven, 5. mu.L of GoldView (available from Zhongke Thailand Biotech Co., Ltd.) was added, and the mixture was shaken well and poured into a slab rubber. After the agarose gel is solidified, a mixture of the PCR product and a Loading buffer (purchased from Beijing all-purpose gold biotechnology Co., Ltd.) is added into a gel hole for electrophoresis detection, and a cDNA fragment with the length of 324bp is recovered.

Example 2:PagARGOSoverexpression vector construction of genes

1) Analysis by Primer Premier 5PagARGOSGenes and primers with Kpn I and BamH I restriction sites were designed (bold italics as restriction sites):

PagARGOS-F2: CGGGGTACCATGGATGTGAGAGCAAG（SEQ ID NO.5）

PagARGOS-R2:CGGGATCCTTACACATATGCAAAAGTT （SEQ ID NO.6）

2) amplification with restriction sitesPagARGOSAnd recovering the PCR product (as described in example 1).

3) Construction of pCAMBIA2300-35S:PagARGOS: double-restriction enzyme with restriction enzyme sites using restriction enzymes Kpn I and BamH IPagARGOSThe product and expression vector pCAMBIA 2300. The structure of the pCAMBIA2300 vector is shown in figure 1, and the pCAMBIA2300 vector has a 35S promoter and can realize excessive expression of a transgene, and a product after enzyme digestion is respectively recovered, and a target gene fragment and an expression vector are connected by using T4-DNA ligase (purchased from Taorigi technology Beijing GmbH, and the specific operation method and the dosage refer to the instruction).

4) The ligation product was transformed into E.coli competent TOP 10 (purchased from Beijing Ederly Biotech Co., Ltd., for which the detailed procedures and amounts were described in the specification), 600. mu.L of LB medium was added, and then placed in a constant temperature shaker and allowed to resuscitate at 37 ℃ and 150 rpm for 60 min. And centrifuging the recovered escherichia coli at 5,000 rpm for 5 min, sucking 500 mu L of supernatant, and discarding. And sucking and uniformly mixing the residual 100 muL by using a gun head, uniformly coating the mixture on an LB solid culture medium containing 50 mug/mL Kan, inverting the culture medium in a constant temperature incubator, and growing for 12-16 h at 37 ℃. After the colonies grow out, the monoclonal colonies are picked and grown for 6 hours at 37 ℃ in 20mL LB liquid medium containing 50 mug/mL Kan. Extraction of plasmid (kit from Tiangen Biotechnology North China)Jing Co Ltd, the specific procedures refer to the specification), sent to the company for sequencing, screened positive clones, and named as 35S:PagARGOS。

5) the carrier 35S:PagARGOStransformation into Agrobacterium GV3101 (available from Huayue, Beijing, the detailed procedures are described in the specification)

Preparing an LB liquid culture medium: weighing 5g tryptone, 5g NaCl, 2.5 g yeast extract, adding ddH₂And O is metered to the total volume of 500 mL. Sterilizing with high pressure steam at 121 deg.C for 20min, and cooling to room temperature. The preparation method of the LB solid culture medium refers to an LB liquid culture medium, 7 g of agar powder is added before constant volume, high-pressure steam sterilization is carried out at 121 ℃ for 20min, and the mixture is cooled to room temperature.

Example 3: genetic transformation of poplar

The overexpression vector 35S:PagARGOSthe method is characterized in that the method is introduced into poplar leaves by an agrobacterium-mediated genetic transformation method and is carried out in a greenhouse after preculture, infection, dark culture, adventitious bud induction, bud rooting induction, propagation expansion and seedling hardening.

1) Tissue culture of 84K Populus: cutting 1-2cm stem of tissue culture seedling of 84K poplar of 5-7 weeks old, and subculturing in subculture medium. The culture temperature is about 25 ℃, the illumination intensity is 50 [ mu ] mol · m-2s-1, and the light cycle is 16 h/8 h.

2) Pre-culturing leaves: cutting 3 rd to 6 th phyllotaxis leaf of the sterile tissue culture seedling, transversely cutting 4-5 cuts on the main vein of the leaf by using a sterilized scalpel, and then placing the leaf with the front face facing downwards on a differentiation culture medium without antibiotics for culturing for 1-2 days.

3) The preparation of the agrobacterium infection solution comprises the steps of sucking 300 mu L of agrobacterium containing 35S:: PagARGOS vector into 100mL LB liquid culture medium containing antibiotics (50 mg/L kanamycin, 50mg/L rifampicin), mixing uniformly, putting into 27 ℃, and shaking and culturing at 180 rpm/min overnight. Bacteria solution OD₆₀₀And when the concentration is 0.6-0.8, infection transformation is carried out.

4) Infection and dark culture: and (3) soaking the pretreated blade in the agrobacterium tumefaciens bacterial liquid for 15-20 min in a super clean bench, and shaking the bacterial liquid once every 3-5 min in order to ensure that the wound part of the blade is in full contact with the bacterial liquid. Taking out the leaves, sucking the bacterial liquid attached to the leaves on sterile filter paper, spreading the leaves with the leaf surfaces facing upwards on a differentiation culture medium without antibiotics, and culturing in the dark for 3-4 days.

5) Induction of resistant adventitious buds: in a clean bench, dark-treated leaves were transferred to a differentiation medium containing kanamycin (50 mg/L) and timentin (200 mg/L) for selective culture under light cycle (16 h/8 h dark) at 25 ℃. Adventitious buds grow out from the wound of the leaf blade for about 2 weeks, and when the adventitious buds grow to 0.5-1 cm, the adventitious buds are transferred to a new differentiation culture medium to promote the adventitious buds to continue growing;

6) rooting culture: and (3) placing the single adventitious bud with the length of more than 1 cm into a rooting culture medium containing 30 mg/L kanamycin and 200 mg/L timentin for inducing rooting so as to enable the single adventitious bud to develop into a complete plant.

Transplanting in a greenhouse: when the seedlings grow to about 7cm and the root systems are developed, the seedlings are trained for 6 to 8 days and then transplanted. Washing off culture medium at poplar root with clear water, transplanting into sterilized nutrient soil, and culturing in greenhouse.

The formula of the poplar culture medium is as follows:

table 3: culture medium formula

Example 4: identification of transgenic poplar, growth rate detection and lignin content detection

1) To identify transgenic poplar: the forward primer was designed based on the 35S promoter sequence and the reverse primer was designed based on the PagARGOS sequence. The poplar genome is used as a template, if the vector is transferred into poplar, the gene segment can be amplified through PCR, and if the vector is not transferred, the gene segment can not be amplified.

Primers were designed as follows:

35S-F：CCTCTGCCGACAGTGGTC （SEQ ID NO.7），

PagARGOS-R：TTACACATATGCAAAAGT（SEQ ID NO.8）。

in a clean bench, leaf of poplar (transgenic poplar obtained in example 3) was cut and genomic DNA was extracted using DN 14-plant genomic DNA rapid extraction kit (purchased from Beijing Ederly Biotech Co., Ltd., the detailed procedures were described in the specification). PCR amplification was performed using genomic DNA as a template, and primers 35SF and PagARGOSR and 2xTaq Mix (purchased from Biotech, Inc., Bomaide, Beijing). And (4) carrying out agarose gel electrophoresis detection on the PCR product, and identifying a positive plant successfully transformed into 35S, namely PagARGOS.

The PCR reaction system and reaction procedure were as follows:

2) detecting the growth rate and the lignin content 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 are domesticated and transplanted to a greenhouse for growing, the plant height is measured after the tissue culture seedlings grow for 2 months as shown in figure 4, and the stalk lignin content is measured after the tissue culture seedlings grow for 4 months as shown in figure 5.

The detection method of the lignin content comprises the following steps:

selecting wild type and transgenic poplar growing for 4 months, taking stems, peeling off barks, and drying in a 50-DEG oven. Pulverizing the dried stalk with a pulverizer, and collecting the wood dust passing through a 40-mesh iron screen. And then extracting the wood chips in a Soxhlet extractor for 7-13 hours by using benzyl alcohol, washing the wood chips for 3 times by using absolute ethyl alcohol, naturally airing, and then drying in a 50-DEG vacuum drying oven for 6-8 hours. Then, 300mg of dried wood chips and 3mL of concentrated sulfuric acid with a mass fraction of 72% were weighed into a hydrolysis flask, and stirred with a glass rod for 1 hour at room temperature. Then 840mL of distilled water is added into the hydrolysis bottle, the bottle cap is screwed down and then the hydrolysis bottle is put into a high-temperature high-pressure autoclave at 121 ℃ for reaction for 1 hour. And taking out the sample after the temperature and the pressure of the sterilization pot are reduced, removing the supernatant, filtering the residual mixture by using a weighed glass crucible, putting the crucible containing the filter residue after filtering into a 105-degree oven, drying for 4 hours, and weighing. The weight difference before and after the crucible is the mass of the acid-insoluble lignin (Klason lignin), and the proportion of the Klason lignin in the dried sample powder is obtained based on the Klason lignin algorithm.

And (3) detection results:

fig. 2 is a phenotype diagram (not to scale) of leaves of a transgenic poplar and a wild-type poplar grown in a greenhouse for 2 months, wherein the leaves from left to right in fig. 2 are respectively a phenotype diagram of 1 st to 8 th leaves of the plant from the top, and the leaves of 3 different plants of the wild-type poplar and the transgenic poplar are from top to bottom, and the result shows that the leaves of the transgenic plant are larger than the wild-type plant, taking the 5 th leaf as an example, the average leaf width of the 5 th leaf of the transgenic plant is 9.9cm, while the leaf width of the corresponding position of the wild-type plant is only 6cm, and the two have significant difference.

FIG. 3 is a schematic diagram (not in proportion) showing the plant height phenotype of PagARGOS transgenic poplar and wild-type poplar grown in greenhouse for 2 months, and it is obvious that the transgenic poplar is higher than the wild-type poplar, and the concrete data statistics are shown in FIG. 4, wherein WT is wild-type from left to right, 35S shows that PagARGOS corresponds to 3 different batches of transgenic poplar, the average tree height of the transgenic poplar can reach 59.17cm, while the wild-type poplar is only 48.83cm, and the two have significant difference.

In order to understand the change of the lignin content of the transgenic fast-growing poplar, the stem component analysis of the transgenic fast-growing poplar is carried out by using the Klason method, and the stem lignin content of the transgenic poplar is found to be lower than that of the wild-type poplar, and the statistical result of the data is shown in figure 5, wherein the lignin accounts for 29.77 percent of the dry weight of the stem in the wild-type poplar, and the lignin accounts for 23.38 percent of the dry weight of the stem in the transgenic poplar, and the two have significant difference.

Example 5: structural functional analysis of PagARGOS protein

As a result of protein hydrophobicity analysis using DNAMAN6.0 software, the protein was analyzed by the present study to further characterize the protein, and as shown in FIG. 6, histidine at position 13, which was the lowest score in hydrophobicity analysis, was-2.85, valine, leucine and leucine at positions 87, 88 and 89, which were the highest scores, were 4.09, and most of the amino acids at positions 45-70 and 75-96 were hydrophobic amino acids. The hydrophobicity analysis has important guiding significance for analyzing the transmembrane region of the membrane protein, and the transmembrane domain is mostly composed of hydrophobic amino acids. The hydrophobicity facilitates the inward folding of the protein to form a secondary structure, further forming a domain, a tertiary structure. Meanwhile, the hydrophobicity is also beneficial to the protein to form alpha helix, so that the stability of the protein is ensured. To further analyze the structure of the PagARGOS protein, the transmembrane domain of the PagARGOS protein was predicted by TMHMM in this study, and the results showed that the PagARGOS protein contains two transmembrane helices, 46-68 amino acids and 78-95 amino acids, which is essentially consistent with the protein hydrophobicity analysis. The PagARGOS1 protein has amino acids 69-77 located on the luminal side (intracellular region), and amino acids 1-45 and amino acids 96-107 predicted to be located on the cytoplasmic side (extracellular region) (see FIGS. 7 and 8).

According to the invention, after PagARGOS is transferred into 8K poplar by a molecular biological means, the growth speed of poplar is accelerated, and the lignin content is reduced, thereby obtaining a novel tree species. Based on the characteristics of the tree species, the problems of shortage of papermaking raw materials and environmental pollution caused by lignin in the papermaking process are expected to be solved. Meanwhile, the invention also provides a new method for improving the poplar.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Sequence listing

<110> Beijing university of forestry

<120> PagARGOS protein, coding gene and application thereof

<130> 1

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 107

<212> PRT

<213> Populus alba

<400> 1

Met Asp Val Arg Ala Arg Arg Ile Thr Ala Asn Asn His Arg Ser Pro

1 5 10 15

Pro Asn Ala Ser Ala Glu Lys Lys Lys Ile Glu Tyr Asn Arg Ser Pro

20 25 30

Ser Gln Gly Ser Thr Gly Arg Leu Leu Thr Thr Ser His Phe Ser Leu

35 40 45

Ala Ser Leu Leu Leu Leu Met Cys Ile Thr Ala Ser Leu Leu Ile Leu

50 55 60

Pro Leu Val Leu Ala Pro Leu Pro Pro Pro Pro Phe Met Leu Leu Met

65 70 75 80

Leu Pro Ile Gly Ile Leu Val Leu Leu Ile Ile Leu Ala Phe Met Pro

85 90 95

Ser Asn Ala Arg Asp Ile Thr Phe Ala Tyr Val

100 105

<210> 2

<211> 324

<212> DNA

<213> Populus alba

<400> 2

atggatgtga gagcaagaag gatcactgct aataatcatc gttcccctcc taatgcaagt 60

gcagagaaaa agaaaattga gtataatcga tcaccctcac aagggagcac cggaaggttg 120

ttgactacaa gccatttcag cttggcatca ttgcttttgc tcatgtgtat cactgcatct 180

ttgttgattc ttcctttggt actagcgcca ttgcctccgc cacctttcat gttacttatg 240

ctaccaatag gtatcttggt attgctcata atcttggctt tcatgccttc taatgcaagg 300

gatataactt ttgcatatgt gtaa 324

<210> 3

<211> 17

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

atggatgtga gagcaag 17

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

ttacacatat gcaaaagt 18

<210> 5

<211> 26

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

cggggtacca tggatgtgag agcaag 26

<210> 6

<211> 27

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

cgggatcctt acacatatgc aaaagtt 27

<210> 7

<211> 18

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

cctctgccga cagtggtc 18

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

ttacacatat gcaaaagt 18