CN119192317A - Transcription factor PtoWRKY11 regulating vascular secondary development in Populus tomentosa stems and its application and method - Google Patents

Abstract

The invention discloses a transcription factor PtoWRKY for regulating and controlling the secondary development of a white poplar stem vascular, an application and a method thereof, wherein the amino acid sequence of the transcription factor PtoWRKY is shown as SEQ ID No.1, the nucleotide sequence is shown as SEQ ID No.2, and after the white poplar plants are transformed by using a PtoWRKY gene and a vector optimized by us, the production speed of xylem cells of the white poplar can be effectively improved, the rapid growth of wood can be improved, a method and a germplasm basis are provided for the selective breeding of good varieties of forest trees, the improvement of the wood yield and economic benefits of economic forests is facilitated, and materials are provided for the construction of protection forests, urban and rural greening and the like.

Description

Transcription factor PtoWRKY for regulating secondary development of populus tomentosa stem and its application and method

Technical Field

The invention relates to the technical field of plant molecular biology, in particular to a transcription factor PtoWRKY11 for regulating the secondary development of a stem vascular of populus tomentosa, an application of PtoWRKY gene in regulating the rapid growth of populus tomentosa wood, and a method for constructing a transgenic populus tomentosa strain with improved rapid growth capacity of the wood.

Background

Wood is one of the most important renewable biological resources in nature and is widely used in many fields such as pulping and papermaking, construction industry, biological energy sources and the like. In recent years, wood biomass has been developed as a green, renewable energy source for the production of liquid biofuels (Sarkanen, 1976; behr et al, 2019). The forest coverage rate of China is low, the contradiction between supply and demand of wood is prominent. Therefore, improving the wood properties and optimizing the wood quality has important significance in fast-growing high-quality forest breeding. Poplar (populus. Spp) is one of the most widely planted fast-growing tree species in the world, is also one of main tree species of artificial forestation and wood economy forestation in China, is an important supply source of wood in China, and has important application value in cultivating fast-growing wood species. Meanwhile, along with the completion of whole genome sequencing and the establishment of a genetic transformation regeneration system, poplar has become one of mode plants for researching the growth and development of forests. Therefore, the wood formation regulation and control research taking poplar as an object is a key for breaking through the current forestry economy and breeding bottleneck.

Wood is mainly derived from secondary activities of the vascular cambium, and the division and differentiation activities of the vascular cambium can be regulated and controlled by various endogenous development signals and exogenous environmental cues so as to keep the dynamic balance of plant growth. In the prior art, WOX4 is reported to be specifically and highly expressed in the area of the vitamin tube cambium, and is also a central factor for regulating and controlling the division activity of the vitamin tube cambium cells, and the expression of the WOX4 is influenced by various factors including mechanical stress, drought and other environmental stresses, auxin, TDIF-PXY small peptide and the like. However, few studies on the transcriptional regulation of WOX4 are reported, and only few transcription factors such as LBD3 and ARF5 are reported to regulate WOX4 and further influence vascular development. Therefore, the method for excavating which key transcription factors on the upstream of the poplar WOX4 participate in regulating and controlling the expression of the key transcription factors to influence the vascular development and exploring the molecular mechanism of the poplar growth adaptation environment has important significance, and can provide a theoretical basis for precise genetic breeding of the forest.

Functional reports of WRKY family transcription factors are mainly focused on plant response to environmental stress, but whether the WRKY family transcription factors are involved in regulation of the tree stem-canal cambium is unclear. The applicant found a new transcription factor PtoWRKY11 in the secondary vascular tissue of populus tomentosa which can directly bind to WOX4 promoter by screening. The Arabidopsis thaliana homologous protein of the transcription factor is reported to be involved in the disease resistance response of Arabidopsis thaliana, but the function in developmental regulation is unclear. In poplar, ptoWRKY11 has never been reported as having the ability to regulate Yang Shuwei tube-forming activity and its effect on poplar wood yield, wood properties and mechanical properties has yet to be explored.

Disclosure of Invention

In view of the above, one of the purposes of the present invention is to provide a transcription factor PtoWRKY for regulating the secondary development of the stem vascular of aspen, another purpose of the present invention is to provide a gene encoding the transcription factor PtoWRKY for regulating the secondary development of the stem vascular of aspen, another purpose of the present invention is to provide a plant expression vector containing the PtoWRKY gene, a fourth purpose of the present invention is to provide an application of the PtoWRKY gene or the plant expression vector in regulating the rapid growth of aspen wood, and a fifth purpose of the present invention is to provide a method for constructing a transgenic aspen strain with improved rapid growth capability of wood.

In order to achieve the above purpose, the present invention provides the following technical solutions:

A transcription factor PtoWRKY11 for regulating the secondary development of the stem vascular of populus tomentosa is disclosed, and the amino acid sequence of the transcription factor PtoWRKY is shown as SEQ ID No. 1.

The nucleotide sequence of the gene for encoding the populus tomentosa stem vascular secondary development regulation transcription factor PtoWRKY is shown as SEQ ID No. 2.

The plant expression vector containing the PtoWRKY gene is obtained by constructing a PtoWRKY gene sequence shown in SEQ ID No.2 between two XcmI enzyme cleavage sites of a pCXSN-flag vector.

The PtoWRKY gene or the plant expression vector is applied to regulating and controlling the rapid growth of aspen wood.

In some embodiments of the present invention, the vascular cambium division activity of poplar is promoted by over-expression of PtoWRKY gene in aspen, and the formation of secondary xylem is accelerated, thereby improving the xylem cell production rate of aspen and the rapid growth ability of wood.

A method of constructing a transgenic aspen line with improved rapid growth of wood, comprising the steps of:

(1) Cloning the gene encoding PtoWRKY11, and connecting to a plant expression vector to obtain a plant expression vector 35S of which the gene PtoWRKY is overexpressed, wherein PtoWRKY11;

(2) Transforming agrobacterium, and introducing the 35S constructed in the step (1) into wild aspen leaves by using an agrobacterium-mediated leaf disc transformation method, wherein PtoWRKY11 vector is introduced into the wild aspen leaves;

(3) Through co-culture, selective culture, obtaining cluster buds, obtaining rooting transgenic plants and positive plant identification, the aspen strain with improved rapid growth capability of wood is finally obtained.

In some embodiments of the invention, the PtoWRKY gene has a nucleotide sequence shown as SEQ ID No. 2.

In some embodiments of the invention, the plant expression vector is a pCXSN-flag vector and the PtoWRKY gene sequence is constructed between the two XcmI cleavage sites of the pCXSN-flag vector.

In some embodiments of the invention, the agrobacterium is GV3101.

The invention has the beneficial effects that the PtoWRKY gene and the vector after optimization can effectively improve the xylem cell production speed of the aspen and the rapid growth of wood after transforming aspen plants, provide a method and a germplasm basis for breeding good varieties of forest, are beneficial to improving the wood yield and economic benefit of economic forest, and also provide materials for building protection forest, urban and rural greening and the like.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1 shows 35S of populus tomentosa, ptoWRKY S of transgenic plant, ptoWRKY plasmid schematic, B of positive transgenic plant, ptoWRKY of over-expressed gene expression of C. PtoWRKY 11.

FIG. 2 shows the growth phenotype analysis of 35S of PtoWRKY11 transgenic plant, the photographs of wild type and 35S of PtoWRKY transgenic plant of 3 month old populus tomentosa, the plant height statistics of wild type and transgenic plant, the diameter statistics of 10 internode of stem of wild type and transgenic plant, and the different letters indicate p <0.05.

FIG. 3 shows 35S of populus tomentosa, analysis of the division activity of the vascular cambium of PtoWRKY11 transgenic lines, imaging of transverse sections of vascular cambium (internode 8), statistics of the number of cell layers of vascular cambium, analysis of the division activity of vascular cambium, and p <0.05 indicated by different letters.

FIG. 4 shows 35S of the transgenic line PtoWRKY A. Xylem development control phenotype analysis, A. Xylem cross-section imaging (internode 10), B. Xylem cell layer count, C. Xylem area ratio count, and different letters indicate p < 0.05.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.

EXAMPLE 1 cloning of the aspen PtoWRKY Gene

The amino acid sequence of the PtrWRKY transcription factor of populus tomentosa (SEQ ID No. 1) and the CDS sequence (SEQ ID No. 2) were obtained by searching the amino acid sequence of the PtrWRKY transcription factor of populus tomentosa in phytozome database (https:// phytozome-next. Jgi. Gov /) (SEQ ID No. 006G072400) through amino acid sequence homology, and then aligning the amino acid sequence with the populus tomentosa library through TBtools software according to the obtained amino acid sequence in the database. We designed and synthesized the full coding frame upstream and downstream primers according to the CDS of PtoWRKY, as follows:

an upstream primer F-PtoWRKY11:5'-ATGGCTGTGGATCTAGTTAG-3' (SEQ ID No. 3);

the downstream primer R-PtoWRKY11:5'-TCAAGTTGAGTGAAACACGT-3' (SEQ ID No. 4).

The cDNA sequence of wild aspen was used as a template, cloning PtoWRKY gene was amplified by PCR using 5X PRIMESTAR BUFFER MIX, and sequence correctness and integrity were verified by sequencing. The PCR conditions were 98℃3 min, 98℃10 sec,56℃20 sec,72℃extension time 15 sec/kb for 35 cycles, 72℃10 min.

Agarose gel electrophoresis showed that the target size gene band was obtained, and sequencing results also confirmed that we cloned the correct PtoWRKY gene.

Example 2 construction of a plant expression vector for over-expression PtoWRKY11 (35S:: ptoWRKY 11)

In order to construct an over-expression PtoWRKY S11 vector, firstly, a pCXSN-flag vector with a CaMV35S promoter is selected, the vector is digested by restriction enzyme XcmI, agarose gel electrophoresis is carried out after the vector is digested at 37 ℃ overnight, a gel-cut linearization vector strip is cut, and a Biospin gel recovery kit is used for gel recovery to obtain a digested linearization vector fragment.

Then, a PtoWRKY gene primer with a carrier homology arm is designed at a carrier enzyme cutting site, a PtoWRKY gene with a pCXSN-flag carrier homology arm is amplified by using PtoWRKY amplified in the embodiment 1 as a template, agarose gel is subjected to gel recovery by using a Biospin gel recovery kit after amplification is completed, and finally PtoWRKY linearized gene fragment with a homology arm is obtained.

And (3) carrying out homologous recombination connection on the cut vector fragment and PtoWRKY with a homology arm by using a DNA fragment homologous recombination kit to obtain a connected pCXSN-flag vector with a PtoWRKY11 fragment. And (3) transforming the ligation product into escherichia coli by using a competent cell method, and detecting a monoclonal colony obtained by transformation by PCR. Inoculating the detected positive colony with LB liquid medium, shaking overnight at 37 ℃, obtaining a vector plasmid by using a Biospin plasmid extraction kit, and obtaining a recombinant plant expression vector containing PtoWRKY gene by enzyme digestion and sequencing verification, which is named pCXSN-35S: ptoWRKY11-flag vector (figure 1, A), hereinafter abbreviated as 35S: ptoWRKY11.

EXAMPLE 3 Agrobacterium tumefaciens-mediated 35S PtoWRKY genetic transformation of Populus tomentosa 11

The genetic transformation of poplar in this study was performed using wild aspen, and agrobacterium-mediated leaf disc dip-dyeing.

1) Agrobacterium transformation

PtoWRKY S PtoWRKY A was introduced into the Agrobacterium GV3101 strain by heat shock (37 ℃, 5min +liquid nitrogen, 2 min; three times repeatedly), transformation positive clones were selected by Kan resistance, and the colony of Agrobacterium GV3101 containing the recombinant plasmid was identified by colony PCR method (F-PtoWRKY primer and R-PtoWRKY primer amplification). Preserving the strain.

2) Cultivation of Agrobacterium

Streaking agrobacterium GV3101 strain containing recombinant plasmid on YEP solid culture medium (containing 20mg/ml Rif and 50mg/ml Kan), inversely culturing at 28 ℃, picking up monoclonal bacteria, inoculating the monoclonal bacteria into YEP liquid culture medium containing corresponding antibiotics, shake culturing at 28 ℃ until logarithmic growth OD600 is 0.6-0.8, inoculating activated agrobacterium in the same 50ml YEP liquid culture medium according to the proportion of 1:100-1:50, continuing culturing at 28 ℃ until OD ₆₀₀ is 0.6-0.8, adding the two-stage agrobacterium bacterial liquid into a 50ml centrifuge tube, centrifuging at 4 ℃ and 4000rpm for 10min, collecting bacterial precipitate, adding 10ml WPM heavy suspension (WPM powder+2.14 g+30 g sucrose+100 mu mol AS), mixing uniformly by a liquid-transferring gun, adding 20-30mlWPM heavy suspension, shake culturing at 28 ℃ and 200rpm for 40min-1h in a round mouth bottle.

3) Agrobacterium-mediated leaf disc dip-dyeing

And (3) taking tissue culture Miao Mojun blades, cutting the tissue culture Miao Mojun blades into leaf discs with the size of 0.5X0.5 cm ² on an ultra-clean workbench, putting the leaf discs into the re-suspended agrobacterium liquid, and dip-dying the leaf discs by 10min, and slightly shaking the bacterial liquid every 2-3 min to fully dip-dye the leaf discs.

4) Co-culture of populus tomentosa

The impregnated leaf discs are clamped out by sterilized tweezers, placed on sterilized filter paper, dried with bacterial liquid, laid flat on WPM co-culture medium (WPM powder+2.14 g+30 g sucrose+100 mu mol AS+1.0 mg NAA+2.0 mg ZT), and subjected to dark culture at 25 ℃ for 2 d.

5) Selective cultivation of populus tomentosa

After co-culturing for 2d, transferring the transformed explant to a callus-inducible selection medium (WPM powder+2.14g+30g sucrose+ 9 mg Hyg+1.0 mgNAA+2.0 mg ZT+400 mg Cef)), and culturing for 3-5 weeks at 25 ℃ in a dark place, wherein the selection medium is replaced with a new selection medium every 5 d days.

6) Populus tomentosa induced bud culture

When white blocky loose callus appears around the leaf margin, the callus is moved into WPM germination medium (WPM powder+2.14g+30g sucrose+ 9 mg Hyg+0.1 mg NAA+2.0 mg ZT+400 mg Cef) on an ultra-clean workbench, and the culture is carried out at 25 ℃ for about 4-5 weeks under illumination, and a new WPM germination medium is replaced every 10 d.

7) Rooting culture of populus tomentosa

When the adventitious bud is about 5 cm, the adventitious bud is transferred into WPM rooting medium (WPM+30 g sucrose+ 9 mg Hyg+0.1 mg NAA+400 mg Cef) containing corresponding antibiotics, and rooting is induced.

8) Transplanting of populus tomentosa

When the root system of the seedling is developed, the seedling is taken out, agar at the root is washed away, the seedling is transplanted into a greenhouse for culture, and the seedling is covered with a preservative film for heat preservation and moisture preservation, and the film is uncovered after the seedling grows for one week.

Yeast extract 10 g g, peptone 10 g, naCl 5 g, pH 7.0,121 ℃and high-temperature and high-pressure sterilization for 20 minutes. And adding 10-12 g of agar powder into the solid culture medium before sterilization.

LB medium, yeast extract 5 g, peptone 10 g, naCl 10 g, pH=7.0, 121℃high temperature sterilization 20 min. And adding 10-12 g of agar powder into the solid culture medium before sterilization.

EXAMPLE 4 selection and identification of transgenic line PtoWRKY11 of aspen 35S overexpressing PtoWRKY11

1.35S PtoWRKY identification of transgenic positive plants

The transgenic plants obtained after tissue culture need to extract the genomic DNA of each plant for positive identification to confirm whether the vector is successfully transferred into the poplar plant.

The extraction method of the poplar genome DNA by the CTAB method comprises the following steps:

1) Taking one 10mL centrifuge tube, adding 3 mL CTAB and 90 mu L of beta-mercaptoethanol, and preheating in a 65 ℃ water bath;

2) Grinding fresh leaves of aspen of about 0.6 g into powder in liquid nitrogen, transferring into the CTAB extract, mixing by vortex, or placing the leaves into a 2mL centrifuge tube, adding CTAB extract and small steel balls (3) and crushing by a tissue crusher;

3) Preheating 45 min in a 65 ℃ water bath kettle, and uniformly mixing three times at intervals (violently) in a shaking way;

4) After the time is up, the mixture is taken out to cool 5min and then is subjected to 12000 and rpm, and is centrifuged to 10 and min;

5) Carefully sucking the supernatant into another clean 1.5 mL centrifuge tube, standing at room temperature for 5min, adding equal volume of chloroform/isophenol (24:1), mixing, emulsifying at 18deg.C after emulsifying 10min, and centrifuging at 12000 rpm for 10 min;

6) Repeating the step 5 for one time to remove protein better;

7) Sucking the supernatant into another clean 10 mL centrifuge tube, adding isopropyl alcohol precooled at equal volume-20 ℃, reversing and uniformly mixing to obtain white flocculent precipitate, 12000 rpm, and centrifuging 10 min;

8) Rinsing twice with 500 μl of 75% (V/V) ethanol, rinsing again with 500 μl of anhydrous ethanol, pipetting the ethanol with a pipette, and drying the precipitate in a 37 ℃ oven until transparent;

9) Dissolving the precipitate with 50-100 μl of sterile water containing RNaseA to obtain genomic DNA of Populus tomentosa.

Next, using genomic DNA of each strain as a template and the wild type as a control,

PCXSN-F:5’ -TCATCGCAAGACCGGCAACA-3’(SEQ ID No.5);

R-PtoWRKY11:5'-TCAAGTTGAGTGAAACACGT-3'(SEQ ID No.4)

And amplifying the specific fragment of the recombinant plasmid by PCR. The amplified product was detected by agarose gel electrophoresis, and the presence or absence of a band at the corresponding position was observed. As a result, it was revealed that the multiple regenerated strains each contained the exogenously introduced gene fragment of interest, demonstrating that the 35S:: ptoWRKY11 transgenic aspen strain was successfully obtained by genetic transformation (FIG. 2, A).

2.35S PtoWRKY screening of the overexpressed lines

To screen PtoWRKY11 over-expressed transgenic lines, plants which were over-expressed PtoWRKY were selected from 35S: ptoWRKY11 transgenic positive lines by quantitative PCR (qRT-PCR) for subsequent analysis of wood development.

Firstly, extracting 35S by using a Biospin polysaccharide polyphenol plant total RNA extraction kit, wherein the total RNA of PtoWRKY11 transgenic positive aspen plants is extracted by the following specific steps:

490 μ L BGLysis and 10 μL of β -mercaptoethanol were added to a 1.5mLRNase-Free EP tube to a final concentration of 2% and mixed well.

Liquid nitrogen grinding, namely grinding a proper amount of tissue into powder in liquid nitrogen, weighing 50-100mg of the powder, putting the powder into the centrifuge tube which is provided with BGLysis and 1.5mL of beta-mercaptoethanol, and immediately and severely oscillating until no obvious particulate matters exist.

The samples were centrifuged in a centrifuge at 12,000 rpm for 5 minutes and the supernatant carefully aspirated into a new 1.5mL centrifuge tube of RNase-Free.

The supernatant volume of the lysate is estimated more precisely, and 0.5 times of absolute ethanol is added, at which time precipitation may occur, but the extraction process is not affected, and immediately shaking and mixing are performed.

The mixture was all aspirated into Spin column and centrifuged at 12,000rpm for 1 min to discard the liquid in the receiver tube.

600 Mu LPG Bufer was added to Spin column and centrifuged at 12,000rpm for 30 seconds, and the liquid in the receiver tube was discarded.

500 Mu L Wash Bufier was added to Spin column and centrifuged at 12,000pm for 30 seconds, and the liquid in the receiver tube was discarded. The Wash was repeated once with the addition of 250. Mu.L Wash Buffer.

The column was then centrifuged at 12000rpm for 1 minute to remove the Wash Buffer as much as possible, so as to prevent the residual ethanol in the Wash Buffer from inhibiting downstream reactions.

The Spin column was transferred to a new 1.5mL RNase-Free centrifuge tube, RElution Buffer. Mu.L was added to the center of the membrane, and the membrane was allowed to stand at room temperature for 2 minutes, at 12,000 rpm for 1 minute, and the first eluate was returned to the Spin column membrane, and was allowed to stand at room temperature for 2 minutes, and at 12,000 rpm for 1 minute, to obtain total RNA.

Next, cDNA was obtained by reverse transcription, and the procedure was as follows according to the instructions of the long-chain RNA reverse transcription kit provided by TaKaRa Corp:

(1) Adding 2 mu L of 5× GDNA ERASER Buffer and 1 mu L GDNA ERASER into an EP tube, adding RNA, calculating the amount of RNA according to 1000 ng/concentration, adding RNASE FREE DDH O, supplementing to 10 mu L, and uniformly mixing;

(2) After heat preservation at 42 ℃ for 2 min, rapidly placing the mixture on ice for cooling;

(3) Sequentially adding 4 μL of 5× PRIME SCRIPT Buffer 2, 1 μ L PRIME SCRIPT RT Enzyme Mix I, specific stem-loop primer and U6 RT primer 0.5 μL each, 1 μ L RT PRIMER Mix, 4 μ L RNASE FREE DDH2O to the EP tube in step 2, and mixing;

(4)42°C,15 min;85°C,5 sec;

(5) Finally, the mixture is stored in a refrigerator at the temperature of-20 ℃.

Finally, specific quantitative primers were designed to reverse transcribe poplar cDNA templates for qRT-PCR amplification. RT-qPCR was performed according to the operation instructions of the fluorescent quantitative kit GoTaq QPCR MASTER Mix, thereby detecting the expression level of PtoWRKY gene. The PCR conditions were 95℃for 30 sec, 40 cycles of 95℃for 5 sec, 60℃for 1 min, 95℃for 15 sec, 60℃for 30 sec, 95℃for 15 sec.

The quantitative primers were as follows:

RT-PtoWRKY11-F:5’-TGAGGGGATGTCCCGCAAGG-3’(SEQ ID No.6);

RT-PtoWRKY11-R:5’-GCCGTGACGTCTTCCGGCAA-3’(SEQ ID No.7)。

Experimental results show that in the 35S PtoWRKY transgenic aspen strain, compared with the wild type, a plurality of strains are obviously over-expressed PtoWRKY. Of these, the expression levels of L1 and L2 plants were highest, 8.7-fold and 16.1-fold, respectively, in the wild type (fig. 1, c).

EXAMPLE 5 analysis of Wood development regulatory function of the PtoWRKY11 transgenic line of Populus tomentosa 35S

In order to explore the regulation and control of PtoWRKY11 over-expression on the development of the secondary vascular of poplar, two strains with the highest relative expression amount are selected for subsequent experiments, namely an L1 strain and an L2 strain in a wild type background. In order to elucidate the function of PtoWRKY11 in stem vascular secondary development, selected positive plants were sectioned after about 3 months of cultivation, the development of the plant cambium and xylem was observed after staining with toluidine blue dye solution, and related indexes were analyzed by Image J software. The results are shown in fig. 3 and 4.

The invention screens the regulatory factor PtoWRKY11 from poplar to WOX4 upstream and clones the gene. An over-expression vector with PtoWRKY.sup.11 sequence was constructed by genetic engineering (35S:: ptoWRKY, FIG. 1, A). Through genetic transformation of populus tomentosa and screening to obtain transgenic over-expression lines, two over-expression lines of PtoWRKY11 with up-regulated expression levels 8.7 times and 16.1 times compared with wild type are obtained, wherein 35S is PtoWRKY-L1 and 35S is PtoWRKY-L2 (figure 1, C). Over-expressed plants 35S:: ptoWRKY11-L1 and 35S:: ptoWRKY-L2 grown for 3 months showed no significant difference in plant height compared to wild type plants, but the stem diameter was significantly thicker, increased by 18.9% and 22.7%, respectively (FIGS. 2, B and C). Further analysis of wood development of the overexpressing strain showed that 35S:: ptoWRKY11 transgenic aspen strain grown for 3 months had significantly improved cambium division activity compared to wild type (FIGS. 3, A and C), 35S::: ptoWRKY11-L1 and 35S:: ptoWRKY-L2 plants had 18.6% and 28.0% increase in cambium cell number, respectively, compared to wild type plants (FIG. 3, B). Meanwhile, the number of xylem cells is obviously increased in 35S of PtoWRKY11-L1 and 35S of PtoWRKY-L2 transgenic populus tomentosa compared with wild plants, 56.1% and 72.9% of the xylem area of the transgenic populus tomentosa are respectively increased (figures 4 and B), and 27.4% and 45.3% of the xylem area of the transgenic populus tomentosa are respectively increased in 35S of PtoWRKY compared with wild plants in L1 and L2 (figures 4 and C), the differences are high in significance (p < 0.001), and the poplar variety for promoting wood formation is obtained by adopting a method for transforming PtoWRKY genes, so that the method provides superior raw materials for industrial production of paper and the like.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (9)

1. The transcription factor PtoWRKY for regulating the secondary development of the stem vascular of populus tomentosa is characterized in that the amino acid sequence of the transcription factor PtoWRKY11 is shown as SEQ ID No. 1.

2. The gene for encoding the populus tomentosa stem vascular secondary development regulation transcription factor PtoWRKY as set forth in claim 1, wherein the nucleotide sequence is shown in SEQ ID No. 2.

3. A plant expression vector containing PtoWRKY gene as defined in claim 2, wherein PtoWRKY gene sequence shown in SEQ ID No.2 is constructed between two XcmI cleavage sites of pCXSN-flag vector.

4. Use of the PtoWRKY gene according to claim 2 or the plant expression vector according to claim 3 for regulating the rapid growth of aspen wood.

5. The method of claim 4, wherein the vascular cambium division activity of the poplar is enhanced by over-expression of PtoWRKY gene in the poplar, and the formation of secondary xylem is accelerated, thereby improving the xylem cell production rate of the poplar and the rapid growth ability of wood.

6. A method for constructing a transgenic aspen strain with improved rapid growth capability of wood is characterized by comprising the following steps:

(1) Cloning the gene encoding PtoWRKY11, and connecting to a plant expression vector to obtain a plant expression vector 35S of which the gene PtoWRKY is overexpressed, wherein PtoWRKY11;

(2) Transforming agrobacterium, and introducing the 35S constructed in the step (1) into wild aspen leaves by using an agrobacterium-mediated leaf disc transformation method, wherein PtoWRKY11 vector is introduced into the wild aspen leaves;

(3) Through co-culture, selective culture, obtaining cluster buds, obtaining rooting transgenic plants and positive plant identification, the aspen strain with improved rapid growth capability of wood is finally obtained.

7. The method according to claim 6, wherein in the step (1), the nucleotide sequence of PtoWRKY gene is shown in SEQ ID No. 2.

8. The method according to claim 6, wherein in the step (1), the plant expression vector is a pCXSN-flag vector, and the PtoWRKY gene is constructed between two XcmI cleavage sites of the pCXSN-flag vector.

9. The method of claim 6, wherein in step (2), the Agrobacterium is GV3101.