Application and method of PtobZIP29 gene in regulating and controlling wood development of populus tomentosa

Abstract

The invention discloses application and a method of a PtobZIP29 gene in regulating and controlling wood development of populus tomentosa, the PtobZIP29 gene is excessively expressed in the populus tomentosa, so that the vascular cambium division activity of the populus tomentosa is promoted, and the formation of secondary xylem is accelerated, thereby obtaining transgenic populus tomentosa germplasm with increased wood biomass and improved wood fast-growing ability. A method and a germplasm basis are provided for breeding of forest fast-growing good varieties, the wood yield and economic benefits of economic forests can be improved, and materials are provided for building of protection forests, urban and rural greening and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application and method of a PtobZIP29 gene in regulating the development of white poplar wood. Background Art

[0002] Wood is a valuable asset for human society and has been used to make building materials and furniture, as well as paper, packaging materials and many other materials. Wood is now also considered a viable bioenergy alternative to fossil fuels and an ideal raw material to replace plastics and chemical products. In addition, forests fix about 82% of terrestrial biomass, or about 450 billion tons of carbon in wood (Bar-On et al., 2018), making it an important carbon dioxide sink for mitigating global warming. The high capacity of wood carbon storage and net zero carbon emissions make wood utilization an effective strategy to reduce the increase in global carbon dioxide concentrations. However, based on the low forest coverage rate in my country, the supply and demand of wood is tight. Therefore, cultivating fast-growing trees with excellent wood quality is of great significance to forest breeding. Poplar (Populus. spp) is one of the most widely distributed and adaptable fast-growing tree species in the world. Therefore, poplar, as an important source of wood supply in my country, has played an important role in economic development and ecological environment improvement. At the same time, since the completion of the whole genome sequencing of Populus trichocarpa in 2006, a stable genetic transformation and regeneration system has been established. Poplar has also become a model plant for molecular biology and other research, playing an important role in various molecular pathways and transgenic breeding research.

[0003] In trees such as poplar, wood is derived from secondary growth of stem vascular tissue, which is based on the division activity of the vascular cambium. The activity of the vascular cambium produces secondary phloem and secondary xylem. Therefore, the ability of cambium cells to divide and differentiate during secondary vascular development is crucial for the diameter growth of stems, branches and trunks of woody plants, and is also one of the key control factors for the rate of wood production. Therefore, understanding how plants regulate cambium activity is important for producing trees with improved wood growth.

[0004] In recent years, the molecular regulatory mechanism of vascular cambium proliferation and differentiation has become a hot topic in the fields of biology and forestry, and great progress has been made in the molecular mechanism of vascular cambium growth and development. Studies have shown that in the process of cambium growth and development, some plant hormones, small peptides and related transcription factors work together to tightly regulate the activity of vascular cambium. The activity of vascular cambium is regulated by several hormones, including auxin (IAA), cytokinin (CK), ethylene (ET), brassinolide (BR), gibberellin (GA), etc. Their coordination can promote cambium proliferation and wood development. Many evidences show that the peak accumulation of auxin mainly occurs in poplar cambium stem cells, and the polar flow of auxin is related to its output transporter PIN1 protein, indicating that the up-regulation of PIN1 gene expression promotes cambium activity and secondary growth. In addition, transcription factors also play a vital role in regulating vascular development. WOX4, a member of the WOX gene family, acts as a regulatory hub for cambium activity and regulates cambium activity. WOX4 protein positively regulates the proliferation and differentiation of poplar cambium cells.

[0005] The bZIP gene family is widely present in plants and is involved in a variety of biological processes, including growth and development and stress response. The transcription factor proteins encoded by the bZIP gene family contain a highly conserved bZIP domain. The structure consists of 60-80 amino acids, including a DNA binding region and an adjacent leucine zipper structure. The binding region contains a nuclear localization signal and an N-X7-R / K motif, which precisely binds to the target DNA. Studies have found that the Arabidopsis bZIP family includes 13 branches, from AM, each branch has its own characteristics, and a less distinctive bZIP branch is family I, which contains 13 members in Arabidopsis, sharing a lysine residue in the basic domain, replacing a highly conserved arginine residue. In several homologous plants, studies have shown that bZIP I subfamily proteins are involved in plant root development, environmental stress response, etc. However, the regulatory function in wood formation in woody plants is still unclear. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide an application of the PtobZIP29 gene in regulating the wood development of Populus tomentosa; a second object of the present invention is to provide a plant expression vector for overexpressing the PtobZIP29 gene; and a third object of the present invention is to provide a method for constructing a transgenic Populus tomentosa strain with increased wood biomass.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: Application of PtobZIP29 gene in regulating wood development of Populus tomentosa By overexpressing the PtobZIP29 gene in Populus tomentosa, the vascular cambium division activity of the poplar is promoted and the formation of secondary xylem is accelerated, thereby obtaining transgenic Populus tomentosa germplasm with increased wood biomass and improved wood fast-growing ability; the amino acid sequence of the PtobZIP29 gene is shown in SEQ ID No.1.

[0008] In some embodiments of the present invention, the nucleotide sequence of the PtobZIP29 gene is shown as SEQ ID No.2.

[0009] The plant expression vector used for overexpressing the PtobZIP29 gene is constructed by inserting the sequence shown in SEQ ID No. 1 between the Bam HI and Xba I restriction sites of the pCAMBIA1300-GFP vector.

[0010] A method for constructing a transgenic Populus tomentosa strain with increased wood biomass comprises the following steps: (1) After cloning the PtobZIP29 gene, it was connected to a plant expression vector to obtain a plant expression vector 35S::PtobZIP29 that overexpresses the PtobZIP29 gene; (2) Transforming 35S::PtobZIP29 into Agrobacterium, and introducing the 35S::PtobZIP29 vector constructed in step (1) into leaves of wild-type Populus tomentosa by using the Agrobacterium-mediated leaf disc transformation method; (3) Through the process of co-cultivation, selective cultivation, obtaining clustered shoots, obtaining rooted transgenic plants, and positive plant identification, we finally obtained a transgenic Populus tomentosa strain with increased wood biomass.

[0011] In some embodiments of the present invention, in step (1), the nucleotide sequence of the PtobZIP29 gene is as shown in SEQ ID No.2.

[0012] In some embodiments of the present invention, in step (1), the plant expression vector is a pCAMBIA1300-GFP vector, which is connected via BamhI and XbaI restriction sites.

[0013] In some embodiments of the present invention, in step (2), the Agrobacterium is GV3101.

[0014] The beneficial effect of the present invention is that after the PtobZIP29 gene and vector are transformed into Populus tomentosa plants, the radial growth of Populus tomentosa stems can be effectively improved, the vascular cambium division activity of Populus tomentosa is promoted, the formation of secondary xylem is accelerated, and the fast-growing ability and biomass of wood are improved. This provides a method and germplasm basis for the selection and breeding of fast-growing and excellent varieties of forest trees, is conducive to improving the wood yield and economic benefits of economic forests, and also provides materials for the construction of protective forests and urban and rural greening. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration: Figure 1 It is a 35S::PtobZIP29 overexpression vector.

[0016] Figure 2 Screening and identification of 35S::PtobZIP29 transgenic plants of Populus tomentosa; A. Identification of transgenic positive lines; B. Screening of overexpression lines.

[0017] Figure 3 Figure 2 shows the stem growth phenotype analysis of 35S::PtobZIP29 transgenic lines of Populus tomentosa. A. Photos of 3-month-old wild-type and 35S::PtobZIP29 transgenic plants of Populus tomentosa. B. Plant height statistics of wild-type and transgenic plants. C. Statistics of stem internode 10 diameter of wild-type and transgenic plants. “ns” means no significant difference. p<0.01. “**” means p<0.01.

[0018] Figure 4 Analysis of vascular cambium division activity in Populus tomentosa 35S::PtobZIP29 transgenic lines; A. Imaging of cross-section of vascular cambium (internode 8); B. Analysis of vascular cambium division activity; C. Statistics of vascular cambium cell layers; "***" indicates p<0.001; "****" indicates p<0.0001.

[0019] Figure 5 Phenotypic analysis of xylem development regulation in the transgenic line of Populus tomentosa 35S::PtobZIP29; A. Stem cross section imaging (internode 8); B. Xylem cross section (enlarged); C. Xylem width statistics; D. Xylem cell layer statistics; "**" indicates p<0.01; "***" indicates p<0.001; "****" indicates p<0.0001. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0021] Example 1: Cloning of Populus tomentosa PtobZIP29 gene Through amino acid sequence homology, the amino acid sequence of the bZIP29 transcription factor of Populus tomentosa was found in the phytozome database (https: / / phytozome-next.jgi.doe.gov / ) (sequence number Potri.004G163800), and then the amino acid sequence obtained in the database was compared with the Populus tomentosa library using TBtools software to obtain the amino acid sequence of the Potri.004G1638001 transcription factor of Populus tomentosa (as shown in SEQ ID No.1) and the CDS sequence (as shown in SEQ ID No.2). We designed and synthesized the upstream primer and downstream primer of the complete coding frame based on the CDS of PtobZIP29, and the specific operations were as follows: Upstream primer F-PtobZIP29: 5′-ATGGGTGATACTGAGGAAGCAAA-3′ (SEQ ID No. 3); Downstream primer R-PtobZIP29: 5′-CTATTGATTTGAATCTGGCTTTGG-3′ (SEQ ID No. 4).

[0022] The wild-type Populus tomentosa cDNA sequence was used as a template, and the PtobZIP29 gene was cloned by PCR amplification using 5× PrimeSTAR Buffer Mix, and the sequence was verified to be correct and complete by sequencing. The PCR reaction conditions were: 98℃ for 3 min; 98℃ for 10 sec, 56℃ for 20 sec, 72℃ for 15 sec / kb extension time, for a total of 35 cycles; 72℃ for 10 min.

[0023] Agarose gel electrophoresis showed that a gene band of the target size was obtained, and sequencing results also verified that we had cloned the correct PtobZIP29 gene.

[0024] Example 2: Construction of a plant expression vector for overexpressing PtobZIP29 (35S::PtobZIP29) (1) To construct the overexpression PtobZIP29 vector, we first selected the pCAMBIA1300-GFP vector with the CaMV35S promoter and double-digested the vector with restriction endonucleases Bam HI and Xba I. After the vector was digested overnight at 37°C, it was subjected to agarose gel electrophoresis. The linearized vector band was cut and recovered using a Biospin gel recovery kit to obtain the linearized vector backbone fragment.

[0025] (2) Then, PtoWRKY11 gene primers with vector homology arms were designed at the vector restriction site, which are: Upstream primer PtobZIP29-F-BamHI: 5'-agctcggtacccggggatccATGGGTGATACTGAGGAAGCAAA -3' (SEQ ID No. 5); Downstream primer PtobZIP29-R-XbaI: 5′-ctcaccatgtcgactctagaTTGATTTGAATCTGGCTTTGGG-3′ (SEQ ID No. 6).

[0026] (3) Using the above primers and the PtobZIP29 gene amplified in Example 1 as a template, PCR amplified the PtobZIP29 gene with the pCAMBIA1300-GFP homology arm. Then, agarose gel electrophoresis and gel recovery using a Biospin gel recovery kit were performed to finally obtain the linearized gene fragment of PtobZIP29 with the homology arm.

[0027] (4) Use a DNA fragment homologous recombination kit to homologously recombinantly connect the restriction-cut vector backbone fragment and the PtobZIP29 fragment with homologous arms to obtain the connected pCAMBIA1300-GFP with the PtobZIP29 fragment. The ligation product is transformed into Escherichia coli using the competent cell method, and the transformed monoclonal colony is tested by PCR. The detected positive colony is inoculated with LB liquid culture medium and shaken at 37°C overnight.

[0028] (5) The vector plasmid was then obtained using the Biospin plasmid extraction kit. The recombinant plant expression vector containing the PtobZIP29 gene was obtained by restriction digestion and sequencing verification and was named 35S::PtobZIP29 vector ( Figure 1 ).

[0029] Example 3: Agrobacterium tumefaciens-mediated genetic transformation of Populus tomentosa with 35S::PtobZIP29 The material used for poplar genetic transformation in this study was wild-type Populus tomentosa, and the Agrobacterium-mediated leaf disc infection method was used for Populus tomentosa genetic transformation.

[0030] 1) Pseudomonas aeruginosa transformation The 35S::PtoWRKY11 vector in Example 2 was introduced into the Agrobacterium GV3101 strain by heat shock method (37°C, 5 min + liquid nitrogen, 2 min; repeated three times), and the transformation positive clones were screened by Kan resistance; and the Agrobacterium GV3101 colonies containing the recombinant plasmid were identified by colony PCR method (F-PtobZIP29 and R-PtobZIP29 primers amplification). The strain was preserved.

[0031] 2) Cultivation of Agrobacterium The Agrobacterium GV3101 strain containing the recombinant plasmid was streaked on YEP solid medium (containing 20 mg / ml Rif and 50 mg / ml Kan) and inverted at 28°C for culture; a single clone was selected and inoculated into YEP liquid medium containing the corresponding antibiotics and cultured at 28°C with shaking until the logarithmic growth OD600 was 0.6-0.8; then the activated Agrobacterium was inoculated into the same 50 ml YEP liquid medium at a ratio of 1:100~1:50 and cultured at 28°C until the OD 600 The pH value is 0.6-0.8. Add the diactive Agrobacterium culture liquid into a 50ml centrifuge tube. Centrifuge at 4°C, 4000rpm for 10min. Collect the bacterial precipitate. Add 10ml WPM resuspension (WPM powder + 2.14g + 30g sucrose + 100μmol AS). Mix well with a pipette. Add 20-30ml WPM resuspension. Transfer into a round-mouth bottle and culture at 28°C, 200rpm for 40min-1h.

[0032] 3) Agrobacterium-mediated leaf disc infection Take the sterile leaves of the tissue culture seedlings and cut them into 0.5×0.5 cm pieces on the clean bench. 2 Place leaf discs of different sizes into the resuspended Agrobacterium solution and infect for 10 minutes. Gently shake the solution every 2 to 3 minutes to ensure that the leaf discs are fully infected.

[0033] 4) Co-cultivation of Populus tomentosa Pick out the infected leaf disc with sterilized tweezers, place it on sterilized filter paper, absorb the bacterial solution, spread the leaf disc flat on WPM co-cultivation medium (WPM powder + 2.14 g + 30 g sucrose + 100 μmol AS + 1.0 mg NAA + 2.0 mg ZT), and culture it in the dark at 25°C for 2 days.

[0034] 5) Selective cultivation of Populus tomentosa After 2 days of co-cultivation, the transformed explants were transferred to a selective medium for inducing callus (WPM powder + 2.14 g + 30 g sucrose + 9 mg Hyg + 1.0 mg NAA + 2.0 mg ZT + 400 mg Cef) and cultured at 25°C in the dark for 3 to 5 weeks, during which time the selective medium was replaced every 5 days.

[0035] 6) Populus tomentosa induced bud culture When white loose callus tissue appears around the leaf margin, move the callus tissue to WPM budding medium (WPM powder + 2.14 g + 30 g sucrose + 9 mg Hyg + 0.1 mg NAA + 2.0 mg ZT + 400 mg Cef) on the clean bench and culture at 25°C under light for about 4 to 5 weeks, replacing new WPM budding medium every 10 days.

[0036] 7) Rooting culture of Populus tomentosa When the adventitious buds grew to about 5 cm, they were transferred to WPM rooting medium (WPM + 30 g sucrose + 9 mg Hyg + 0.1 mg NAA + 400 mg Cef) containing corresponding antibiotics to induce rooting.

[0037] 8) Transplantation of Populus tomentosa When the root system of the seedlings is relatively developed, take out the seedlings, rinse off the agar on the roots, transplant them into the greenhouse for cultivation, and cover them with plastic wrap to keep warm and moist. Remove the film after one week of growth.

[0038] YEP medium: 10 g yeast extract, 10 g peptone, 5 g NaCl, pH 7.0, sterilized at 121°C for 20 minutes. For solid medium, add 10-12 g agar powder before sterilization.

[0039] LB medium: 5 g yeast extract, 10 g peptone, 10 g NaCl, pH = 7.0, sterilized at 121 °C for 20 min. For solid medium, add 10-12 g agar powder before sterilization.

[0040] Example 4 Screening and identification of Populus tomentosa 35S::PtobZIP29 transgenic strains 1. Identification of 35S::PtobZIP29 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 has been successfully transferred into the poplar plant.

[0041] The CTAB method for extracting poplar genomic DNA is as follows: 1) Take a 10 mL centrifuge tube, add 3 mL CTAB and 90 μL β-mercaptoethanol, and preheat in a 65°C water bath; 2) Take about 0.6 g of fresh leaves of Populus tomentosa, grind them into powder in liquid nitrogen, transfer them into the above CTAB extract, and vortex to mix, or place the leaves in a 2 mL centrifuge tube, add CTAB extract and small steel balls (3 pieces), and then crush them with a tissue disruptor; 3) Preheat in a 65°C water bath for 45 min, shaking (vigorously) three times to mix; 4) After the time is up, take it out and cool it for 5 minutes, then centrifuge it at 12000 rpm for 10 minutes; 5) Carefully pipette the supernatant into another clean 1.5 mL centrifuge tube, let stand at room temperature for 5 min, then add an equal volume of chloroform:isoform alcohol (24:1), invert vigorously to mix, then place flat to emulsify for 10 min, then centrifuge at 18°C, 12,000 rpm for 10 min; 6) Repeat step 5 once to better remove the protein; 7) Pipette the supernatant into another clean 10 mL centrifuge tube, add an equal volume of -20°C pre-cooled isopropanol, invert and mix until a white flocculent precipitate is visible, and centrifuge at 12,000 rpm for 10 min; 8) Rinse twice with 500 μL 75% (V / V) ethanol, rinse once with 500 μL anhydrous ethanol, drain the ethanol with a pipette, and dry the precipitate in a 37°C oven until transparent; 9) Dissolve the precipitate with 50-100 μL of sterile water containing RNaseA to obtain the genomic DNA of Populus tomentosa.

[0042] Next, using the genomic DNA of each strain as a template and the wild type as a control, pCAMBIA1300-F:5'-GGGACTGAGCTCGGTACCC-3' (SEQ ID No. 7); R-PtobZIP29: 5'-CTATTGATTTGAATCTGGCTTTGG-3' (SEQ ID No. 4) PCR amplified the specific fragment of the recombinant plasmid. The amplified product was detected by agarose gel electrophoresis to observe the presence of the corresponding position band. The results showed that multiple regenerated strains contained the exogenously introduced target gene fragment, proving that the transgenic Populus tomentosa strain of 35S::PtobZIP29 was successfully obtained through genetic transformation ( Figure 2 , A).

[0043] 2. Screening of 35S::PtobZIP29 overexpression strains In order to screen transgenic lines with overexpression of PtobZIP29, it is planned to screen plants with overexpression of PtobZIP29 from 35S::PtobZIP29 transgenic positive lines by quantitative PCR (qRT-PCR) for subsequent analysis of wood development.

[0044] First, the total RNA of the 35S::PtobZIP29 transgenic positive Populus tomentosa plants was extracted using the Biospin polysaccharide and polyphenol plant total RNA extraction kit. The specific steps are as follows: Add 490 μL BGLysis and 10 μL β-mercaptoethanol to a 1.5 mL RNase-Free EP tube to a final concentration of 2% and mix well.

[0045] Liquid nitrogen grinding: Grind an appropriate amount of tissue into powder in liquid nitrogen, weigh 50-100 mg and put it into the above-mentioned 1.5 mL centrifuge tube containing BGLysis and β-mercaptoethanol, and immediately shake vigorously until there are no obvious particles.

[0046] Centrifuge the above samples at 12,000 rpm for 5 minutes and carefully pipette the supernatant into a new RNase-Free 1.5 mL centrifuge tube.

[0047] Estimate the volume of the lysate supernatant more accurately and add 0.5 times the volume of anhydrous ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Shake and mix immediately.

[0048] The entire mixture was pipetted into the Spin column, centrifuged at 12,000 rpm for 1 minute, and the liquid in the collecting tube was discarded.

[0049] Add 600 μL PG Bufer to the spin column, centrifuge at 12,000 rpm for 30 seconds, and discard the liquid in the collecting tube.

[0050] Add 500μL Wash Buffer to the spin column, centrifuge at 12,000pm for 30 seconds, discard the liquid in the tube, and add 250μL Wash Buffer to repeat the wash.

[0051] Then centrifuge the empty column at 12000rpm for 1 minute to remove the Wash Buffer as much as possible to prevent the residual ethanol in the Wash Buffer from inhibiting the downstream reaction.

[0052] Move the spin column into a new 1.5mL RNase-Free centrifuge tube, add 25μL of RElutionBuffer to the center of the membrane, let it stand at room temperature for 2 minutes, centrifuge at 12,000rpm for 1 minute, add the first eluate back to the spincolumn membrane, let it stand at room temperature for 2 minutes, centrifuge at 12,000rpm for 1 minute to obtain total RNA.

[0053] Next, cDNA was obtained by reverse transcription according to the instructions of the long-chain RNA reverse transcription kit provided by TaKaRa. The specific steps are as follows: (1) Add 2 µL 5×gDNA Eraser Buffer and 1 µL gDNA Eraser to the EP tube, then add RNA (the amount of RNA is calculated as 1000 ng / concentration), and finally add RNase Free ddH2O to make up to 10 µL and mix well; (2) After keeping at 42°C for 2 min, quickly cool on ice; (3) Add 4 µL 5× Prime Script Buffer 2, 1 µL Prime Script RT EnzymeMix I, 0.5 µL each of the specific stem-loop primer and U6 RT primer, 1 µL RT Primer Mix, and 4 µL RNase FreeddH2O to the EP tube in step 2 and mix well. (4) 42℃, 15 min; 85℃, 5 sec; (5) Finally, store in a -20℃ refrigerator.

[0054] Finally, specific quantitative primers were designed and qRT-PCR amplification was performed with the reverse transcribed poplar cDNA template. RT-qPCR was performed according to the instructions of the fluorescence quantitative kit GoTaq® qPCR Master Mix to detect the expression level of the PtobZIP29 gene. The PCR reaction conditions were: 95℃, 30 sec; 40 cycles: 95℃, 5 sec; 60℃, 1 min; 95℃, 15 sec; 60℃, 30 sec; 95℃, 15 sec.

[0055] The quantitative primers are as follows: PtobZIP29-qPCR-F: 5'-TCTTTTACAGGTAGCGATGTGA-3' (SEQ ID No. 8); PtobZIP29-qPCR-R: 5'-AATGCCTCGTTTAAAGCATCTC - 3' (SEQ ID No. 9).

[0056] The experimental results showed that among the transgenic Populus tomentosa lines of 35S::PtobZIP29, many lines significantly overexpressed PtobZIP29 compared with the wild type. Among them, the expression levels of L1 and L2 plants were the highest, reaching 2.65 times and 3.94 times that of the wild type, respectively ( Figure 2 , B).

[0057] Example 5: Analysis of wood development regulation function of Populus tomentosa 35S::PtobZIP29 transgenic strain In order to explore the regulation of PtobZIP29 overexpression on secondary vascular development in poplars, the two strains with the highest relative expression levels were selected for subsequent experiments, namely the L1 strain and the L2 strain under the wild-type background. In order to clarify the function of PtobZIP29 in the secondary development of stem vascular, the selected positive plants were cultured for about 3 months and then sectioned. The development of the plant cambium and xylem was observed after staining with toluidine blue dye, and the relevant indicators were analyzed using Image J software. Figure 4 and Figure 5 shown.

[0058] The present invention screened the regulatory factor PtobZIP29 upstream of WOX4 from poplar and cloned its gene. Using genetic engineering methods, an overexpression vector (35S: PtobZIP29, Figure 1 ). By genetically transforming Populus tomentosa and screening to obtain transgenic overexpression strains ( Figure 2 , A), obtained two overexpression strains of PtobZIP29, whose expression levels were upregulated by 2.65 and 3.94 times compared with the wild type: 35S::PtobZIP29-L1 and 35S::PtobZIP29-L2 ( Figure 2 , B). Further analysis of the wood development of the overexpression strains showed that there was no significant difference in plant height between the 35S::PtobZIP29 transgenic white poplar strains grown for 3 months and the wild type ( Figure 3 , B), while the stem diameter increased significantly, with L1 and L2 plants increasing by 9.4% and 12.0%, respectively, compared with the wild type ( Figure 3 , C). Slice observation revealed that overexpression of PtobZIP29 promoted the division activity of the stem vascular cambium ( Figure 4 , A and B), and resulted in an increase of 37.7% and 48.8% in the number of cambium cells in the L1 and L2 overexpression lines, respectively, compared with the wild type ( Figure 4, C). At the same time, the xylem width of 35S::PtobZIP29 transgenic white poplar increased by 33.5% and 36.6% in L1 and L2 plants compared with the wild type, respectively ( Figure 5 , A and C), the differences were highly significant (p < 0.01); the number of xylem cell layers in L1 and L2 transgenic white poplars was also significantly increased compared with the wild type, increasing by 10.6% and 12.6%, respectively ( Figure 5 , B and D). The method of transforming the PtobZIP29 gene was used to obtain a poplar variety that promotes wood formation, providing excellent raw materials for industrial production such as paper production.

[0059] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Application of PtobZIP29 gene in regulating wood development of Populus tomentosa, characterized by: By overexpressing the PtobZIP29 gene in Populus tomentosa, the vascular cambium division activity of the poplar is promoted and the formation of secondary xylem is accelerated, thereby obtaining transgenic Populus tomentosa germplasm with increased wood biomass and improved wood fast-growing ability; the amino acid sequence of the PtobZIP29 gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the PtobZIP29 gene is shown as SEQ ID No.

2.

3. A plant expression vector for overexpressing the PtobZIP29 gene, characterized in that: The sequence shown in SEQ ID No. 1 was constructed between the Bam HI and Xba I restriction sites of the pCAMBIA1300-GFP vector.

4. A method for constructing a transgenic Populus tomentosa strain with increased wood biomass, characterized in that: The steps include: (1) After cloning the PtobZIP29 gene, it was connected to a plant expression vector to obtain a plant expression vector 35S::PtobZIP29 that overexpresses the PtobZIP29 gene; (2) Transforming 35S::PtobZIP29 into Agrobacterium, and introducing the 35S::PtobZIP29 vector constructed in step (1) into leaves of wild-type Populus tomentosa by using the Agrobacterium-mediated leaf disc transformation method; (3) Through the process of co-cultivation, selective cultivation, obtaining clustered shoots, obtaining rooted transgenic plants, and positive plant identification, we finally obtained a transgenic Populus tomentosa strain with increased wood biomass.

5. The method according to claim 4, characterized in that: In step (1), the nucleotide sequence of the PtobZIP29 gene is shown as SEQ ID No.

2.

6. The method according to claim 4, characterized in that: In step (1), the plant expression vector is a pCAMBIA1300-GFP vector, which is connected via BamhI and XbaI restriction sites.

7. The method according to claim 4, characterized in that: In step (2), the Agrobacterium is GV3101.

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