Application and method of PtobZIP29 gene in regulating wood development of Populus tomentosa

Abstract

The present invention discloses the application and method of the PtobZIP29 gene in regulating the wood development of white poplar. By overexpressing the PtobZIP29 gene in white poplar, the division activity of the vascular cambium of the poplar is promoted, and the formation of secondary xylem is accelerated, thereby obtaining transgenic white poplar germplasm with increased wood biomass and improved wood fast-growing ability. This provides a method and germplasm basis for the breeding of fast-growing and superior forest varieties, is conducive to improving the wood yield and economic benefits of economic forests, and also provides materials for the construction of shelter forests and urban and rural greening.

Description

Technical Field

[0001] The present 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, used to manufacture building materials and furniture, as well as paper, packaging, and many other materials. It is also now considered a viable bioenergy alternative to fossil fuels and an ideal raw material for replacing plastics and chemical products. Furthermore, forests sequester approximately 82% of terrestrial biomass, representing approximately 450 billion tons of carbon in wood (Bar-On et al., 2018), making them a crucial carbon dioxide sink for mitigating global warming. Wood's high carbon storage capacity and net-zero carbon emissions make its use an effective strategy for mitigating global CO2 concentrations. However, due to my country's low forest cover, the supply and demand for wood are tight. Therefore, cultivating fast-growing trees with excellent wood quality is of paramount importance in forest breeding. Poplars (Populus spp.) are one of the most widespread and adaptable fast-growing tree species in the world. Therefore, as a key source of timber in my country, poplars play a vital role in economic development and ecological 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. Vascular cambium activity 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 in woody plants and is one of the key factors controlling 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 mechanisms underlying vascular cambium proliferation and differentiation have become a hot topic in biology and forestry, and significant progress has been made in understanding the molecular mechanisms of vascular cambium growth and development. Studies have shown that during cambium growth and development, several plant hormones, small peptides, and related transcription factors work synergistically to tightly regulate vascular cambium activity. Vascular cambium activity is regulated by several hormones, including auxin (IAA), cytokinin (CK), ethylene (ET), brassinosteroids (BR), and gibberellins (GA). These hormones coordinate to promote cambium proliferation and wood development. Numerous lines of evidence indicate that peak auxin accumulation occurs primarily in poplar cambium stem cells, and that polarized auxin flow is associated with its export transporter, PIN1, suggesting that upregulation of PIN1 promotes cambium activity and secondary growth. Furthermore, transcription factors play a crucial role in regulating vascular development. WOX4, a member of the WOX gene family, acts as a regulatory hub for cambium activity, positively regulating the proliferation and differentiation of poplar cambium cells.

[0005] The bZIP gene family is widely present in plants and participates in a variety of biological processes, including growth, development, and stress responses. The transcription factor proteins encoded by this bZIP gene family contain a highly conserved bZIP domain. This structure, composed of 60-80 amino acids, includes a DNA-binding region and an adjacent leucine zipper. The binding region contains a nuclear localization signal and an N-X7-R / K motif, enabling precise binding to target DNA. Studies have found that the Arabidopsis bZIP family comprises 13 branches, each with its own distinct characteristics, starting from AM. A less well-characterized bZIP branch is group I, which comprises 13 members in Arabidopsis thaliana. These members share a single lysine residue in the basic domain, replacing a highly conserved arginine residue. Studies in several homologous plants have shown that bZIP I subfamily proteins are involved in plant root development and environmental stress responses. However, their regulatory function in wood formation in woody plants remains unclear. Summary of the Invention

[0006] In view of this, one object of the present invention is to provide the use 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:

[0008] Application of PtobZIP29 gene in regulating wood development of Populus tomentosa

[0009] 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 growth rate; the amino acid sequence of the PtobZIP29 gene is shown in SEQ ID No. 1.

[0010] In some embodiments of the present invention, the nucleotide sequence of the PtobZIP29 gene is shown as SEQ ID No. 2.

[0011] A plant expression vector for overexpressing the PtobZIP29 gene was 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.

[0012] A method for constructing a transgenic Populus tomentosa strain with increased wood biomass comprises the following steps:

[0013] (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;

[0014] (2) 35S::PtobZIP29 was transformed into Agrobacterium, and the 35S::PtobZIP29 vector constructed in step (1) was introduced into wild-type Populus tomentosa leaves using the Agrobacterium-mediated leaf disc transformation method;

[0015] (3) Through the process of co-cultivation, selective cultivation, obtaining clustered buds, obtaining rooted transgenic plants and positive plant identification, a transgenic Populus tomentosa strain with increased wood biomass was finally obtained.

[0016] In some embodiments of the present invention, in step (1), the nucleotide sequence of the PtobZIP29 gene is shown as SEQ ID No. 2.

[0017] 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.

[0018] In some embodiments of the present invention, in step (2), the Agrobacterium is GV3101.

[0019] The beneficial effects of the present invention are that, after transforming Populus tomentosa plants with the PtobZIP29 gene and vector, they can effectively increase the radial growth of Populus tomentosa stems, promote the division activity of the vascular cambium, and accelerate the formation of secondary xylem, thereby increasing the fast-growing ability and wood biomass. This provides a method and germplasm foundation for the selection and breeding of fast-growing and high-quality forest varieties, is conducive to increasing the timber yield and economic benefits of economic forests, and also provides materials for the establishment of shelter forests and urban and rural greening. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0021] Figure 1 It is a 35S::PtobZIP29 overexpression vector.

[0022] Figure 2 Screening and identification of 35S::PtobZIP29 transgenic plants of Populus tomentosa; A. Identification of transgenic positive lines; B. Screening of overexpressing lines.

[0023] Figure 3 Analysis of stem growth phenotypes of 35S::PtobZIP29 transgenic lines of Populus tomentosa; A. Photographs 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. Diameter statistics of the 10th internode of the stem of wild-type and transgenic plants; "ns" indicates no significant difference; p < 0.01; "**" indicates p < 0.01.

[0024] Figure 4 Analysis of vascular cambium division activity in the Populus tomentosa 35S::PtobZIP29 transgenic line; A. Imaging of vascular cambium cross-section (internode 8); B. Analysis of vascular cambium division activity; C. Statistical analysis of vascular cambium cell layers; "***" indicates p < 0.001; "****" indicates p < 0.0001.

[0025] Figure 5 Phenotypic analysis of xylem development regulation in the 35S::PtobZIP29 transgenic line of Populus tomentosa; 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

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0027] Example 1: Cloning of the Populus tomentosa PtobZIP29 gene

[0028] Through amino acid sequence homology, we found the amino acid sequence of the bZIP29 transcription factor of Populus tomentosa (sequence number Potri.004G163800) in the phytozome database (https: / / phytozome-next.jgi.doe.gov / ). We then used TBtools software to align the amino acid sequence obtained in the database with the Populus tomentosa database, obtaining the amino acid sequence (shown in SEQ ID No. 1) and CDS sequence (shown in SEQ ID No. 2) of the Potri.004G1638001 transcription factor of Populus tomentosa. Based on the CDS of PtobZIP29, we designed and synthesized upstream and downstream primers for the entire coding frame as follows:

[0029] Upstream primer F-PtobZIP29: 5′-ATGGGTGATACTGAGGAAGCAAA-3′ (SEQ ID No. 3);

[0030] Downstream primer R-PtobZIP29: 5′-CTATTGATTTGAATCTGGCTTTGG-3′ (SEQ ID No. 4).

[0031] The PtobZIP29 gene was amplified and cloned by PCR using a wild-type Populus tomentosa cDNA sequence as a template using 5× PrimeSTAR Buffer Mix. Sequence accuracy and integrity were verified by sequencing. PCR reaction conditions were: 98°C for 3 min; 98°C for 10 sec, 56°C for 20 sec, 72°C with an extension time of 15 sec / kb for 35 cycles; and 72°C for 10 min.

[0032] 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.

[0033] Example 2: Construction of a plant expression vector overexpressing PtobZIP29 (35S::PtobZIP29)

[0034] (1) To construct the overexpression PtobZIP29 vector, the pCAMBIA1300-GFP vector with the CaMV35S promoter was first selected and double-digested with the restriction endonucleases Bam HI and Xba I. The vector was digested overnight at 37°C and then 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.

[0035] (2) Then, PtoWRKY11 gene primers with vector homology arms were designed at the vector restriction site, which are:

[0036] Upstream primer PtobZIP29-F-BamHI: 5'-agctcggtacccggggatccATGGGTGATACTGAGGAAGCAAA -3' (SEQ ID No. 5);

[0037] Downstream primer PtobZIP29-R-XbaI: 5′-ctcaccatgtcgactctagaTTGATTTGAATCTGGCTTTGGG-3′ (SEQ ID No. 6).

[0038] (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 arms. Agarose gel electrophoresis was then performed and gel recovery was performed using a Biospin gel recovery kit to obtain a linearized PtobZIP29 gene fragment with the homology arms.

[0039] (4) Using a DNA fragment homologous recombination kit, the enzyme-cut vector backbone fragment and the PtobZIP29 fragment with homologous arms were homologously ligated to obtain pCAMBIA1300-GFP with the PtobZIP29 fragment. The ligation product was transformed into Escherichia coli using the competent cell method, and the transformed monoclonal colonies were detected by PCR. The positive colonies detected were inoculated with LB liquid medium and shaken at 37°C overnight.

[0040] (5) The vector plasmid was then obtained using the Biospin plasmid extraction kit. After enzyme digestion and sequencing verification, a recombinant plant expression vector containing the PtobZIP29 gene was obtained and named 35S::PtobZIP29 vector ( Figure 1 ).

[0041] Example 3: Agrobacterium tumefaciens-mediated genetic transformation of Populus tomentosa with 35S::PtobZIP29

[0042] 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.

[0043] 1) Pseudomonas aeruginosa transformation

[0044] The 35S::PtoWRKY11 vector from Example 2 was introduced into Agrobacterium tumefaciens GV3101 using the heat shock method (37°C, 5 min + liquid nitrogen, 2 min; repeated three times). Positive clones were screened for Kan resistance. Agrobacterium tumefaciens GV3101 colonies harboring the recombinant plasmid were identified using colony PCR (amplification with primers F-PtobZIP29 and R-PtobZIP29). The strain was preserved.

[0045] 2) Cultivation of Agrobacterium

[0046] 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. A single clone was picked and inoculated into YEP liquid medium containing the corresponding antibiotics and cultured at 28°C with shaking until the logarithmic growth OD600 reached 0.6-0.8. The activated Agrobacterium was then 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 was 0.6-0.8. The two-living Agrobacterium liquid was added into a 50ml centrifuge tube. The tube was centrifuged at 4000rpm for 10min at 4℃. The bacterial precipitate was collected and added with 10ml WPM resuspension (WPM powder + 2.14g + 30g sucrose + 100μmol AS). The tube was mixed with a pipette. 20-30ml WPM resuspension was added and the tube was transferred into a round-mouth bottle and cultured at 28℃ and 200rpm for 40min-1h.

[0047] 3) Agrobacterium-mediated leaf disc infection

[0048] 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.

[0049] 4) Co-cultivation of Populus tomentosa

[0050] The infected leaf disc was picked out with sterilized tweezers, placed on sterilized filter paper, and the bacterial solution was absorbed dry. The leaf disc was spread flat on WPM co-culture medium (WPM powder + 2.14 g + 30 g sucrose + 100 μmol AS + 1.0 mg NAA + 2.0 mg ZT) and cultured in the dark at 25°C for 2 days.

[0051] 5) Selective cultivation of Populus tomentosa

[0052] After 2 days of co-cultivation, the transformed explants were transferred to a callus-inducing selection medium (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 selection medium was replaced every 5 days.

[0053] 6) Populus tomentosa bud induction culture

[0054] When white, loose callus tissue appeared around the leaf margin, the callus tissue was transferred 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 a clean bench and cultured at 25°C under light for about 4 to 5 weeks, with new WPM budding medium replaced every 10 days.

[0055] 7) Rooting culture of Populus tomentosa

[0056] 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.

[0057] 8) Transplanting Populus tomentosa

[0058] 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 them warm and moist. Remove the film after one week of growth.

[0059] YEP medium: 10 g yeast extract, 10 g peptone, 5 g NaCl, pH 7.0, sterilize at 121°C for 20 minutes. For solid culture medium, add 10–12 g agar powder before sterilization.

[0060] LB medium: 5 g yeast extract, 10 g peptone, 10 g NaCl, pH = 7.0, autoclave at 121°C for 20 min. For solid culture medium, add 10–12 g agar powder before sterilization.

[0061] Example 4: Screening and identification of Populus tomentosa 35S::PtobZIP29 transgenic lines

[0062] 1. Identification of 35S::PtobZIP29 transgenic positive plants

[0063] 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.

[0064] The CTAB method for extracting poplar genomic DNA is as follows:

[0065] 1) In a 10 mL centrifuge tube, add 3 mL of CTAB and 90 μL of β-mercaptoethanol and preheat in a 65°C water bath.

[0066] 2) Grind approximately 0.6 g of fresh Populus tomentosa leaves in liquid nitrogen into a powder. Transfer the powder to the CTAB extract and vortex to mix thoroughly. Alternatively, place the leaves in a 2 mL centrifuge tube, add the CTAB extract and three small steel balls, and then crush using a tissue disruptor.

[0067] 3) Preheat the mixture in a 65°C water bath for 45 min, shaking vigorously three times to mix thoroughly.

[0068] 4) After the time is up, take out the tube and cool it for 5 minutes, then centrifuge it at 12,000 rpm for 10 minutes;

[0069] 5) Carefully transfer the supernatant to another clean 1.5 mL centrifuge tube. Let stand at room temperature for 5 minutes, then add an equal volume of chloroform:isopropyl alcohol (24:1). Mix thoroughly by inverting the tube, then place it flat to emulsify for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes at 18°C.

[0070] 6) Repeat step 5 once to remove the protein better;

[0071] 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 appears, and centrifuge at 12,000 rpm for 10 min.

[0072] 8) Rinse twice with 500 μL 75% (v / v) ethanol and once with 500 μL absolute ethanol. Remove the ethanol with a pipette and dry the pellet in a 37°C oven until transparent.

[0073] 9) Dissolve the precipitate in 50-100 μL of sterile water containing RNase A to obtain the genomic DNA of Populus tomentosa.

[0074] Next, using the genomic DNA of each strain as a template and the wild type as a control,

[0075] pCAMBIA1300-F:5'-GGGACTGAGCTCGGTACCC-3' (SEQ ID No. 7);

[0076] R-PtobZIP29: 5'-CTATTGATTTGAATCTGGCTTTGG-3' (SEQ ID No. 4)

[0077] 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 band. The results showed that multiple regenerated lines contained the exogenously introduced target gene fragment, proving that the 35S::PtobZIP29 transgenic Populus tomentosa lines were successfully obtained through genetic transformation ( Figure 2 , A).

[0078] 2. Screening of 35S::PtobZIP29 overexpressing strains

[0079] In order to screen transgenic lines overexpressing PtobZIP29, quantitative PCR (qRT-PCR) was used to screen PtobZIP29-overexpressing plants from 35S::PtobZIP29 transgenic positive lines for subsequent wood development analysis.

[0080] 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:

[0081] Add 490 μL of BGLysis and 10 μL of β-mercaptoethanol to a final concentration of 2% in a 1.5 mL RNase-Free EP tube and mix well.

[0082] Liquid nitrogen grinding: Grind an appropriate amount of tissue into powder in liquid nitrogen, weigh 50-100 mg and place it into the 1.5 mL centrifuge tube containing BGLysis and β-mercaptoethanol, and immediately shake vigorously until no obvious particles are present.

[0083] Centrifuge the above samples at 12,000 rpm for 5 minutes and carefully transfer the supernatant to a new RNase-Free 1.5 mL centrifuge tube.

[0084] More accurately estimate the volume of the lysate supernatant and add 0.5 times the volume of anhydrous ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Immediately shake and mix.

[0085] The entire mixture was pipetted into the spin column, and centrifuged at 12,000 rpm for 1 minute to discard the liquid in the collecting tube.

[0086] 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.

[0087] Add 500μL Wash Buffer to the spin column, centrifuge at 12,000 rpm for 30 seconds, discard the liquid in the tube, and add 250μL Wash Buffer to repeat the wash.

[0088] Then centrifuge the empty column at 12000 rpm for 1 minute to remove as much Wash Buffer as possible to prevent the residual ethanol in the Wash Buffer from inhibiting downstream reactions.

[0089] Transfer the spin column to a new 1.5 mL RNase-Free centrifuge tube, add 25 μL of RElution Buffer to the center of the membrane, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, add the first eluate back to the spin column membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain total RNA.

[0090] 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:

[0091] (1) Add 2 µL of 5× gDNA Eraser Buffer and 1 µL of 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.

[0092] (2) After keeping warm at 42°C for 2 min, quickly cool on ice;

[0093] (3) Add 4 µL 5× Prime Script Buffer 2, 1 µL Prime Script RT Enzyme Mix I, 0.5 µL each of the specific stem-loop primer and U6 RT primer, 1 µL RT Primer Mix, and 4 µL RNase Free ddH2O to the EP tube in step 2 and mix well.

[0094] (4) 42℃, 15 min; 85℃, 5 sec;

[0095] (5) Finally, store in a -20℃ refrigerator.

[0096] Finally, specific quantitative primers were designed and used for qRT-PCR amplification using the reverse-transcribed poplar cDNA template. RT-qPCR was performed according to the instructions for the GoTaq® qPCR Master Mix kit to measure the expression level of the PtobZIP29 gene. The PCR reaction conditions were: 95°C for 30 seconds, followed by 40 cycles of: 95°C for 5 seconds, 60°C for 1 minute, 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds.

[0097] The quantitative primers are as follows:

[0098] PtobZIP29-qPCR-F: 5'-TCTTTTACAGGTAGCGATGTGA-3' (SEQ ID No. 8);

[0099] PtobZIP29-qPCR-R: 5'-AATGCCTCGTTTAAAGCATCTC - 3' (SEQ ID No. 9).

[0100] The experimental results showed that among the 35S::PtobZIP29 transgenic Populus tomentosa lines, many lines significantly overexpressed PtobZIP29 compared to the wild type. Among them, the expression levels in L1 and L2 plants were the highest, reaching 2.65 times and 3.94 times that of the wild type, respectively ( Figure 2 , B).

[0101] Example 5: Analysis of Wood Development Regulation Function of Populus tomentosa 35S::PtobZIP29 Transgenic Line

[0102] 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, and the relevant indicators were analyzed using Image J software. The results are shown in Figure 2. Figure 4 and Figure 5 shown.

[0103] 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 transgenic overexpression strains ( Figure 2 , A), obtained two overexpression lines of PtobZIP29, 35S::PtobZIP29-L1 and 35S::PtobZIP29-L2, whose expression levels were upregulated by 2.65-fold and 3.94-fold compared with the wild type, respectively ( Figure 2 , B). Further analysis of wood development in the overexpression lines showed that there was no significant difference in plant height between the 3-month-old 35S::PtobZIP29 transgenic white poplar lines 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 PtobZIP29 gene transformation method was used to obtain a poplar variety that promotes wood formation, providing excellent raw materials for industrial production such as paper production.

[0104] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. PtobZIP29 The application of genes in regulating the development of Populus tomentosa wood is characterized by: By overexpressing in Populus tomentosa PtobZIP29 Gene, promotes the division activity of the vascular cambium of poplar, accelerates the formation of secondary xylem, thereby obtaining transgenic white poplar germplasm with increased wood biomass and improved wood growth rate; PtobZIP29 The amino acid sequence of the gene is shown in SEQ ID No.

1.

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

2.

3. A method for constructing a transgenic Populus tomentosa strain with increased wood biomass, characterized by: The steps include: (1) Cloning PtobZIP29 After the gene is generated, it is connected to a plant expression vector to obtain overexpression PtobZIP29 Plant expression vectors for genes 35S::PtobZIP29 The nucleotide sequence of the PtobZIP29 gene is shown in SEQ ID No. 2; (2) 35S::PtobZIP29 was transformed into Agrobacterium, and the Agrobacterium-mediated leaf disc transformation method was used to transform the 35S::PtobZIP29 The vector was introduced into leaves of wild-type Populus tomentosa; (3) Through the process of co-cultivation, selective cultivation, obtaining clustered buds, obtaining rooted transgenic plants and positive plant identification, a transgenic Populus tomentosa strain with increased wood biomass was finally obtained.

4. The method according to claim 3, wherein: In step (1), the plant expression vector is a pCAMBIA1300-GFP vector, which is connected through the BamhI and XbaI restriction sites.

5. The method according to claim 3, wherein: In step (2), the Agrobacterium is GV3101 .

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