Use of poplar ptrcob11 gene in increasing wood density

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of a poplar PtrCOB11 gene in increasing wood density. The application solves the problem of insufficient natural wood density and mechanical strength. The application relates to application of a recombinant vector or engineering bacteria of the poplar PtrCOB11 gene in increasing wood density. The application makes the stem segment xylem fiber cell wall S layer (secondary wall) thicken through overexpression of a PtrCOB11 gene strain, while not affecting the morphology of xylem cells. Overexpression of the PtrCOB11 gene of the application does not produce negative effects on the growth and development of plants. The application significantly promotes the development of the xylem fiber cell S layer of a poplar, and uniformly improves the density and mechanical strength of wood.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of the poplar PtrCOB11 gene in increasing wood density. Background Technology

[0002] Timber, as an important natural renewable resource, is widely used in construction, furniture, paper, and other fields, and plays an increasingly important role in bioenergy and environmental protection. With continuous economic development and the constant improvement of people's living standards, the demand for timber is increasing year by year, showing a rapid growth trend. High-density timber typically has stronger compressive, bending, and tensile mechanical properties, thus having a significant advantage in fields such as construction and structural materials where high load-bearing capacity is required.

[0003] Furthermore, denser wood generally exhibits better corrosion resistance and effectively resists external factors such as moisture and insect infestation, thus performing superiorly in outdoor or humid applications and extending the lifespan of the wood. Increasing wood density not only improves its mechanical properties and durability but also enhances its fire resistance, rot resistance, and carbon storage capacity. However, naturally occurring wood still has significant limitations in density and mechanical strength. Genetic engineering technology offers potential for improving wood density, but candidate genes for effectively regulating wood density remain scarce. This bottleneck stems primarily from the complex biosynthetic processes of wood and its highly diverse genetic basis. Therefore, in-depth exploration of key genes related to wood characteristics is crucial for achieving direct regulation of wood density.

[0004] Therefore, it is urgent to propose the application of the poplar PtrCOB11 gene in increasing wood density to solve the above-mentioned technical problems. Summary of the Invention

[0005] This invention addresses the problem of insufficient density and mechanical strength in naturally occurring wood. A brief overview of the invention is provided below to offer a basic understanding of certain aspects thereof. It should be understood that this overview is not an exhaustive summary of the invention. It is not intended to identify key or essential parts of the invention, nor is it intended to limit the scope of the invention.

[0006] The technical solution of the present invention:

[0007] Application of the poplar PtrCOB11 gene in increasing wood density, and application of the recombinant vector or engineered bacteria of the poplar PtrCOB11 gene in increasing wood density.

[0008] Preferred: The nucleotide sequence of poplar PtrCOB11 is shown in SEQ ID No. 1.

[0009] Preferred: The amino acid sequence of the protein encoded by the poplar PtrCOB11 gene is shown in SEQ ID No. 2.

[0010] Preferred method: Overexpression of the PtrCOB11 gene increases the thickness of the S layer of cell walls in the developing xylem and mature xylem fibers of poplar.

[0011] Preferred: The recombinant vector is the poplar PtrCOB11 gene recombined into the plant expression vector pGWB2.

[0012] Preferably, the engineered bacteria is one of Escherichia coli or Agrobacterium.

[0013] A method for increasing wood density involves constructing a recombinant vector containing the poplar PtrCOB11 gene as described in claim 1, and transforming poplar trees to obtain transgenic plants.

[0014] Preferably, the plant expression vector used to construct the recombinant vector is pGWB2.

[0015] Preferred method: Agrobacterium-mediated transformation.

[0016] The present invention has the following beneficial effects:

[0017] This invention thickens the S layer (secondary wall) of xylem fiber cell walls in stem segments by overexpressing the PtrCOB11 gene in the strain, without affecting the morphology of xylem cells.

[0018] Overexpression of the PtrCOB11 gene in this invention did not have a negative impact on plant growth and development.

[0019] This invention significantly promotes the development of the S layer of fiber cells in poplar wood, and uniformly improves the density and mechanical strength of the wood. Attached Figure Description

[0020] Figure 1 This is a diagram showing the transcriptional levels of the PtrCOB11 gene in different tissues.

[0021] Figure 2 This is a graph of PtrCOB11 gene expression levels based on the Aspwood transcription database.

[0022] Figure 3 This is a diagram identifying the transcriptional level of 35S::PtrCOB11 overexpression lines;

[0023] Figure 4 This is a graph identifying the protein levels in the 35S::PtrCOB11 overexpression line.

[0024] Figure 5This is a phenotypic diagram of wild-type and 35S::PtrCOB11 overexpression trees, with the scale bar representing 5 cm.

[0025] Figure 6 These are optical microscopic images of stem cross sections from wild-type and 35S::PtrCOB11 overexpressing plants. The scale bar represents 20 μm.

[0026] Figure 7 These are scanning electron microscope and transmission electron microscope images of the stem cross sections of wild-type and 35S::PtrCOB11 overexpressing plants. The white scale bar represents 50 μm, the gray scale bar represents 10 μm, and the black scale bar represents 2 μm.

[0027] Figure 8 This is a statistical graph of xylem fiber cell wall thickness in wild-type and 35S::PtrCOB11 overexpressing transgenic plants.

[0028] Figure 9 This is a statistical graph showing the wood density and mechanical strength of wild-type and 35S::PtrCOB11 overexpressing transgenic plants. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described below with reference to specific embodiments shown in the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0030] Specific implementation method one: Combining Figures 1-9 This embodiment describes the application of the poplar PtrCOB11 gene in increasing wood density, and the application of the recombinant vector or engineered bacteria of the poplar PtrCOB11 gene in increasing wood density. By regulating the wood density of poplar through genetic engineering, the needs of different industries for wood characteristics can be met.

[0031] For example, this technology can be used to obtain ideal timber for the furniture and construction industries that require high-strength and high-density wood.

[0032] Specific Implementation Method Two: Combining Figures 1-9 This embodiment describes the application of the poplar PtrCOB11 gene in increasing wood density. The nucleotide sequence of poplar PtrCOB11 is shown in SEQ ID No. 1.

[0033] Specific implementation method three: Combining Figures 1-9This embodiment describes the application of the poplar PtrCOB11 gene in increasing wood density. The amino acid sequence of the protein encoded by the poplar PtrCOB11 gene is shown in SEQ ID No. 2.

[0034] Specific implementation method four: Combination Figures 1-9 This embodiment describes the application of the poplar PtrCOB11 gene in increasing wood density. By overexpressing the PtrCOB11 gene, the thickness of the S layer of the fiber cell wall in the developing xylem and mature xylem of poplar is increased. This gene regulation technology can improve the quality of wood without affecting the growth rate and adaptability of poplar, which helps to achieve efficient and sustainable utilization of forestry resources.

[0035] Specific Implementation Method Five: Combining Figures 1-9 This embodiment describes the application of the poplar PtrCOB11 gene in increasing wood density. The recombinant vector is the plant expression vector pGWB2, in which the poplar PtrCOB11 gene is recombined into the plant expression vector pGWB2. By recombining the poplar PtrCOB11 gene into the plant expression vector pGWB2 and overexpressing the gene, researchers are able to effectively increase the wood density of poplar trees.

[0036] This genetic engineering technology can increase the thickness of the fiber cell walls in poplar wood, improve wood density, and thus enhance its mechanical properties and quality.

[0037] Specific Implementation Method Six: Combination Figures 1-9 This embodiment describes the application of the poplar PtrCOB11 gene in increasing wood density. The engineered bacteria is one of Escherichia coli or Agrobacterium.

[0038] Specific implementation method seven: Combination Figures 1-9 This embodiment describes a method for increasing wood density by constructing a recombinant vector containing the poplar PtrCOB11 gene as described in claim 1, transforming poplar trees to obtain transgenic plants, thereby achieving regulation of wood density.

[0039] This method uses genetic engineering to enhance the expression level of the PtrCOB11 gene, thereby improving the density and quality of wood.

[0040] Specific implementation method eight: Combination Figures 1-9 This embodiment describes a method for increasing wood density. The plant expression vector used in the construction of the recombinant vector is pGWB2. By constructing the pGWB2 vector and transforming the PtrCOB11 gene into poplar, the wood density can be increased.

[0041] Specific Implementation Method Nine: Combining Figures 1-9 This embodiment describes a method for increasing wood density. The conversion method is Agrobacterium-mediated transformation, which not only has high precision and efficiency but also provides a new approach for the functional improvement of wood.

[0042] Example 1: Acquisition and tissue expression level of the PtrCOB11 gene in Populus tomentosa

[0043] 1. Referring to the Populus tomentosa genome database provided by the phytozome website, the full-length CDS sequence of the PtrCOB11 gene was obtained, and primers were designed at both ends of it. The designed primers are as follows:

[0044] PtrCOB11-CDS-F:ATGGAATTCGATAAGTCTGCCA

[0045] PtrCOB11-CDS-R: TACCCCATTGACAGCAATATC

[0046] Total RNA was extracted from the xylem of wild-type (WT) Populus trichocarpa Nisqually-1 genotype plants using the pBIOZOL plant RNA extraction reagent provided by BioFlux. Subsequently, cDNA was obtained by reverse transcription using a Takara reverse transcription kit (RR047A). This cDNA was used as a template for cloning the PtrCOB11 gene. The PCR amplification reaction mixture consisted of: 33 μL ddH2O, 1 μL each of forward and reverse primers, 1 μL template cDNA, 5 μL 10×KOD buffer, 5 μL dNTPs, 2 μL MgSO4, and 1 μL KOD enzyme. The above 50 μL reaction mixture was added to a PCR tube, and the PCR program was set as follows: 94℃ for 2 min; 30 cycles: 94℃ for 15 s, 60℃ for 30 s, 68℃ for 1 min; 68℃ for 5 min.

[0047] PCR products were recovered using a gel extraction kit provided by Qiagen. The recovered products were ligated into the PJET1.2 vector. The ligation mixture consisted of: 1 μL ddH2O, 0.25 μL PJET1.2 blunt, 2.5 μL 2×Rection Buffer, 0.25 μL T4 Ligase, and 1 μL of the target fragment. The 5 μL reaction mixture was incubated at 22°C for 2 hours. The ligation product was then transformed into *E. coli* for screening for positive clones. The plasmid was extracted and sequenced. The nucleotide sequence is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2. This gene fragment was named PtrCOB11 and consists of 1365 bases.

[0048] 2. Intra-tissue expression level of the PtrCOB11 gene in Populus hairy-fruited tamarisk

[0049] Designed PtrCOB11 gene-specific quantitative primers and PtrActin2 internal control primers, including:

[0050] PtrCOB11-Quantitative-F:CAGGATCCTACAGCTTCAGTCTC

[0051] PtrCOB11-Quant-R:GCACAGGTTGGGCAAGGAGTGAT

[0052] PtrActin2-F:TTGGTGCAGAGTGTTTCCGTTG

[0053] PtrActin2-R: GACGCCAGTATAGACCCTCCG

[0054] We used quantitative RT-PCR to detect the expression patterns of the PtrCOB11 gene in the xylem, phloem, leaf, and root tissues of Populus tomentosa. The results showed that PtrCOB11 exhibited significantly high expression levels in the xylem tissue and possessed specific transcriptional expression (PtCOB11). Figure 1 Further analysis of AspWood gene expression data showed that the PtrCOB11 gene had the most significant preferential expression in the xylem, with its expression level increasing significantly from the cambium to the xylem region, and maintaining a high level in the secondary cell wall forming zone. Figure 2 These results indicate that the PtrCOB11 gene is highly expressed in xylem tissues.

[0055] Example 2: Obtaining Populus tomentosa plants overexpressing PtrCOB11

[0056] Construction of 1.35S::PtrCOB11 plant expression vector

[0057] For the overexpression construct, using the PtrCOB11-PJET1.2 plasmid from Example 1 as a template, the CDS sequence of PtrCOB11 was amplified, and the purified fragment was ligated into the pENTR / D-TOPO vector. The ligation system consisted of: 0.5 μl SaltSolution, 0.5 μl TOPO vector, 1 μL target fragment, and 1 μL ddH2O. The 3 μL reaction tube was placed in a 25°C water bath and reacted overnight. The ligation product was transformed into E. coli, positive clones were identified, plasmids were extracted, and sequencing analysis was performed.

[0058] The target fragment was ligated into the pGWB2 vector using the LR reaction to generate the 35S::PtrCOB11 plant expression vector. The reaction system consisted of 0.5 μl of LR enzyme, 1.5 μl of pGWB2 vector, and 0.5 μl of PtrCOB11-TOPO plasmid. The 3 μl reaction tube was placed in a 25°C water bath and reacted overnight. The ligation product was transformed into *E. coli*, and positive clones were identified. The plasmid was extracted and identified. The correctly identified 35S::PtrCOB11 plant expression vector plasmid was transformed into *Agrobacterium* and stored at -80°C.

[0059] Genetic transformation of Populus tomentosa using the 2.35S::PtrCOB11 plant expression vector

[0060] The 35S promoter-driven PtrCOB11 gene vector obtained above was genetically transformed into Populus hairy fruit using Agrobacterium-mediated transformation to obtain 35S::PtrCOB11 transgenic plants. The specific steps are as follows:

[0061] Step 1: Agrobacterium culture: The bacterial culture stored at -80℃ was streaked onto a solid medium containing the appropriate antibiotic and cultured for 48 hours. A single colony was picked from the plate and inoculated into 20 mL of liquid medium containing the appropriate antibiotic, and incubated overnight at 28℃ with shaking at 200 rpm. When OD... 600 When the OD value is 1.0, add 2 mL of bacterial culture to 50 mL of liquid culture medium containing the corresponding antibiotic, and incubate at 28°C with shaking at 200 rpm for approximately 6 hours. 600 When the concentration is 0.6, centrifuge at 2200 rpm for 10 min at 4℃, discard the supernatant, resuspend the bacterial cells in an equal volume of suspension, and let stand for 2 h for infection of plant stem segments.

[0062] Step 2: Infection and Transformation: Select healthy 25-30 day old Populus tomentosa tissue culture seedlings from the greenhouse, cut them into stem segments about 1 cm long in a sterile ultra-clean workbench, about 100 segments in total, and put the cut stem segments into the resuspended bacterial solution. Infect the stem segments for 20 minutes, gently shaking them during the process to ensure that the stem segments are in full contact with Agrobacterium.

[0063] Step 3: Co-cultivation: Gently remove the stem segments from the bacterial culture with tweezers, spread the stem segments flat on the differentiation medium (pH 5.8) with added acetylsylsyringone, and incubate at 25°C in the dark for 48 hours.

[0064] Step 4: Selection Culture: Transfer the co-cultured plant explants to a selection differentiation medium and culture them in a greenhouse with a photoperiod of 16h / 8h and a temperature of 25℃.

[0065] Step 5: Rooting culture: When the resistant buds grow to about 1cm, cut them off in a sterile workbench and place them in a rooting medium containing antibiotics for rooting culture. The culture conditions are a photoperiod of 16h / 8h and a temperature of 25℃.

[0066] Identification of plants overexpressing 3.35S::PtrCOB11

[0067] Following the genetic transformation method described above for *Populus tomentosa*, a total of 19 resistant plants were obtained. Using extracted genomic DNA from the leaves of WT and resistant plants as templates, PCR molecular identification was performed using kanamycin resistance primers. The results showed that all 19 plants were transgenic lines. Further, total RNA was extracted from the xylem of WT and 35S::PtrCOB11 transgenic plants and reverse transcribed into cDNA. Using cDNA as a template, quantitative RT-PCR was performed using PtrCOB11 and PtrActin2 primers to molecularly identify the expression levels of transgenic plants. The quantitative RT-PCR results are shown below. Figure 3 As shown, the relative expression levels were normalized using the geometric mean of PtrActin2. The error bars represent the standard errors of three biological replicates, and the asterisks indicate the t-test results: *P<0.05, **P<0.01, ***P<0.001. The results show that this embodiment obtained multiple overexpression lines with high expression levels.

[0068] Further protein level analysis was performed on the top seven lines with high transcriptional expression. Total xylem protein was extracted from WT and OE lines, and Western blotting was performed using Anti-PtrCOB11 antibody. The results are as follows: Figure 4 As shown, the protein levels of all seven strains were significantly increased compared to WT. We selected the overexpression strains 35S::PtrCOB3-8 (OE8) and 12 (OE12) with the highest protein levels for further functional identification.

[0069] Example 3: Application of the PtrCOB11 gene in increasing wood density in Populus tomentosa

[0070] External morphological phenotype of plants overexpressing 1.35S::PtrCOB11

[0071] Phenotypes of wild-type and 35S::PtrCOB11 overexpressing saplings grown in a greenhouse for 4 months were as follows: Figure 5As shown, the scale bar represents 5 cm. Compared to WT, there were no significant changes in the growth and development of plants overexpressing 35S::PtrCOB3-8 and 12. Morphological observation of the xylem of the stems of plants overexpressing 35S::PtrCOB11 under a light microscope showed no significant differences in the morphology and size of xylem fibers and vessels compared to WT, but the cell wall thickness of the xylem fibers was significantly increased. Figure 6 ).

[0072] 2.35S::PtrCOB11 overexpression increased wood density in plants.

[0073] Further scanning electron microscopy observation of the stem cross-section showed that overexpression of the PtrCOB11 gene increased the thickness of the S layer of the xylem fiber cell wall in both developing and mature xylem, far exceeding the thickness of the WT xylem fiber cell wall. Figure 7 Transmission electron microscopy revealed that the S1 layer, S2 layer, and total S content of xylem fibers in OE-8 and OE-12 transgenic plants were approximately twice as thick. Figure 8 Consistently, the transgenic plants showed a significant increase in wood density and mechanical strength. Figure 9 The error bars represent the standard errors of three biological replications, and the asterisks indicate the results of the t-test: *P<0.05, **P<0.01.

[0074] SEQ ID No.1

[0075]

[0076] MEFDKSAKDCYQSLQRSLLAEMKFIFLIALVFMIVPHAAAYDPLDPNGNITIKWDVMSWTPDGYQTATVTMSNFQMYRHIISPGWTLSWSWAKKEVLWSMVGAQTTEQGDCSK FKGNIPHCCKKTPTVVDLLPGVPYNQQFSNCCKGGVMAAWGQDPTASVSAFQVSVGLAGTSNKTVKLPKNFTLLGPGPGYTCGPAKVVPSTVFLTPDRRRKTQALMTWNVTCTY SQFLASKNPTCCVSFSSFYNETITPCPTCACGCQNKNSCVKSNSKESHKKGINTPKKDNTPLLQCTHHMCPIRVHWHVKVNYRDYWRAKVAVTNFNYRMNYTEWTLVVQHPNL NNVTQVFSFDYKPLVPYESINDTGMFYGMKFYNDLLMEAGPFGNVQSEVLLQKDKNTFSLKQGWAFPRKVYFNGDECMLPPPPDTYPYLPNSAYANPTSILSMAASLLLILLSMG

[0077] It should be noted that in the above embodiments, as long as the technical solutions are not contradictory, they can be permuted and combined. Those skilled in the art can exhaust all possibilities based on the mathematical knowledge of permutation and combination. Therefore, the present invention will not describe the technical solutions after permutation and combination one by one, but it should be understood that the technical solutions after permutation and combination have been disclosed by the present invention.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of the poplar PtrCOB11 gene in increasing the wood density of Populus tomentosa, wherein the application of the poplar PtrCOB11 gene in increasing the wood density of Populus tomentosa is based on a recombinant vector or engineered bacteria, characterized in that, The nucleotide sequence of the poplar PtrCOB11 gene is shown in SEQ ID No. 1; the amino acid sequence of the protein encoded by the poplar PtrCOB11 gene is shown in SEQ ID No.

2.

2. The application of the poplar PtrCOB11 gene in increasing wood density according to claim 1, characterized in that: Overexpression of the PtrCOB11 gene in poplar increased the thickness of the S layer of the cell wall of the fiber cells in both the developing and mature xylem of Populus tomentosa.

3. The application of the poplar PtrCOB11 gene in increasing wood density according to claim 1, characterized in that: The recombinant vector is a plant expression vector pGWB2 in which the poplar PtrCOB11 gene is recombined.

4. The application of the poplar PtrCOB11 gene in increasing wood density according to claim 1, characterized in that: The engineered bacteria is one of Escherichia coli or Agrobacterium.

5. A method for increasing the density of Populus tomentosa wood, characterized in that: A recombinant vector containing the poplar PtrCOB11 gene as described in claim 1 was constructed, and the vector was used to transform hairy poplar to obtain transgenic plants.

6. A method for increasing the density of Populus tomentosa wood according to claim 5, characterized in that: The plant expression vector used to construct the recombinant vector was pGWB2.

7. A method for increasing the density of Populus tomentosa wood according to claim 5, characterized in that: The transformation method is the Agrobacterium-mediated transformation method.

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