Application of poplar PtrCOB11 gene in increasing wood density

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a poplar PtrCOB11 gene to increase of wood density. The problem that naturally generated wood is insufficient in density and mechanical strength is solved. The recombinant vector or the engineering bacterium of the poplar PtrCOB11 gene is applied to increase of wood density. According to the method, the S layer (secondary wall) of the xylem fiber cell wall of a stem segment is thickened through a strain of overexpressing the PtrCOB11 gene, meanwhile, the form of the xylem cell is not influenced, overexpression of the PtrCOB11 gene has no negative influence on growth and development of the plant, development of the S layer of the xylem fiber cell of the poplar is remarkably promoted, and the yield of the xylem fiber cell of the poplar is increased. And the density and the mechanical strength of the wood are uniformly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to the application of the poplar PtrCOB11 gene in increasing wood density. Background Art

[0002] As an important natural renewable resource, wood is widely used in fields such as construction, furniture, and paper, and is playing an increasingly important role in bioenergy and environmental protection. With the continuous development of the economy and the continuous improvement of people's living standards, the demand for wood has been increasing year by year, showing a rapid growth trend. High-density wood usually has stronger mechanical properties such as compressive, flexural, and tensile strength, so it has obvious advantages in fields such as construction and structural materials that have high requirements for bearing capacity.

[0003] In addition, wood with a larger density usually has better corrosion resistance and can effectively resist the invasion of external factors such as moisture and pests. Therefore, it has excellent performance in outdoor or humid areas and can extend the service life of wood. By increasing the wood density, not only can its mechanical properties and durability be improved, but also its fire resistance, corrosion resistance, and carbon storage capacity can be enhanced. However, the naturally generated wood density and mechanical strength still have obvious deficiencies. Although genetic engineering technology provides potential for wood density improvement, the candidate genes for effectively regulating wood density are still relatively scarce. This bottleneck mainly stems from the complex biosynthesis process of wood and its highly diverse genetic basis. Therefore, deeply exploring the key genes related to wood characteristics has become the key to directly regulating wood density.

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

[0005] The present invention is to solve the problem of insufficient density and mechanical strength of naturally generated wood. A brief overview of the present invention is given below to provide a basic understanding of certain aspects of the present invention. It should be understood that this overview is not an exhaustive overview of the present invention. It is not intended to identify the key or important parts of the present invention, nor is it intended to limit the scope of the present invention.

[0006] Technical Solution of the Present Invention:

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

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

[0009] Preferably, the amino acid sequence of the protein encoded by the poplar PtrCOB11 gene is as shown in SEQ ID No. 2.

[0010] Preferably, overexpression of the PtrCOB11 gene increases the thickness of the S layer of the cell walls of developing xylem and mature xylem fibers in poplar.

[0011] Preferably, the recombinant vector is obtained by recombining the poplar PtrCOB11 gene into the plant expression vector pGWB2.

[0012] Preferably, the engineered bacterium is one of Escherichia coli and Agrobacterium.

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

[0014] Preferably, the plant expression vector used for constructing the recombinant vector is pGWB2.

[0015] Preferably, the transformation method is the Agrobacterium transformation method.

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

[0017] In the lines overexpressing the PtrCOB11 gene of the present invention, the S layer (secondary cell wall) of the xylem fiber cell walls of the stem segments is thickened, and at the same time, the morphology of the xylem cells is not affected;

[0018] Overexpression of the PtrCOB11 gene of the present invention has no negative impact on the growth and development of plants;

[0019] The present invention significantly promotes the development of the S layer of xylem fiber cells in poplar, and uniformly increases the density and mechanical strength of wood. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a transcription level map of the PtrCOB11 gene in different tissues;

[0021] Figure 2 is a PtrCOB11 gene expression level map based on the Aspwood transcription database;

[0022] Figure 3 is a transcription level identification map of the 35S::PtrCOB11 overexpression line;

[0023] Figure 4 is a protein level identification map of the 35S::PtrCOB11 overexpression line;

[0024] Figure 5Phenotype diagrams of wild-type and 35S::PtrCOB11 overexpressing trees, with the scale bar representing 5 cm;

[0025] Figure 6 Optical microscope observation diagrams of the stem cross-sections of wild-type and 35S::PtrCOB11 overexpressing plants, with the scale bar representing 20 μm;

[0026] Figure 7 Scanning electron microscope and transmission electron microscope observation diagrams of the stem cross-sections of wild-type and 35S::PtrCOB11 overexpressing plants, with the white scale bar representing 50 μm, the gray scale bar representing 10 μm, and the black scale bar representing 2 μm;

[0027] Figure 8 Statistical graph of the xylem fiber cell wall thickness of wild-type and 35S::PtrCOB11 overexpressing transgenic plants;

[0028] Figure 9 Statistical graph of the wood density and mechanical strength of wild-type and 35S::PtrCOB11 overexpressing transgenic plants. Detailed implementation manners

[0029] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be described below through specific examples shown in the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention.

[0030] Detailed implementation manner one: Combining Figures 1 - 9 To illustrate this implementation manner, the application of the Populus PtrCOB11 gene of this implementation manner in increasing wood density, and the application of the recombinant vector or engineered bacteria of the Populus PtrCOB11 gene in increasing wood density. By means of genetic engineering to regulate the wood density of Populus, the requirements of different industries for wood properties can be met.

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

[0032] Detailed implementation manner two: Combining Figures 1 - 9 To illustrate this implementation manner, the application of the Populus PtrCOB11 gene of this implementation manner in increasing wood density, and the nucleotide sequence of the Populus PtrCOB11 is shown as SEQ ID No.1.

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

[0034] Specific Embodiment 4: Figures 1 - 9 This embodiment describes the application of the Populus PtrCOB11 gene in increasing wood density. By overexpressing the PtrCOB11 gene, the thickness of the S layer of the fiber cell walls in the developing xylem and mature xylem of Populus is increased. This gene regulation technology can improve the quality of wood without affecting the growth rate and adaptability of Populus, contributing to the efficient and sustainable utilization of forestry resources.

[0035] Specific Embodiment 5: Figures 1 - 9 This embodiment describes the application of the Populus PtrCOB11 gene in increasing wood density. The recombinant vector is obtained by recombining the Populus PtrCOB11 gene into the plant expression vector pGWB2. By recombining the Populus PtrCOB11 gene into the plant expression vector pGWB2 and overexpressing this gene, researchers can effectively increase the wood density of Populus.

[0036] Through this genetic engineering technology, the thickness of the fiber cell walls of Populus wood can be increased, the wood density can be improved, and thus its mechanical properties and quality can be enhanced.

[0037] Specific Embodiment 6: Figures 1 - 9 This embodiment describes the application of the Populus PtrCOB11 gene in increasing wood density. The engineered bacterium is one of Escherichia coli and Agrobacterium.

[0038] Specific Embodiment 7: Figures 1 - 9 This embodiment describes a method for increasing wood density. A recombinant vector containing the Populus PtrCOB11 gene as claimed in claim 1 is constructed and transformed into Populus to obtain transgenic plants, thereby enabling the regulation of wood density.

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

[0040] Specific Embodiment 8: Figures 1 - 9 This embodiment describes a method for increasing wood density. The plant expression vector used for constructing the recombinant vector is pGWB2. By constructing the pGWB2 vector and transforming the PtrCOB11 gene into Populus, the wood density can be increased.

[0041] Specific Embodiment 9:Figures 1 - 9 Regarding this embodiment, a method for increasing the wood density in this embodiment, the transformation method is the Agrobacterium transformation method. This method not only has high precision and efficiency but also provides a new approach for the functional improvement of wood.

[0042] Example 1: Obtaining the PtrCOB11 gene of Populus trichocarpa and its expression level in tissues

[0043] 1. Referring to the Populus trichocarpa 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. The designed primers are as follows:

[0044] PtrCOB11-CDS-F: ATGGAATTCGATAAGTCTGCCA

[0045] PtrCOB11-CDS-R: TCACCCCATTGACAGCAATATC

[0046] The total RNA of the xylem of wild-type (WT) plants of Populus trichocarpa Nisqually-1 genotype was extracted using the plant RNA extraction reagent pBIOZOL provided by BioFlux. Subsequently, reverse transcription was carried out using the reverse transcription kit (RR047A) of Takara to obtain cDNA. Using this cDNA as a template, the PtrCOB11 gene was cloned. The PCR amplification reaction system was: 33 μL of ddH2O, 1 μL of each upstream and downstream primer, 1 μL of template cDNA, 5 μL of 10×KOD Buffer, 5 μL of dNTP, 2 μL of MgSO4, and 1 μL of KOD enzyme. The above 50 μL reaction system was added to a PCR tube, and the PCR instrument program was set: 94°C for 2 min; 30 cycles: 94°C for 15 s, 60°C for 30 s, 68°C for 1 min; 68°C for 5 min.

[0047] The PCR product was recovered using the gel recovery kit provided by Qiagen. The recovered product was ligated to the PJET1.2 vector. The ligation system was: 1 μL of ddH2O, 0.25 μL of PJET1.2 blunt, 2.5 μL of 2×Rection Buffer, 0.25 μL of T4 Ligase, and 1 μL of the target fragment. The above 5 μL reaction system was reacted in a 22°C water bath for 2 hours. Subsequently, the ligation product was transformed into Escherichia coli, and positive clones were screened. The plasmid was extracted and sequenced. The obtained nucleotide sequence was SEQ ID NO.1, and the amino acid sequence was SEQ ID NO.2. This gene fragment was named PtrCOB11 and consisted of 1365 bases.

[0048] 2. Expression level of PtrCOB11 gene in Populus trichocarpa tissues

[0049] Specific quantitative primers for PtrCOB11 gene and internal reference primer PtrActin2 were designed, including:

[0050] PtrCOB11 - Quantitative - F: CAGGATCCTACAGCTTCAGTCTC

[0051] PtrCOB11 - Quantitative - R: GCACAGGTTGGGCAAGGAGTGAT

[0052] PtrActin2 - F: TTGGTGCAGAGTGTTTCCGTTG

[0053] PtrActin2 - R: GACGCCAGTATAGACCCTCCG

[0054] We detected the expression pattern of PtrCOB11 gene in xylem, phloem, leaf and root tissues of Populus trichocarpa by quantitative RT - PCR method. The results showed that PtrCOB11 showed a significantly high expression level in xylem tissue and had specific transcriptional expression( Figure 1 ). Further analysis of AspWood gene expression data showed that PtrCOB11 gene had the most significant preferential expression in xylem, and its expression level increased significantly from the cambium to the xylem region and maintained a high level in the secondary wall formation region( Figure 2 ). These results indicate that PtrCOB11 gene is highly and specifically abundantly expressed in xylem tissue.

[0055] Example 2: Obtaining of Populus trichocarpa PtrCOB11 over - expression plants

[0056] 1. Construction of 35S::PtrCOB11 plant expression vector

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

[0058] The target fragment was ligated to the pGWB2 vector using the LR reaction to generate the 35S::PtrCOB11 plant expression vector. The reaction system was as follows: 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 of the above system was placed in a water bath at 25 °C and reacted overnight. The ligation product was transformed into Escherichia coli, positive clones were identified, plasmids were extracted and identified. The correctly identified 35S::PtrCOB11 plant expression vector plasmid was transferred into Agrobacterium and stored at -80 °C.

[0059] 2.35S::PtrCOB11 Genetic Transformation of Populus trichocarpa

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

[0061] Step 1: Cultivation of Agrobacterium: The bacterial liquid stored in the -80 °C refrigerator was streaked on a solid medium containing the corresponding antibiotics and cultured for 48 h. A single colony was picked from the petri dish and inoculated into 20 mL of liquid medium containing the corresponding antibiotics, and cultured overnight at 28 °C with shaking at 200 rpm. When the OD 600 reached 1.0, 2 mL of the bacterial liquid was aspirated and added to 50 mL of liquid medium containing the corresponding antibiotics, and cultured with shaking at 28 °C for about 6 h. When the OD 600 reached 0.6, it was centrifuged at 2200 rpm at 4 °C for 10 min, the supernatant was discarded, and the cells were resuspended with an equal volume of suspension and placed for 2 h for use in infecting plant stem segments.

[0062] Step 2: Infection and transformation: Select healthy Populus trichocarpa tissue culture seedlings grown in the greenhouse for 25 - 30 days, cut them into stem segments about 1 cm long in a sterile laminar flow bench, with about 100 segments. The cut stem segments were placed in the resuspended bacterial liquid and infected for 20 min, gently shaken during this period to allow the stem segments to come into full contact with Agrobacterium.

[0063] Step 3: Co-cultivation: The stem segments in the bacterial liquid were gently fished out with forceps and placed flat on a differentiation medium added with acetosyringone (pH 5.8), and cultured for 48 h under dark conditions at 25 °C.

[0064] Step 4: Selection culture: The co-cultured plant explants were transferred to a selection and differentiation medium and cultured in a greenhouse with a light cycle of 16 h / 8 h and a temperature of 25 °C.

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

[0066] 3.35S::PtrCOB11 Overexpression Plant Identification

[0067] According to the above Populus trichocarpa genetic transformation method, a total of 19 resistant plants were obtained. Using the genomic DNA extracted from the leaves of WT and resistant plants as templates, PCR molecular identification was carried out using kanamycin resistance primers. The results showed that all 19 plants were transgenic lines. Further, the total RNA of the xylem of WT and 35S::PtrCOB11 transgenic plants was extracted and reverse transcribed into cDNA as a template. Quantitative RT-PCR molecular identification of the expression level of transgenic plants was carried out using PtrCOB11 quantitative primers and PtrActin2 primers. The results of quantitative RT-PCR are as Figure 3 shown. The geometric mean of PtrActin2 was used to normalize the relative expression level. The error bars represent the standard error calculated from three biological replicates, and the asterisks indicate the results of the t-test: *P < 0.05, **P < 0.01, ***P < 0.001. The results showed that multiple overexpression lines with high expression levels were obtained in this example.

[0068] Furthermore, protein level analysis was performed on the top 7 high-transcription expression lines. The total proteins of the xylem of WT and OE were extracted respectively, and Western blotting hybridization was carried out with Anti-PtrCOB11 antibody. The results are as Figure 4 shown. Compared with WT, the protein levels of the 7 lines all increased significantly. We selected the overexpression lines 35S::PtrCOB3-8 (OE8) and 12 (OE12) with the highest protein levels for the next functional identification.

[0069] Example 3: Application of Populus trichocarpa PtrCOB11 Gene in Increasing Wood Density

[0070] 1. External Morphological Phenotypes of 35S::PtrCOB11 Overexpression Plants

[0071] The phenotypes of 4-month-old wild-type and 35S::PtrCOB11 overexpression saplings grown in the greenhouse are as Figure 5As shown, the scale represents 5 cm. Compared with WT, there were no obvious changes in the growth and development of 35S::PtrCOB3-8 and 12 overexpression plants. Optical microscopy was performed on the xylem of the stems of 35S::PtrCOB11 overexpression plants. Compared with WT, there were no obvious differences in the morphology and size of xylem fibers and vessels in 35S::PtrCOB11 overexpression plants, but the cell wall thickness of xylem fibers increased significantly( Figure 6 ).

[0072] 2. The wood density of 35S::PtrCOB11 overexpression plants increased

[0073] Furthermore, scanning electron microscopy of stem cross-sections showed that overexpression of the PtrCOB11 gene increased the thickness of the S layer of the cell walls of developing and mature xylem fibers, far exceeding the thickness of the xylem fiber cell walls in WT( Figure 7 ). Transmission electron microscopy showed that the S1 layer, S2 layer, and total S of xylem fibers in OE-8 and OE-12 transgenic plants were thickened by about 2-fold( Figure 8 ). Consistently, the wood density of transgenic plants increased significantly, and the mechanical strength of the wood also increased significantly( Figure 9 ). Error bars represent the standard error calculated from three biological replicates, and asterisks indicate the results of the t-test: *P<0.05, **P<0.01

[0074] SEQ ID No.1

[0075]

[0076] MEFDKSAKDCYQSLQRSLLAEMKFIFLIALVFMIVPHAAAYDPLDPNGNITIKWDVMSWTPDGYQTATVTMSNFQMYRHIISPGWTLSWSWAKKEVLWSMVGAQTTEQGDCSKFKGNIPHCCKKTPTVVDLLPGVPYNQQFSNCCKGGVMAAWGQDPTASVSAFQVSVGLAGTSNKTVKLPKNFTLLGPGPGYTCGPAKVVPSTVFLTPDRRRKTQALMTWNVTCTYSQFLASKNPTCCVSFSSFYNETITPCPTCACGCQNKNSCVKSNSKESHKKGINTPKKDNTPLLQCTHHMCPIRVHWHVKVNYRDYWRAKVAVTNFNYRMNYTEWTLVVQHPNLNNVTQVFSFDYKPLVPYESINDTGMFYGMKFYNDLLMEAGPFGNVQSEVLLQKDKNTFSLKQGWAFPRKVYFNGDECMLPPPDTYPYLPNSAYANPTSILSMAASLLLILLSMG

[0077] It should be noted that in the above embodiments, as long as the technical solutions do not conflict, they can be arranged and combined. Those skilled in the art can exhaust all possibilities based on the mathematical knowledge of permutations and combinations. 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 are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of Populus trichocarpa PtrCOB11 gene in increasing wood density, characterized in that: Use of the recombinant vector or engineered bacteria of the poplar PtrCOB11 gene in increasing wood density.

2. Use of the Populus PtrCOB11 gene according to claim 1 in increasing wood density, characterized in that: The nucleotide sequence of the poplar PtrCOB11 is as shown in SEQ ID No.

1.

3. Use of the poplar PtrCOB11 gene according to claim 1 in increasing wood density, characterized in that: The amino acid sequence of the protein encoded by the poplar PtrCOB11 gene is as shown in SEQ ID No.

2.

4. Use of the poplar PtrCOB11 gene according to claim 1 in increasing wood density, characterized in that: Overexpression of the PtrCOB11 gene increased the thickness of the S layer of the fiber cell walls in the developing xylem and mature xylem of poplar.

5. Use of the poplar PtrCOB11 gene according to claim 1 in increasing wood density, characterized in that: The recombinant vector is obtained by recombining the poplar PtrCOB11 gene into the plant expression vector pGWB2.

6. Use of the poplar PtrCOB11 gene according to claim 1 in increasing wood density, characterized in that: The engineered bacteria are one of Escherichia coli and Agrobacterium.

7. A method for increasing the wood density, characterized in that: Construct a recombinant vector containing the poplar PtrCOB11 gene as claimed in claim 1, and transform poplar to obtain transgenic plants.

8. A method for increasing the density of wood according to claim 7, characterized in that: The plant expression vector used for the construction of the recombinant vector is pGWB2.

9. A method for increasing the density of wood according to claim 7, characterized in that: The transformation method is the Agrobacterium transformation method.

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