Application of the PtoNAC028 gene in regulating the ratio of paulownia pectin monomers

Abstract

This invention discloses PtoNAC028 The gene is used to regulate the ratio of pectin monomers in poplar trees. The coding region of this gene has the nucleotide sequence shown in SEQ ID NO:1. This is achieved through overexpression or silencing of the gene in poplar trees. PtoNAC028 Genes capable of increasing or decreasing the proportion of lignin monomers in poplar provide key genes for the genetic improvement of poplar processing traits. This invention also discloses a method for cultivating poplars with a high lignin monomer ratio, through overexpression of [gene name missing] in poplar trees. PtoNAC028 The gene can significantly increase the proportion of lignin monomers in poplar trees, achieving targeted regulation of monomeric lignin metabolism, which helps to rapidly cultivate new poplar varieties with excellent wood properties and fast growth.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving PtoNAC028 Genes, especially those involving PtoNAC028 Application of genes in regulating the ratio of pectin monomers in poplar. Background Technology

[0002] Poplar ( Populus Poplar (L.) is a general term for tree species in the genus Populus of the family Salicaceae. It is an important species for short-cycle industrial timber forests and ecological protection forests worldwide. Poplars generally possess biological characteristics such as rapid growth, strong adaptability, and convenient asexual reproduction, and are widely used in fields such as wood-based panel manufacturing, pulp and paper making, bioenergy, and ecological restoration. With the continuous growth in demand for timber resources, the targeted breeding of new poplar varieties that combine excellent wood properties (such as high density, suitable fiber morphology, and ideal chemical composition) with rapid growth through genetic improvement has become an important way to alleviate the contradiction between timber supply and demand and ensure timber security.

[0003] The industrial value of wood largely depends on the chemical composition of its secondary metabolites—lignin monomers. Syringyl lignin (S-type lignin), with its numerous ether bonds and linear structure, is more easily degraded during chemical pulping and biorefining, thus significantly improving production efficiency and reducing energy consumption. In contrast, guaiac-based lignin (G-type lignin), with its high degree of cross-linking and stable structure, is less suitable for processing, leading to decreased production efficiency and increased pollution during pulping. Based on these characteristics of lignin monomers, increasing the proportion of syringyl lignin while decreasing the proportion of guaiac-based lignin is of great significance for reducing energy consumption and improving wood quality.

[0004] In the timber industry, lignin removal has long relied on chemical methods, leading to numerous drawbacks such as high energy consumption, significant pollution, and low efficiency. Currently, there is a lack of precise methods for regulating the proportion of lignin monomers in poplar wood, resulting in slow progress in the genetic improvement of poplar wood processing quality. Therefore, it is necessary to utilize molecular biology techniques to identify key regulatory factors related to the proportion of lignin monomers in poplar wood, achieving targeted regulation of monomeric lignin metabolism. This will provide important theoretical basis and genetic resources for cultivating new poplar germplasm with significantly optimized wood processing traits. Summary of the Invention

[0005] To overcome the above problems, the inventors conducted intensive research and identified a key gene that affects the ratio of two lignin monomers in poplar wood. PtoNAC028 By cloning the gene and verifying its transgenic function, this study confirmed for the first time that the gene is a key regulator of the ratio of syringyl lignin and guaiacyl lignin in poplar, providing a key gene for the genetic improvement of poplar processing traits. Simultaneously, by regulating the ratio of syringyl lignin and guaiacyl lignin in poplar, this study also demonstrated that the gene is a key regulator of the ratio of syringyl lignin and guaiacyl lig PtoNAC028Gene expression enables targeted regulation of monomeric lignin metabolism, which is of great significance for cultivating new poplar germplasm with significantly optimized wood processing traits, thus completing this invention.

[0006] Specifically, the object of the present invention is to provide the following aspects:

[0007] Firstly, a gene is provided that regulates the ratio of pectin monomers in poplar wood. The gene is... PtoNAC028 The gene, whose coding region has a nucleotide sequence as shown in SEQ ID NO:1.

[0008] Secondly, a biomaterial is provided, wherein the biomaterial is any one of A1) to A4):

[0009] A1) The protein encoded by the gene described in the first aspect;

[0010] A2) A recombinant vector containing the genes described in the first aspect;

[0011] A3) Recombinant microorganisms containing the genes described in the first aspect or recombinant microorganisms containing the recombinant vector described in A2);

[0012] A4) A transgenic plant containing the gene described in the first aspect or a transgenic plant containing the recombinant vector described in A2).

[0013] Thirdly, the application of the gene described in the first aspect in regulating the ratio of poplar pectin monomers is provided.

[0014] Fourthly, the application of the biomaterials described in the second aspect in increasing the proportion of genus resin monomers in poplar is provided.

[0015] Fifthly, a method for cultivating poplar trees with a high lignin monomer ratio is provided, the method comprising the step of overexpressing the gene described in the first aspect in the poplar trees.

[0016] Sixthly, a method for regulating the lignin monomer ratio in poplar is provided, the method comprising the steps of overexpressing the gene described in the first aspect in poplar to increase the lignin monomer ratio, and / or

[0017] The step of silencing the expression of the genes described in the second aspect in poplar to reduce the proportion of lignin monomers.

[0018] The beneficial effects of this invention include:

[0019] (1) This invention is the first to demonstrate PtoNAC028The gene plays a regulatory role in the ratio of syringyl lignin to guaiac lignin in poplar, and is a key regulatory factor in the ratio of the two lignin monomers in poplar. This discovery lays a theoretical foundation for the genetic improvement of the processing quality of poplar wood and provides key gene resources.

[0020] (2) The present invention provides PtoNAC028 The application of genes and biomaterials in regulating the ratio of pectin monomers in poplar can target and intervene in the pectin content of poplar. PtoNAC028 Gene expression enabled targeted regulation of monomeric lignin metabolism, significantly increasing the S-type lignin / G-type lignin ratio in poplar, providing new ideas and methods for improving the quality of poplar wood.

[0021] (3) The method for cultivating poplar trees with a high lignin monomer ratio provided by the present invention involves overexpressing lignin monomers in poplar trees. PtoNAC028 This method, which utilizes genetics, is simple to operate, highly efficient, and helps to quickly cultivate new poplar varieties with excellent wood properties and fast growth, thereby alleviating the contradiction between timber supply and demand and ensuring timber security. Attached Figure Description

[0022] Figure 1 (a) shows the embodiment 1 of the present invention. PtoNAC028 and PtoNAC028- Gel electrophoresis results of RNAi; Figure 1 (b) shows the embodiment 3 of the present invention. PtoNAC028 Gel electrophoresis results of vector sequence detection at both ends of the cloning site in transgenic plants; Figure 1 (c) shows the embodiment 3 of the present invention. PtoNAC028 Gel electrophoresis image of Agrobacterium sequence detection in transgenic plants;

[0023] Figure 2 The relative expression levels of overexpressing poplar plants, wild-type plants, and silent-expressing poplar plants are shown in Example 2 of the present invention.

[0024] Figure 3 The growth phenotypes of poplar plants with overexpression, wild-type plants, and silent expression were shown.

[0025] Figure 4 The results of microspectral analysis of overexpressing poplar plants, wild-type plants, and silent-expressing poplar plants are shown. Detailed Implementation

[0026] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the features and advantages of the present invention will become clearer and more apparent.

[0027] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0028] NAC transcription factors are a class of key transcription factors unique to plants. The inventors have discovered that, as the core regulatory hub in the plant secondary cell wall synthesis network, NAC transcription factors can directly or indirectly regulate the expression of multiple rate-limiting enzymes in the lignin monomer synthesis pathway by initiating the transcriptional cascade regulatory network, thereby affecting the lignin S / G monomer ratio (S-type lignin content / G-type lignin content). In other words, the lignin monomer ratio is precisely regulated by NAC transcription factors.

[0029] This invention comprehensively utilizes molecular genetics and genetic engineering techniques to target the expression of key NAC transcription factors in poplar, thereby achieving directional regulation of monomeric lignin metabolism.

[0030] In a first aspect, the present invention provides a gene for regulating the ratio of pectin monomers in poplar trees, said gene being... PtoNAC028 Genes whose coding regions have the nucleotide sequence shown in SEQ ID NO:1, preferably PtoNAC028 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO:1.

[0031] In this invention, the lignin monomer ratio is the ratio of the content of S-type lignin monomers to the content of G-type lignin monomers, i.e., S-type lignin content / G-type lignin content.

[0032] Preferably, the poplar is a white poplar, more preferably a hairy white poplar and / or an 84K poplar.

[0033] In this invention, based on extensively targeted metabolomics data of xylem from a national germplasm resource population (303 trees) of Populus tomentosa, the inventors used a combined approach of population genetics, metabolomics, transcriptomics, and molecular biology to identify key genes affecting the ratio of monomers of two types of lignin (S-type lignin and G-type lignin) in the xylem of Populus tomentosa. PtoNAC028 Its coding region is 1185bp in length.

[0034] Preferably, the PtoNAC028 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0035] Among them, the PtoNAC028 The gene-encoded protein consists of 394 amino acid residues. The predicted molecular weight of the protein is approximately 43.72 kDa, and the theoretical isoelectric point (pI) is 5.302.

[0036] This invention is the first to demonstrate PtoNAC028The gene plays a regulatory role in the ratio of syringyl lignin to guaiacyl lignin in poplar, and is a key regulatory factor in the proportion of lignin monomers in poplar. This discovery lays a theoretical foundation for the genetic improvement of poplar wood processing quality and provides key gene resources.

[0037] A second aspect of the present invention is a biomaterial, said biomaterial being any one of A1) to A4):

[0038] A1) The protein encoded by the gene described in the first aspect;

[0039] A2) A recombinant vector containing the gene described in the first aspect;

[0040] A3) Recombinant microorganisms containing the genes described in the first aspect or recombinant microorganisms containing the recombinant vector described in A2);

[0041] A4) A transgenic plant containing the gene described in the first aspect or a transgenic plant containing the recombinant vector described in A2).

[0042] Preferably, the transgenic plant is a transgenic poplar, more preferably a white poplar, and even more preferably a white poplar and / or a 84K poplar.

[0043] More preferably, the amino acid sequence of the protein encoded by the gene described in the first aspect is shown in SEQ ID NO:2.

[0044] A third aspect of the invention provides the first aspect described above. PtoNAC028 Application of genes in regulating the ratio of pectin monomers in poplar.

[0045] The poplar tree is preferably Populus tomentosa, and more preferably Populus tomentosa and / or Populus 84K.

[0046] Preferably, the regulation involves increasing or decreasing the proportion of poplar pectin monomers.

[0047] More preferably, the regulation is achieved through overexpression or silencing of expression in poplar trees. PtoNAC028 Genetic realization;

[0048] Among them, by overexpressing in poplar trees PtoNAC028 Genes are used to increase the proportion of syringyl lignin (S-type lignin) monomers and decrease the proportion of guaiacyl lignin (G-type lignin) monomers, that is, to increase the proportion of poplar pectin monomers.

[0049] Expressing through silence within the poplar tree PtoNAC028 Genes are used to reduce the proportion of syringyl lignin (S-type lignin) monomers and increase the proportion of guaiacyl lignin (G-type lignin) monomers, that is, to reduce the proportion of poplar pectin monomers.

[0050] According to a preferred embodiment of the present invention, the overexpression in poplar PtoNAC028 Genes were introduced into poplar trees containing... PtoNAC028 Gene overexpression was achieved using a recombinant vector; the silencing of gene expression in poplar trees was also achieved. PtoNAC028 Genes were introduced into poplar trees containing... PtoNAC028 Gene silencing recombinant vectors have been implemented.

[0051] Preferably, the PtoNAC028 Gene overexpression recombinant vectors, through PtoNAC028 The coding region sequence of a gene is constructed onto a basic vector to obtain the gene; PtoNAC028 Gene silencing recombinant vectors, through PtoNAC028 The silent gene fragments are obtained by constructing them onto a basic vector.

[0052] More preferably, the PtoNAC028 The coding region sequence of the gene is shown in SEQ ID NO:1, and the silenced fragment is shown in SEQ ID NO:3.

[0053] In this invention, PtoNAC028 The higher the gene expression level in poplar, the higher the content of S-type lignin monomers and the lower the content of G-type lignin monomers, the higher the S / G monomer ratio of lignin, and the better the wood processing quality. PtoNAC028 The lower the gene expression level in poplar, the lower the content of S-type lignin monomers and the higher the content of G-type lignin monomers. The lower the S / G monomer ratio of lignin, the worse the wood processing quality.

[0054] A fourth aspect of the present invention provides the application of the biomaterials described in the second aspect in increasing the proportion of genus resin monomers in poplar.

[0055] The poplar tree is preferably Populus tomentosa, and more preferably Populus tomentosa and / or Populus 84K.

[0056] Preferably, the increase in the proportion of poplar lignin monomers is achieved by increasing the amount of lignin in the poplar plant. PtoNAC028 It is achieved through the expression level of genes.

[0057] A fifth aspect of the invention provides a method for cultivating poplar trees with a high lignin monomer ratio, the method comprising overexpressing lignin monomers in the poplar trees. PtoNAC028 The steps of gene generation.

[0058] The poplar tree is preferably Populus tomentosa, and more preferably Populus tomentosa and / or Populus 84K.

[0059] Preferably, the method includes the following steps:

[0060] Step 1, obtain PtoNAC028 Gene coding region sequence, construction PtoNAC028 Gene overexpression recombinant vector.

[0061] According to an embodiment of the present invention, using the cDNA of Populus tomentosa 1316 as a template, primers were used... PtoNAC028 -F and primers PtoNAC028 -R was used for PCR amplification to obtain PtoNAC028 The coding region sequence of a gene.

[0062] Among them, primers PtoNAC028 The -F sequence is shown in SEQ ID NO:4, primer PtoNAC028 The sequence of -R is shown in SEQ ID NO:5.

[0063] In this invention, PtoNAC028 Gene overexpression recombinant vectors, through PtoNAC028 The coding region sequence of the gene (as shown in SEQ ID NO:1) was constructed into the PBI121 vector.

[0064] The PBI121 vector drives the expression of the target gene with a strong 35S promoter and carries a GFP tag, which facilitates molecular identification and gene function studies.

[0065] Step 2: Perform genetic transformation to obtain... PtoNAC028 Transgenic poplar plants with overexpressed genes.

[0066] Preferably, the successfully constructed PtoNAC028 Gene overexpression recombinant vectors were transformed into Agrobacterium, and 84K poplar was transformed using the Agrobacterium-mediated leaf disc method. Positive transgenic plants were then obtained through kanamycin resistance screening.

[0067] According to a preferred embodiment of the present invention, step 2 further includes the step of identifying positive transgenic plants, wherein the identification includes the step of verifying the integration of exogenous genes and excluding Agrobacterium contamination by using PCR amplification.

[0068] Preferably, PtoNAC028 The criteria for identifying positive transgenic poplar plants are: the target band can be amplified using vector boundary-specific primers, but the target band cannot be amplified using Agrobacterium-specific primers.

[0069] According to a preferred embodiment of the present invention, the verification of the integration of the exogenous gene is achieved by detecting the presence of vector sequences at both ends of the cloning site. The primers for detecting the vector sequences at both ends of the cloning site are pBI121-F and pBI121-R, and their primer sequences are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0070] The exclusion of Agrobacterium contamination is achieved by detecting the presence of Agrobacterium sequences. The primers for detecting Agrobacterium sequences are GV3101-VirD2-F and GV3101-VirD2-R, and their primer sequences are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0071] Preferably, in the detection results, the target bands of the vector sequences at both ends of the cloning site are present, while the Agrobacterium sequence band is absent. PtoNAC028 Gene overexpression positive transgenic plants.

[0072] If the vector sequences at both ends of the cloning site can amplify the expected bands, it indicates that the exogenous gene has been successfully integrated. If the Agrobacterium sequence does not amplify any bands, Agrobacterium contamination is ruled out.

[0073] According to a preferred embodiment of the present invention, the PtoNAC028 The screening of positive transgenic poplar plants also includes testing. PtoNAC028 Steps for determining gene expression levels in transgenic poplar plants.

[0074] Preferably, the PtoNAC028 The expression level of the gene in transgenic poplar plants was determined using real-time quantitative PCR (RT-PCR).

[0075] More preferably, the primers for the real-time quantitative PCR are... PtoNAC028 -qPCR-F and PtoNAC028 -qPCR-R, whose primer sequences are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0076] in, PtoNAC028 The expression level of the gene in the overexpressed transgenic poplar plants was significantly higher than that in the wild type.

[0077] In this invention, PtoNAC028 The screening of transgenic plants is simple to operate, highly specific, and accurate, and can quickly and effectively identify transgenic positive plants.

[0078] Step 3: Perform phenotypic analysis on the overexpressing transgenic poplar plants.

[0079] Preferably, the phenotype includes plant height and lignin monomer ratio.

[0080] More preferably, compared to wild-type plants, PtoNAC028 Plants with gene overexpression showed significantly increased plant height and an increased proportion of lignin monomers.

[0081] According to an embodiment of the present invention, in the characteristic wavelength range of syringyl lignin (1320-1340 cm⁻¹) -1In the Raman spectroscopy analysis of the plant, compared with the wild-type plant, PtoNAC028 Plants that overexpressed the gene exhibited significantly enhanced spectral intensity;

[0082] PtoNAC028 Gene-overexpressing plants, wild-type plants and PtoNAC028 The lignin monomer ratios (the ratio of syringyl lignin to guaiacyl lignin) in gene-silenced plants were 1.15, 1.07, and 1.03, respectively.

[0083] PtoNAC028 Overexpression of the gene increased the proportion of syringyl lignin monomers in poplar while decreasing the proportion of guaiac lignin monomers; and PtoNAC028 Silent plants exhibit the opposite phenotype to overexpressed plants.

[0084] The method for cultivating poplar trees with a high proportion of lignin monomers provided by this invention can significantly accelerate the genetic improvement process of poplar wood processing quality.

[0085] A sixth aspect of the present invention provides a method for regulating the lignin monomer ratio in poplar trees, the method comprising the steps of overexpressing the gene described in the first aspect in poplar trees to increase the lignin monomer ratio, and / or

[0086] The steps of silencing the gene described in the first aspect in poplar to reduce the proportion of lignin monomers.

[0087] Preferably, the poplar is a white poplar, more preferably a hairy white poplar and / or an 84K poplar.

[0088] The step of overexpressing the gene described in the first aspect in poplar trees is carried out in accordance with the method described in the fifth aspect.

[0089] According to a preferred embodiment of the present invention, the silencing expression in poplar trees... PtoNAC028 Genes were introduced into poplar trees containing... PtoNAC028 Gene silencing recombinant vectors have been implemented.

[0090] Preferably, the silent expression recombination vector, through... PtoNAC028 The silenced gene fragment was constructed into a base vector, as shown in SEQ ID NO:3. The base vector was the PBI121 vector.

[0091] More preferably, the successfully constructed PtoNAC028 The gene silencing expression recombinant vector was transformed into Agrobacterium, and 84K poplar was transformed using the Agrobacterium-mediated leaf disc method. Positive silent transgenic plants were then obtained through kanamycin resistance screening.

[0092] According to embodiments of the present invention, compared with wild-type plants, the silent transgenic plants have significantly reduced plant height and a reduced proportion of lignin monomers.

[0093] In the characteristic wavelength range of guaiac lignin (1250-1290 cm⁻¹) -1 In the Raman spectral analysis, the spectral intensity of the silent transgenic plants was higher than that of the overexpressing transgenic plants and wild-type plants, and the proportion of lignin monomers was significantly lower than that of the overexpressing transgenic plants and wild-type plants.

[0094] Example

[0095] The present invention is further described below through specific examples; however, these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present invention.

[0096] Unless otherwise specified, the reagents involved in the following examples are all commercially available conventional reagents, and the methods used are all methods commonly used in this technical field.

[0097] In this embodiment, all primers were synthesized by Sangon Biotech Co., Ltd.

[0098] Example 1 PtoNAC028 Construction of gene overexpression recombinant vectors and silencing recombinant vectors

[0099] Based on extensive targeted metabolomics data of xylem from a national germplasm resource population (303 Populus tomentosa) (Li P, Xiao L, Du Q, et al. Genomic insights into selection for heterozygous alleles and woody traits in Populus tomentosa[J]. Plant Biotechnology Journal, 2023, 21:2002-2018.), a key gene regulating the ratio of two lignin monomers in poplar was identified using a combination of population genetics, metabolomics, transcriptomics, and molecular biology methods. PtoNAC028 Using the cDNA of Populus tomentosa 1316 as a template, the full-length CDS sequence and the silenced fragment of this gene were cloned. PtoNAC028- RNAi sequences, and nucleotide sequences are shown in SEQ ID NO:1 and SEQ ID NO:3, respectively. PtoNAC028 Gel electrophoresis results of CDS fragments and silenced fragments of the gene are as follows: Figure 1 As shown in (a) of the diagram.

[0100] in, PtoNAC028The full-length coding region of the gene is 1185 bp, encoding a protein composed of 394 amino acid residues, the amino acid sequence of which is shown in SEQ ID NO:2. The predicted molecular weight of this protein is approximately 43.72 kDa, and the theoretical isoelectric point (pI) is 5.302.

[0101] PtoNAC028 The full length of the gene-silenced fragment is 295 bp.

[0102] Specifically: the leaves of Populus tomentosa 1316 were thoroughly ground in a sterile, enzyme-free mortar, and total RNA was extracted from the leaves using a total RNA extraction kit. DNase I was used to remove genomic DNA contamination, and then the RNA was reverse transcribed into cDNA using the Promega GoScript™ ReverseTranscription System.

[0103] according to PtoNAC028 Specific amplification primers were designed for the CDS fragment and the silenced fragment of the gene, and high-fidelity enzyme amplification was used. The amplification primers for the CDS fragment were as follows: PtoNAC028 -F (SEQ ID NO:4) and PtoNAC028 -R (SEQ ID NO:5) indicates that the primer sequence contains the vector homologous arm sequence (lowercase part) used for cloning, as detailed below:

[0104] PtoNAC028 -F (SEQ ID NO:4):

[0105] 5'-cgggggactctagaggatccATGCTCTTGCGAGCAATTCCA-3';

[0106] PtoNAC028 -R (SEQ ID NO:5):

[0107] 5'-tgctcaccatggtaccGAAGAAGAAGTAAAATGGAACGAAGCACAG-3'.

[0108] PtoNAC028 The primers for amplifying the silent gene fragment are: PtoNAC028 -RNAi-F and PtoNAC028 -RNAi-R (as shown in SEQ ID NO:12 and SEQ ID NO:13 respectively), the specific sequences (lowercase parts are vector homologous arm sequences) are as follows:

[0109] PtoNAC028 -RNAi-F (SEQ ID NO:12):

[0110] 5'-cgcggatccGAGGAGCAGTTCAAGGAGAT-3';

[0111] PtoNAC028 -RNAi-R (SEQ ID NO:13):

[0112] 5'-cgcctcgagCTTGTTGGCATTGATGTTCTC-3'.

[0113] The CDS fragment and the silencing fragment were respectively ligated into the PBI121-eGFP vector. The PBI121-eGFP overexpression vector drives the expression of the target gene with a strong 35S promoter and carries a GFP tag, which facilitates molecular identification and gene function studies.

[0114] The ligation products were then heat-shocked into *E. coli* DH5α competent cells, and single colonies were picked and sequenced after plating. The correctly sequenced plasmids were then heat-shocked into *Agrobacterium* GV3101 cells, plated on LB agar containing 17 mg / L rifampin and 50 mg / L kanamycin, and incubated overnight at 28°C. Single colonies with the correct bands were identified by PCR and stored in 100 mL LB liquid medium (17 mg / L rifampin and 50 mg / L kanamycin) by shaking. The PCR program was as follows: 95°C denaturation for 5 minutes; (95°C, denaturation for 30 seconds; 56°C annealing for 30 seconds; 72°C, extension for 90 seconds) 35 cycles; 72°C, extension for 5 minutes.

[0115] After the above steps, complete PtoNAC028 Construction of gene overexpression recombinant vectors and silencing recombinant vectors.

[0116] Example 2 PtoNAC028 Obtaining transgenic plants with overexpression and those with silenced expression

[0117] (1) Pretreatment: Select healthy and uncontaminated 84K tissue culture seedlings and make uniform incisions on their unfolded leaves using a sterile scalpel. Inoculate the wounded explants into 84K-specific differentiation medium (MS basal medium + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 7 g / L agar + 30 g / L sucrose, pH 5.8) without any antibiotics and pre-culture for 2-3 days under a photoperiod of 25±1℃ and 16h light / 8h dark to enhance the sensitivity of the explants to Agrobacterium.

[0118] (2) Preparation of bacterial culture: from newly activated bacterial culture containing PtoNAC028 Agrobacterium with gene overexpression recombinant vector and containing PtoNAC028Single, well-formed colonies were picked from Agrobacterium tumefaciens plates containing the gene-silencing recombinant vector and inoculated into Erlenmeyer flasks containing 100 mL of liquid LB medium (containing 25 mg / L kanamycin and 25 mg / L rifampin). The flasks were placed in a shaker at 28°C and 180 rpm for approximately 24 hours, during which the bacterial concentration was monitored periodically using a UV spectrophotometer. 600 Stop culturing when the value reaches the optimal infection concentration of 0.6-0.8.

[0119] (3) Infection and co-culture: Aliquot the cultured Agrobacterium tumefaciens into 50mL sterile centrifuge tubes and centrifuge at 3500-5000 rpm for 7-10 minutes at room temperature. Discard the supernatant and collect the bacterial cells. Add an equal volume of sterile resuspension (10mM MgCl2, 10mM MES, 100μM AS) to the centrifuge tubes and gently shake to completely resuspend the bacterial cells. Completely immerse the pre-cultured leaf explants in the bacterial suspension, gently shake to ensure full contact, and infect for 10-15 minutes. After infection, remove the explants with sterile forceps, absorb excess bacterial suspension on the surface with sterile filter paper, and then spread them evenly on co-culture medium (same composition as pre-culture medium, with an additional 100μM AS). Incubate in the dark at 25℃ for 3-4 days to complete the transfer and initial integration of T-DNA.

[0120] (4) Resistance selection culture: After co-culture, the explants were carefully dried with sterile filter paper to remove any Agrobacterium tumefaciens adhering to their surface, and then transferred to selection medium (MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 7 g / L agar + 30 g / L sucrose + 25 mg / L kanamycin + 200 mg / L timentin). The culture was maintained at 25°C, with a 16 h light / 8 h dark cycle and a light intensity of 40 μmol·m⁻¹. -2 ·s -1 The culture was carried out in a specialized culture room for selection. To prevent Agrobacterium overgrowth and degradation of the selection agent, the culture medium was replaced with fresh selection medium every 10 days, and the culture was continued until healthy resistant adventitious shoots differentiated from the edges of the explants.

[0121] (5) Rooting induction culture: When the adventitious buds grow to a height of 1-2 cm, they are cut off from the mother explant using a sterile scalpel or forceps in a clean bench and vertically inserted into the rooting medium (1 / 2 MS + 0.05 mg / L IBA + 0.05 mg / L NAA + 7 g / L agar + 20 g / L sucrose + 25 mg / L kanamycin + 200 mg / L termethin). Rooting induction is carried out under the same culture room conditions. After about 10 days, new roots can be observed at the base of the adventitious buds, and finally, complete transgenic plants are obtained.

[0122] Example 3 PtoNAC028 Overexpression of transgenic plants and PtoNAC028 Identification of Silent Expression Transgenic Plants

[0123] (1) Plant DNA was extracted using the FastPure Plant DNA Isolation Mini Kit from Nanjing Novizan Biotechnology Co., Ltd. PtoNAC028 Overexpression of transgenic plants and PtoNAC028 Genomic DNA from silent transgenic plants was analyzed using PCR to detect the vector sequences and Agrobacterium sequences at both ends of the cloning site. The primers for detecting the vector sequences at both ends of the cloning site were pBI121-F and pBI121-R, with sequences shown in SEQ ID NO:6 and SEQ ID NO:7, respectively. The primers for detecting the Agrobacterium sequences were GV3101-VirD2-F and GV3101-VirD2-R, with sequences shown in SEQ ID NO:8 and SEQ ID NO:9, respectively. Transgenic plants numbered 3 and 9 (overexpression transgenic plants) were identified as positive transgenic plants (denoted as OE3 and OE9) because the target band (1185 bp) was present in the vector sequences at both ends of the cloning site, while the Agrobacterium sequence band was absent. Silent transgenic plants numbered 1 and 2 (silent expression transgenic plants) were also identified as positive transgenic plants (denoted as RNAi1 and RNAi2) because the target band (295 bp) was present in the vector sequences at both ends of the cloning site, while the Agrobacterium sequence band was absent. PtoNAC028 The results of vector sequence detection at both ends of the transgenic cloning site and the results of Agrobacterium sequence detection are as follows: Figure 1 As shown in (b) and (c) in the figure.

[0124] (2) Further, total RNA was extracted from the plants using the Novozymes Biotechnology Company's centrifuge column RNA extraction kit, and cDNA was synthesized by reverse transcription using the reverse transcription cDNA first-strand synthesis kit (R312-01 / 02). The cDNA was then detected by real-time quantitative PCR. PtoNAC028 Gene expression levels in overexpressing and silent expression plants.

[0125] Specifically as follows:

[0126] ① Design RT-qPCR primers: Primers are PtoNAC028 -qPCR-F and PtoNAC028 -qPCR-R, the sequences are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively;

[0127] ②Using the overexpressing plants OE3 and OE9, the silent expression plants RNAi1 and RNAi2, and the wild-type plants obtained above as experimental materials, RNA was extracted and reverse transcribed into cDNA;

[0128] ③ Measure the cDNA concentration and dilute it to 200 ng / µl;

[0129] ④ Prepare the PCR reaction system on ice, specifically as follows: PtoNAC028 -qPCR-F 0.5μL; PtoNAC028 -qPCR-R 0.5μL; ddH2O 3μL; TBgreen 5μL; cDNA 1μL.

[0130] ⑤ Using QuantStudio 6 Flex Real-Time PCR System software, with 18sRNA as an internal control, RT-qPCR was performed in triplicate for each sample. The program is shown in Table 1:

[0131] Table 1

[0132]

[0133] Test results as follows Figure 2 As shown: In overexpressing plants OE3 and OE9 PtoNAC028 The gene expression level was significantly higher in the silent expression plants than in the wild-type plants (WT). In the silent expression plants, RNAi1 and RNAi2 expression levels were significantly higher. PtoNAC028 The gene expression level was significantly lower than that of the wild-type plant (WT).

[0134] Example 4 PtoNAC028 Phenotypic analysis of transgenic poplar plants

[0135] For evaluation PtoNAC028 To investigate the biological function of the genes, this embodiment involved simultaneously culturing overexpressing plants OE3 and OE9, silent expressing plants RNAi1 and RNAi2, and wild-type 84K poplar trees under greenhouse conditions, with three biological replicates. After three months of culture, plant height was measured and photographs were taken. The results are as follows: Figure 3 As shown in the figure, it can be seen that the plant height of overexpressing plants OE3 and OE9 is significantly increased compared with wild-type plants and silent expression plants.

[0136] Furthermore, using overexpressing plants OE3 and OE9, silent expression plants RNAi1 and RNAi2, and wild-type 84K poplar as experimental materials, three biological replicates were set up for each line for microscopic examination and analysis. According to the method described in the XSP biological microscope manual, secondary xylem tissue from the stem of each line was taken, fixed with FAA fixative, dehydrated with gradient ethanol, and then prepared into section samples using an ultramicrotome.

[0137] Based on the high specificity of Raman spectroscopy in detecting the chemical structure of lignin, this study employed laser confocal Raman microscopy. The principle is that different lignin structural units produce characteristic Raman spectra under laser excitation; the typical vibrational band of syringyl lignin (S-type lignin) is located at 1320-1340 cm⁻¹. -1 The characteristic spectral bands of guaiac lignin (G-type lignin) are concentrated in the 1250-1290 cm⁻¹ range. -1 Interval.

[0138] By performing spectral scanning and data acquisition on the sliced ​​samples, Raman spectral information of the slices in characteristic bands was obtained. Then, by analyzing the intensity differences of the Raman spectra in characteristic bands, the differences in the ratio of the two lignin monomers between transgenic plants and wild-type plants were semi-quantitatively compared.

[0139] The results are as follows Figure 4 As shown: in the characteristic wavelength range of syringyl lignin (1320-1340 cm⁻¹) -1 In Raman spectroscopy analysis, the overexpressing plants exhibited significantly enhanced spectral intensities, indicating a marked increase in the relative content of syringyl lignin. In contrast, the content of syringyl lignin was significantly increased in the characteristic wavelength range of guaiac lignin (1250-1290 cm⁻¹). -1 The spectral intensity of silent expression plants was higher than that of overexpression plants and wild-type plants.

[0140] After comparing the peak values ​​of the transgenic lines and wild-type lines in the two characteristic bands, it was found that the ratios of syringyl lignin to guaiacyl lignin in the overexpressing plants, wild-type plants, and silent plants were 1.15, 1.07, and 1.03, respectively.

[0141] The above results indicate that PtoNAC028 Overexpression of the gene increased the proportion of syringyl lignin monomers in poplar while decreasing the proportion of guaiac lignin monomers, thus increasing the lignin monomer ratio (S-type lignin content / G-type lignin content). Silent expression... PtoNAC028 Genes, when overexpressed, can cause plants to exhibit characteristics similar to those expressed. PtoNAC028 Plants with the same gene exhibit opposite phenotypes. This discovery provides... PtoNAC028 The experimental data supports the finding that genes regulate the processing quality of poplar wood by controlling the ratio of two lignin monomers.

[0142] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. A gene that regulates the ratio of pectin monomers in poplar trees, characterized in that, The gene is PtoNAC028 The gene, whose coding region has a nucleotide sequence as shown in SEQ ID NO:1; The lignin monomer ratio is the ratio of syringyl lignin monomer content to guaiac lignin monomer content; The regulation is achieved through overexpression. PtoNAC028 Genes can be used to increase the proportion of lignin monomers through silencing expression. PtoNAC028 Genes are used to reduce the proportion of lignin monomers.

2. The gene according to claim 1, characterized in that, The PtoNAC028 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. A biomaterial, characterized in that, The biomaterial is any one of A1) to A3): A1) The protein encoded by the gene as described in claim 1; A2) A recombinant vector containing the gene described in claim 1; A3) A recombinant microorganism containing the gene described in claim 1 or a recombinant microorganism containing the recombinant vector described in A2).

4. The application of the gene according to claim 1 or 2 in regulating the ratio of pectin monomers in poplar, characterized in that, The lignin monomer ratio is the ratio of syringyl lignin monomer content to guaiac lignin monomer content; The regulation is achieved through overexpression. PtoNAC028 Genes can be used to increase the proportion of lignin monomers through silencing expression. PtoNAC028 Genes are used to reduce the proportion of lignin monomers.

5. The application according to claim 4, characterized in that, The overexpression in poplar PtoNAC028 Genes were introduced into poplar trees containing... PtoNAC028 Gene overexpression was achieved using a recombinant vector; the silencing of gene expression in poplar trees was also achieved. PtoNAC028 Genes were introduced into poplar trees containing... PtoNAC028 Gene silencing recombinant vectors have been implemented.

6. The application of the biomaterial according to claim 3 in increasing the proportion of phenolic monomers in poplar wood, characterized in that, The increase in the proportion of poplar pectin monomers is achieved by overexpression in poplar trees. PtoNAC028 Achieved through genes; The lignin monomer ratio is the ratio of syringyl lignin monomer content to guaiac lignin monomer content.

7. A method for cultivating poplar trees with a high proportion of lignin monomers, characterized in that, The method includes the step of overexpressing the gene of claim 1 in poplar, wherein the lignin monomer ratio is the ratio of syringyl lignin monomer content to guaiacyl lignin monomer content.

8. The method according to claim 7, characterized in that, The method includes the following steps: Step 1, obtain PtoNAC028 Gene coding region sequence, construction PtoNAC028 Gene overexpression recombinant vectors; Step 2: Perform genetic transformation to obtain... PtoNAC028 Transgenic poplar plants with overexpressed genes; Step 3: Perform phenotypic analysis on the overexpressing transgenic poplar plants.

9. A method for regulating the proportion of resin monomers in poplar wood, characterized in that, The lignin monomer ratio is the ratio of syringyl lignin monomer content to guaiac lignin monomer content; The method for regulating the lignin monomer ratio in poplar trees is to overexpress the gene described in claim 1 or 2 in poplar trees to increase the lignin monomer ratio, or Silencing the gene described in claim 1 or 2 in poplar to reduce the proportion of lignin monomers.

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