A bamboo ubiquitin ligase PeKFB9 and its application

Abstract

The present invention belongs to the field of plant breeding technology, and specifically relates to a bamboo ubiquitin ligase PeKFB9 and its application. Its amino acid sequence comprises the sequence shown in SEQ ID NO: 1. The bamboo ubiquitin ligase protein of the present invention can degrade peroxidase (PRX) involved in the polymerization of lignin monomers in the plant, thereby effectively reducing its activity and thereby directionally changing the amount of lignin synthesized in the plant. Experiments have also verified that the bamboo ubiquitin ligase protein of the present invention can reduce the activity of peroxidase involved in the polymerization of lignin monomers in the plant by at least 60%, thereby affecting its lignin synthesis function.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and particularly relates to a bamboo ubiquitin ligase PeKFB9 and an application thereof. Background Art

[0002] The stems of moso bamboo (Phyllostachysedulis) possess excellent wood properties, with applications spanning furniture, construction, and papermaking, offering broad application prospects and significant market value. Lignin is a key substance influencing wood properties, and a high lignin content in moso bamboo can enhance its wood properties. Lignin is also a key component of the secondary cell walls of vascular plants. Together, lignin and cellulose form a natural physical barrier that effectively prevents the invasion of various pathogens and enhances the plant's defenses against various biotic and abiotic stresses. However, the presence of lignin also has numerous negative impacts on practical applications. For example, lignin degradation has been a major challenge in achieving a green and environmentally friendly bamboo pulp and paper industry.

[0003] In summary, discovering a gene that can regulate lignin synthesis is necessary for the targeted breeding of plants such as bamboo with high or low lignin content. Summary of the Invention

[0004] In response to the above problems, one of the objectives of the present invention is to provide a bamboo ubiquitin ligase encoding gene PeKFB9 and an encoded protein. The protein encoded by the bamboo ubiquitin ligase gene PeKFB9 can degrade peroxidase (PRX) involved in the polymerization process of lignin monomers, thereby directionally inhibiting or promoting its role in lignin synthesis. That is, the bamboo ubiquitin ligase encoding gene PeKFB9 has broad application prospects in the directed breeding of plant lignin improvement, and provides a new gene resource for plant genetic engineering.

[0005] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0006] In one aspect, the present invention provides a bamboo ubiquitin ligase PeKFB9, characterized in that its amino acid sequence comprises the sequence shown in SEQ ID NO: 1.

[0007] Another aspect of the present invention provides a nucleic acid sequence encoding the above-mentioned bamboo ubiquitin ligase PeKFB9.

[0008] On the other hand, the present invention provides an application of a moso bamboo ubiquitin ligase gene PeKFB9 or its encoded protein in reducing the activity of peroxidase involved in the polymerization of lignin monomers in plants, the application comprising: degrading the peroxidase involved in the polymerization of lignin monomers by the protein encoded by the moso bamboo ubiquitin ligase gene PeKFB9, thereby reducing the activity of the peroxidase; wherein, the sequence of the moso bamboo ubiquitin ligase gene PeKFB9 comprises the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the moso bamboo ubiquitin ligase gene PeKFB9 comprises the sequence shown in SEQ ID NO: 1.

[0009] On the other hand, the present invention provides an application of a bamboo ubiquitin ligase gene PeKFB9, its encoded protein, or a biomaterial containing the bamboo ubiquitin ligase gene PeKFB9 in reducing the activity of peroxidases involved in the polymerization of lignin monomers in plants. The application includes: reducing the activity of peroxidases involved in the polymerization of lignin monomers in plants by promoting the overexpression of the bamboo ubiquitin ligase gene PeKFB9 in plants or upregulating the activity of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 in plants; wherein the sequence of the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 1.

[0010] On the other hand, the present invention provides an application of a bamboo ubiquitin ligase gene PeKFB9, its encoded protein, or a biomaterial containing the bamboo ubiquitin ligase gene PeKFB9 in improving the activity of peroxidases involved in the polymerization of lignin monomers in plants. The application includes: down-regulating the expression of the bamboo ubiquitin ligase gene PeKFB9 in the plant or down-regulating the activity of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 in the plant, thereby improving the activity of peroxidases involved in the polymerization of lignin monomers in the plant; wherein the sequence of the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 1.

[0011] On the other hand, the present invention provides an application of a bamboo ubiquitin ligase gene PeKFB9, its encoded protein or a biological material containing the bamboo ubiquitin ligase gene PeKFB9 in plant genetic breeding; the application includes: in plant genetic breeding, by up-regulating the activity of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 in the plant or promoting the overexpression of the bamboo ubiquitin ligase gene PeKFB9 in the plant, screening out plants with low peroxidase activity participating in the polymerization of lignin monomers; or by down-regulating the activity of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 in the plant or down-regulating the expression of the bamboo ubiquitin ligase gene PeKFB9 in the plant, screening out plants with high peroxidase activity participating in the polymerization of lignin monomers; wherein the sequence of the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 1.

[0012] The beneficial effects of the present invention include: the bamboo ubiquitin ligase protein of the present invention can degrade peroxidase (PRX) involved in the polymerization of lignin monomers in the plant, thereby effectively reducing its activity and thus directionally changing the amount of lignin synthesis in the plant; and experiments have verified that the bamboo ubiquitin ligase protein of the present invention can reduce the activity of peroxidase involved in the polymerization of lignin monomers in the plant by at least 60%, thereby affecting its lignin synthesis function. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is an agarose gel electrophoresis image of the CDS sequence amplification of the bamboo ubiquitin ligase gene PeKFB9 in Example 1;

[0014] Figure 2 This is the enzyme digestion map of the yeast two-hybrid vector in Example 2;

[0015] Figure 3 Schematic diagram of the structure of the PeKFB9 protein and its truncated form in Example 2;

[0016] Figure 4 This is the yeast two-hybrid experiment in Example 3;

[0017] Figure 5 This is the enzyme digestion map of the expression vector in Example 4;

[0018] Figure 6 This is the PCR electrophoresis diagram of the Agrobacterium monoclonal colony transformed with the expression vector plasmid in Example 5;

[0019] Figure 7 The peroxidase activity in tobacco leaves was determined in Example 6;

[0020] Figure 8This is the protein immunoblotting experiment in Example 7;

[0021] in, Figure 1 In the figure, M is a DNA molecular weight marker, and lanes 1–4 are the amplification products under 56°C, 59°C, 62°C, and 65°C, respectively;

[0022] Figure 2 In the figure, M1 and M2 are DNA molecular weight markers, 1-3 are three monoclonal colonies transformed with pGBKT7-PeKFB9, and 4-6 are three monoclonal colonies transformed with pGBKT7-K^PeKFB9;

[0023] Figure 5 In the figure, M1 and M2 are DNA molecular weight markers, 1-3 are monoclonal colonies transformed with pCAMBIA1300-PeKFB9, 4-6 are monoclonal colonies transformed with pCAMBIA1300-F^PeKFB9, and 7-9 are monoclonal colonies transformed with pCAMBIA1300-PePRX72-1;

[0024] Figure 6 In the figure, M is a DNA molecular weight marker, 1 is a negative control, 2-3 are two monoclonal colonies transformed with pCAMBIA1300-PeKFB9, 4 is a pCAMBIA1300-PeKFB9 vector plasmid (positive control), 5 is a negative control, 6-7 are two monoclonal colonies transformed with pCAMBIA1300-F^PeKFB, 8 is a pCAMBIA1300-F^PeKFB vector plasmid (positive control), 9 is a negative control, 10-11 are two monoclonal colonies transformed with pCAMBIA1300-PePRX72-1, and 12 is a pCAMBIA1300-PePRX72-1 vector plasmid (positive control);

[0025] Figure 7 In the figure, 1 is the co-transformation of pCAMBIA1300-PeKFB9 and pCAMBIA1300-PePRX72-1, 2 is the co-transformation of pCAMBIA1300-F^PeKFB9 and pCAMBIA1300-PePRX72-1, and 3 is the co-transformation of pCAMBIA1300-3×FLAG and pCAMBIA1300-PePRX72-1;

[0026] Figure 8In the figure, 1 represents the co-transformation of pCAMBIA1300-PeKFB9 and pCAMBIA1300-PePRX72-1, 2 represents the co-transformation of pCAMBIA1300-F^PeKFB9 and pCAMBIA1300-PePRX72-1, and 3 represents the co-transformation of pCAMBIA1300-3×FLAG and pCAMBIA1300-PePRX72-1. DETAILED DESCRIPTION

[0027] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0028] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0029] The term "high lignin content" used in the present invention means that the lignin content of the gene-edited plant is greater than that of the non-gene-edited plant; the term "low lignin content" means that the lignin content of the gene-edited plant is less than that of the non-gene-edited plant.

[0030] The term "low peroxidase activity" used in the present invention means that the peroxidase activity of the gene-edited plant is lower than that of the non-gene-edited plant; the term "low peroxidase activity" means that the peroxidase activity of the gene-edited plant is lower than that of the non-gene-edited plant.

[0031] An embodiment of the present invention provides a bamboo ubiquitin ligase PeKFB9, characterized in that its amino acid sequence comprises the sequence shown in SEQ ID NO: 1.

[0032] Another embodiment of the present invention provides a nucleic acid sequence encoding the aforementioned bamboo ubiquitin ligase PeKFB9.

[0033] In some specific embodiments, the nucleic acid sequence includes the sequence shown in SEQ ID NO: 2.

[0034] It should be noted that ubiquitin ligases are important substances that mediate the ubiquitination process in plants. Specifically, post-translational modifications (PTMs) are central to regulating protein stability and protein activity, affecting many aspects of plant growth and development. Ubiquitination is an important post-translational modification of proteins. Unlike most post-translational modifications, ubiquitinated proteins are targeted for degradation by the 26S proteasome. The ubiquitination process is mediated by three consecutive ubiquitinases: E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase). The latter catalyzes the formation of polypeptides between activated ubiquitin and lysine residues of substrate proteins. Finally, these polypeptides are degraded in the 26S proteasome, while ubiquitin monomers are recovered under the action of deubiquitinating enzymes. The specificity of protein ubiquitination is primarily determined by E3, which provides substrate recognition and binding specificity in a temporally and spatially regulated manner. F-box proteins, along with centromere protein 1 (SKP1), Cullin 1 (CUL1), and Ring-Box 1 (RBX1), form a ubiquitin ligase complex that plays a key role in recruiting substrates to the ubiquitin-proteasome system. F-box proteins are divided into distinct subfamilies based on the presence of protein-protein interaction motifs at their C-termini. One subfamily, designated Kelch Repeat F-box (KFB), contains a C-terminal Kelch repeat sequence in addition to the N-terminal F-box domain. KFBs are involved in the regulation of the phenylpropanoid pathway. By interacting with isoforms of phenylpropanoid pathway enzymes, such as PAL and CCR, KFBs selectively mediate the ubiquitination and degradation of these enzymes via the 26S proteasome, thereby contributing to plant lignification.

[0035] In addition, the research on ubiquitin ligase (KFB) in bamboo is still blank, especially the key enzyme of lignin biosynthesis targeted by this protein for degradation is also a research focus. Therefore, the ubiquitin ligase KFB protein related to the regulation of lignin synthesis in bamboo and the discovery of its direct degradation enzyme have important application prospects for the lignin improvement and targeted breeding of bamboo. In the examples of the present invention, it was verified that the ubiquitin ligase can effectively inhibit the activity of peroxidase involved in the polymerization of lignin monomers by degrading the PePRX72-1 protein, thereby affecting its lignin synthesis function.

[0036] Yet another embodiment of the present invention provides an application of a moso bamboo ubiquitin ligase gene PeKFB9 or its encoded protein in reducing the activity of peroxidases involved in the polymerization of lignin monomers in plants, the application comprising: degrading peroxidases involved in the polymerization of lignin monomers by the protein encoded by the moso bamboo ubiquitin ligase gene PeKFB9, thereby reducing the activity of peroxidases; wherein, the sequence of the moso bamboo ubiquitin ligase gene PeKFB9 comprises the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the moso bamboo ubiquitin ligase gene comprises the sequence shown in SEQ ID NO: 1.

[0037] It should be noted that existing studies generally believe that lignin polymerization is carried out by repeated free radical coupling of monomers with polymers (or oligomers) growing in the cell wall through oxidative coupling catalyzed by class III peroxidases (PRX) and laccases. The present invention discovered that the bamboo ubiquitin ligase PeKFB9 in moso bamboo, as shown in SEQ ID NO: 1, can degrade peroxidases (PRX). Based on this, in some specific embodiments of the present invention, the bamboo ubiquitin ligase degrades the peroxidases (PRX) involved in the polymerization of lignin monomers, thereby reducing their activity and thus affecting the function of lignin synthesis in the plant.

[0038] Yet another embodiment of the present invention provides an application of a bamboo ubiquitin ligase gene PeKFB9, its encoded protein, or a biomaterial containing the bamboo ubiquitin ligase gene PeKFB9 in improving the activity of peroxidases involved in the polymerization of lignin monomers in plants, the application comprising: improving the activity of peroxidases involved in the polymerization of lignin monomers in plants by down-regulating the expression of the bamboo ubiquitin ligase gene PeKFB9 in plants or down-regulating the activity of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 in plants; wherein the sequence of the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 1.

[0039] Yet another embodiment of the present invention provides an application of a bamboo ubiquitin ligase gene PeKFB9, its encoded protein, or a biomaterial containing the bamboo ubiquitin ligase gene PeKFB9 in improving the activity of peroxidases involved in the polymerization of lignin monomers in plants, the application comprising: improving the activity of peroxidases involved in the polymerization of lignin monomers in plants by down-regulating the expression of the bamboo ubiquitin ligase gene PeKFB9 in plants or down-regulating the activity of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 in plants; wherein the sequence of the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 1.

[0040] Similarly, as described above, the present invention discovered that the bamboo ubiquitin ligase PeKFB9 with a sequence as shown in SEQ ID NO: 1 in bamboo can degrade peroxidase (PRX). Based on this, in some specific embodiments of the present invention, the bamboo ubiquitin ligase degrades the peroxidase (PRX) involved in the polymerization of lignin monomers, thereby reducing its activity and thereby affecting the function of lignin synthesis in the plant.

[0041] It should be noted that in the above applications (reducing the activity of peroxidases involved in the polymerization of lignin monomers in plants or increasing the activity of peroxidases involved in the polymerization of lignin monomers in plants), the activity of peroxidase (PRX) in plants is regulated by moso bamboo ubiquitin ligase, thereby affecting lignin synthesis, and plants with high lignin content or low lignin content can be obtained in a targeted manner. In this way, for the application of plants with low lignin content, such as moso bamboo, moso bamboo with low lignin content can be well recycled after secondary processing (such as making paper); for the application of plants with high lignin content, the material properties of the plants can be improved and the utilization rate of the plants can be increased.

[0042] Another embodiment of the present invention provides a moso bamboo ubiquitin ligase gene PeKFB9, its encoded protein, or a biomaterial containing the moso bamboo ubiquitin ligase gene PeKFB9 for use in plant genetic breeding; wherein the sequence of the moso bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the encoded protein of the moso bamboo ubiquitin ligase gene PeKFB9 includes the sequence shown in SEQ ID NO: 1. It should be noted that, based on the above findings, the moso bamboo ubiquitin ligase gene PeKFB9, its encoded protein, or a biomaterial containing the moso bamboo ubiquitin ligase gene PeKFB9 of the present invention can also be used in plant genetic breeding to cultivate plants with high or low lignin content.

[0043] In some specific embodiments, the above application in plant genetic breeding may include: upregulating the activity of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 in the plant, and screening for plants with low peroxidase activity involved in the polymerization of lignin monomers; or promoting the overexpression of the bamboo ubiquitin ligase gene PeKFB9 in the plant, and screening for plants with low peroxidase activity involved in the polymerization of lignin monomers.

[0044] In some specific embodiments, the above application in plant genetic breeding may include: down-regulating the activity of the protein encoded by the bamboo ubiquitin ligase gene PeKFB9 in the plant, and screening for plants with high peroxidase activity involved in the polymerization of lignin monomers; or down-regulating the expression of the bamboo ubiquitin ligase gene PeKFB9 in the plant, and screening for plants with high peroxidase activity involved in the polymerization of lignin monomers.

[0045] In some specific embodiments, in the above applications (including applications in reducing the activity of peroxidases involved in the polymerization of lignin monomers in plants, applications in increasing the activity of peroxidases involved in the polymerization of lignin monomers in plants, and applications in plant genetic breeding), the biological materials are known in the art, including but not limited to gene expression cassettes, expression vectors, cloning vectors or engineered bacteria; in addition, the plants are plants that can be lignified as known in the art, including but not limited to bamboo.

[0046] In some specific embodiments, in the above-mentioned applications (including applications in reducing the activity of peroxidases involved in the polymerization of lignin monomers in plants, applications in increasing the activity of peroxidases involved in the polymerization of lignin monomers in plants, and applications in plant genetic breeding), methods for upregulating the activity of proteins encoded by the moso bamboo ubiquitin ligase gene PeKFB9 in plants or promoting the overexpression of the moso bamboo ubiquitin ligase gene PeKFB9 in plants are known in the art, such as introducing the above-mentioned nucleic acid sequence or biological material into the plant.

[0047] In some specific embodiments, in the above-mentioned applications (including applications in reducing the activity of peroxidases involved in the polymerization of lignin monomers in plants, applications in increasing the activity of peroxidases involved in the polymerization of lignin monomers in plants, and applications in plant genetic breeding), methods for down-regulating the activity of proteins encoded by the moso bamboo ubiquitin ligase gene PeKFB9 in plants or down-regulating the expression of the moso bamboo ubiquitin ligase gene PeKFB9 in plants are known in the art, such as mutating or knocking out the entire sequence or partial sequence of the gene as shown in SEQ ID NO: 2 in the plant; or using interfering RNA to interfere with the expression of the gene as shown in SEQ ID NO: 2 in the plant; or using a gene silencing system to silence the gene as shown in SEQ ID NO: 2 in the plant, etc.

[0048] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0049] Example 1 Obtaining the coding sequence of the bamboo ubiquitin ligase gene PeKFB9

[0050] Using bamboo shoots (Phyllostachysedulis) as the material, RNA was extracted and reverse transcribed into cDNA as a template. Specific primers were designed for the predicted gene PH02Gene23593 in Phyllostachys edulis, and the coding region sequence was amplified by PCR; the primer sequences are as follows:

[0051] Upstream primer: 5′-ATGAGGAGCCCAAAGAGCCG-3′,

[0052] Downstream primer: 5′-TCAGACGCGGACGGAGGC-3′;

[0053] PCR amplification was performed at different annealing temperatures (56°C, 59°C, 62°C, and 65°C) to obtain PCR products. The reaction system (20 μL) was as follows: PrimeSTAR MaxPremix (2×) 10.0 μL, 1.0 μL each of upstream and downstream primers, 1.5 μL template, and 6.5 μL ddH2O; the amplification program was: 98°C for 10 s; 56-71°C for 15 s; 72°C for 2 min 40 s, for 35 cycles. PCR amplification products were detected by agarose gel electrophoresis (detection results are shown in the table). Figure 1 ), the target band was excised, purified, and recovered. The recovered DNA fragment was ligated into the pGEM-TEasy vector and transformed into Escherichia coli DH5α competent cells. After blue-white screening, the positive clone plasmid was extracted and analyzed by enzyme digestion pattern. Then, a single clone was sequenced, and the insert fragment was 1095 bp, as shown in SEQ ID NO:2. The sequenced fragment was compared with the PH02Gene23593 sequence using Blast online software, and the sequences were completely consistent.

[0054] In addition, online comparative analysis using Blast software revealed that the amino acid sequence encoded by this gene (SEQ ID NO: 1) is highly identical to KFB proteins from other monocots. Protein domain analysis revealed that this protein possesses typical structural features of F-box and Kelch domains. Therefore, the cloned gene encodes a KelchRepeat F-box protein, and this gene was named PeKFB9 (SEQ ID NO: 2).

[0055] Example 2 Construction of a yeast two-hybrid vector carrying the bamboo ubiquitin ligase gene PeKFB9

[0056] Using bamboo cDNA as a template, primers were designed based on the sequence shown in SEQ ID NO: 2 to amplify the deoxyribonucleotide sequence of the bamboo PeKFB9 coding region by PCR. EcoRI and BamHI restriction sites were introduced at both ends of the primers, respectively. The primer sequences are as follows:

[0057] Upstream primer: 5′-CGggattcATGAGGAGCCCAAA-3′ (EcoRI site in lowercase letters),

[0058] Downstream primer: 5′-CGgaattcTCAGACGCGGACG-3′ (BamHI site in lowercase);

[0059] The PCR amplification products were detected by agarose gel electrophoresis. The reaction system (20 μL) was as follows: 10.0 μL of PrimeSTAR Max Premix (2×), 1.0 μL of upstream and downstream primers, 1.5 μL of template, and 6.5 μL of ddH2O. The amplification program was as follows: 98°C, 10 s; 65°C, 15 s, 72°C, 2 min 40 s, 35 cycles. The PCR amplification products were detected by agarose gel electrophoresis, and the target band was excised and purified. The recovered DNA fragment was ligated into the pGEM-TEasy vector and transformed into Escherichia coli DH5α competent cells. After blue-white spot screening, the positive clone plasmid was extracted. After the single clone was correctly sequenced, the plasmid pT-PeKFB9-1 containing EcoRⅠ and BamHⅠ restriction sites and the deoxyribonucleotide sequence encoding the bamboo ubiquitin ligase gene PeKFB9 was obtained.

[0060] Plasmid pT-PeKFB9-1 and expression vector pGBKT7 were digested with EcoRI and BamHI at 37℃ for 6h, respectively. The digestion products were analyzed by 1% agarose gel electrophoresis. The target bands were recovered and ligated with T4 DNA ligase at 4℃ overnight. The ligation products were transformed into DH5 competent cells, and kanamycin-resistant (50mg·L -1 ) The single clones grown on the plate were extracted and the enzyme digestion pattern was identified (the experimental results were as follows Figure 2 The obtained recombinant expression vector was named pGBKT7-PeKFB9.

[0061] In addition, according to Figure 3 The PeKFB9 sequence was truncated as shown in the figure and connected to the expression vector pGBKT7 according to the above method. The enzyme digestion pattern was identified (the experimental results are shown in Figure 2 The obtained recombinant expression vectors were named pGBKT7-K^PeKFB9.

[0062] Example 3 Yeast two-hybrid experiment confirms the binding of PeKFB9 and PePRX72-1

[0063] Combining the characteristics of the multiple cloning sites of PePRX72-1 and pGADT7 vectors, the restriction enzyme cleavage sites were determined, and specific primers were designed (upstream primer: 5′-ATGGAGGCCAGTGAATTCATGAGGGGTGCAATGGT-3′, downstream primer: 5′-CTCGAGCTCGATGGATCCTCAGTTGTGATTGACCCT-3′) for PCR amplification. The pGADT7 vector was digested with EcoRI and BamHI, and the target fragment and vector backbone were recovered and ligated using a one-step directional cloning kit. The plasmid was transformed, and the restriction enzyme cleavage map was identified and sequenced for verification. The product was named pGADT7-PePRX72-1.

[0064] The constructed vector was co-transformed into the competent yeast AH109; the bacterial solution was spread on DDO (SD / -Ieu / Trp) screening medium and inverted cultured at 29°C for 2 days. A single colony was picked in DDO liquid medium and cultured at 29°C and 200 rpm until the bacterial solution OD 600 The OD value is between 0.6-0.8, and the bacterial solution is centrifuged and resuspended. 600 1, aspirate 5 μL of bacterial solution and spot it on QDO (SD / -Ieu / Trp / -His / -Ade) containing Xa-gal, invert and culture for 3 days–5 days, and take pictures; the results are as follows; Figure 4 As shown, experimental group 1 (pGADT7-PePRX72-1 and pGBKT7-PeKFB9) grew normally and turned blue in defective medium (QDO), while experimental group 2 (pGADT7-PePRX72-1 and pGBKT7-K^PeKFB9) could not grow, indicating that PePRX72-1 and PeKFB9 bound to each other and the binding site was the Kelch repeat sequence of PeKFB9.

[0065] Example 4 Construction of a plant expression vector carrying the bamboo ubiquitin ligase gene PeKFB9

[0066] Using bamboo cDNA as a template, primers were designed based on the sequence shown in SEQ ID NO: 2 to amplify the deoxyribonucleotide sequence of the coding region of the bamboo ubiquitin ligase gene PeKFB9 by PCR. KpnⅠ and BamHI restriction sites were introduced at both ends of the primers, respectively. The primer sequences are as follows:

[0067] Upstream primer: 5′-GGggtaccATGAGGAGCCCAAA-3′ (Kpn I site in lowercase);

[0068] Downstream primer: 5′-CGgaattcTCAGACGCGGACG-3′ (BamHI site in lowercase);

[0069] The PCR amplification products were detected by agarose gel electrophoresis. The PCR amplification conditions and amplification system were the same as those in Example 2. The target band was excised, purified and recovered, and the recovered DNA fragment was ligated into the pGEM-TEasy vector and transformed into Escherichia coli DH5α competent cells. After blue-white spot screening, the positive clone plasmid was extracted. After the single clone was correctly sequenced, the plasmid pT-PeKFB9-2 containing KpnⅠ and BamHI restriction sites and the deoxyribonucleotide sequence encoding the bamboo ubiquitin ligase gene PeKFB9 was obtained.

[0070] Plasmid pT-PeKFB9-2 and expression vector pCAMBIA1300-3×FLAG plasmid were digested with KpnⅠ and BamHI at 37℃ for 6h, respectively. The digestion products were analyzed by 1% agarose gel electrophoresis, and the target bands were recovered and ligated with T4 DNA ligase at 4℃ overnight. The ligation products were transformed into DH5 competent cells, and kanamycin-resistant (50mg·L -1 ) The single clones grown on the plate were extracted and the enzyme digestion pattern was identified (the experimental results were as follows Figure 5 The obtained recombinant expression vector was named pCAMBIA1300-PeKFB9; Figure 3 PeKFB9 was truncated as shown in the figure and ligated with the expression vector pCAMBIA1300-3×FLAG according to the above method. The enzyme digestion pattern was identified (the experimental results are shown in Figure 5 The obtained recombinant expression vector was named pCAMBIA1300-F^PeKFB9 after verification by sequencing; the pCAMBIA1300-PePRX72-1 recombinant expression vector was constructed by the same method.

[0071] Example 5 Identification of Monoclonal Colonies Containing Expression Vectors

[0072] The expression vectors pCAMBIA1300-PeKFB9 and pCAMBIA1300-F^PeKFB9 constructed in Example 2 were transformed into competent cells of Agrobacterium tumefaciens strain EHA105 by electroporation, and kanamycin-resistant (50 mg·L -1 ) plates were identified by PCR; monoclonal colonies formed after transformation with the PeKFB9 gene recombinant expression vector were used as templates and PCR detection was performed using the primers in Example 2, while the recombinant plasmid and water were used as controls; pCAMBIA1300-PePRX72-1 was verified by the same method.

[0073] The results are as follows Figure 6As shown in the figure, the PCR electrophoresis results of Agrobacterium monoclonal colonies transformed with expression vectors pCAMBIA1300-PeKFB9, pCAMBIA1300-F^PeKFB9 and pCAMBIA1300-PePRX72-1 plasmids showed that the monoclonal colonies contained the target gene fragments and could be used for infection and transformation experiments.

[0074] Example 6 Detection of peroxidase activity

[0075] To investigate whether the interaction between PeKFB9 and PePRX72-1 isozymes affects the activity of peroxidase, an enzyme activity assay was performed as follows: pCAMBIA1300-PeKFB9, pCAMBIA1300-F^PeKFB9, pCAMBIA1300-PePRX72-1, and pCAMBIA1300 were used to form an Agrobacterium suspension, and the cells were collected by centrifugation; the cells were suspended in an infiltration medium (10 mM MgCl2, 10 mM MES, and 100 μM AS) to a concentration of OD 0. 600 When the p-value reaches about 0.8, let it stand at room temperature for 3 hours. Before infection, mix equal volumes of pCAMBIA1300-PeKFB9 and pCAMBIA1300-PePRX72-1, pCAMBIA1300-F^PeKFB and pCAMBIA1300-PePRX72-1, and pCAMBIA1300 and pCAMBIA1300-PePRX72-1, respectively. Take the same volume of the mixed suspension and inject it into tobacco leaves respectively. Place the injected tobacco in a dark incubator for 12 hours, then culture normally for 2-3 days, and store the samples in a -80℃ refrigerator.

[0076] The peroxidase activity of the above samples was determined by visible spectrophotometry. The results showed that the peroxidase activity in the leaves co-expressing pCAMBIA1300-PeKFB9 and pCAMBIA1300-PePRX72-1 decreased by more than 60% compared with the control (see Figure 7 ).

[0077] Example 7 Protein immunoblotting experiment

[0078] To verify that the reduction in peroxidase activity was due to degradation of the PePRX72-1 protein, a protein immunoblot experiment was performed using the above samples to test the stability of the PePRX72-1-FLAG fusion protein. The results showed that in tobacco leaf extracts co-expressing pCAMBIA1300-PeKFB9 and pCAMBIA1300-PePRX72-1, the PePRX72-1-FLAG fusion protein signal was weakened, while the signal remained almost unchanged when pCAMBIA1300-F^PeKFB was co-expressed with pCAMBIA1300-PePRX72-1. This shows that PeKFB9 can inhibit peroxidase activity by ubiquitinating and degrading the PePRX72-1 protein, thereby affecting its lignin synthesis function.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A bamboo ubiquitin ligase PeKFB9, characterized in that The amino acid sequence thereof comprises the sequence shown in SEQ ID NO:

1.

2. A nucleic acid sequence, characterized in that Encodes the bamboo ubiquitin ligase PeKFB9 described in claim 1.

3. The nucleic acid sequence according to claim 2, characterized in that Including the sequence shown in SEQ ID NO: 2.

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