PeLBT1, a transcription factor related to lignin synthesis in moso bamboo, and its application

Abstract

This invention relates to the field of genetic engineering technology, and discloses a transcription factor PeLBT1 related to lignin synthesis in moso bamboo and its applications. The amino acid sequence of PeLBT1 is shown in SEQ ID NO.1. The nucleotide sequence of a gene related to lignin synthesis in moso bamboo is shown in SEQ ID NO.2, and its encoded amino acid sequence is shown in SEQ ID NO.1. This invention utilizes the aforementioned transcription factor PeLBT1 and its applications to overexpress PeLBT1 or its encoding gene in rice, which can effectively reduce the lignin content in rice, providing a new gene resource for plant property improvement genetic engineering.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to PeLBT1, a transcription factor related to lignin synthesis in moso bamboo, and its applications. Background Technology

[0002] Moso bamboo (Phyllostachys edulis), an important plant belonging to the genus Phyllostachys of the subfamily Bambusoideae in the family Poaceae, occupies a vital position in my country's forestry resources. It is characterized by rapid growth, high yield, and strong adaptability, making it an excellent substitute for timber. Lignin, a key component of the cell wall of moso bamboo, is a crucial factor influencing its wood properties. Therefore, studying the regulatory mechanisms of lignin synthesis in moso bamboo is of great significance for improving its wood properties. However, the abundant presence of lignin in the industrial applications of moso bamboo has brought many problems. In the papermaking process, large amounts of chemical agents and energy are required to remove lignin, which not only increases production costs but also generates a large amount of polluting wastewater. Research on the regulation of lignin synthesis by transcription factors is still in its early stages, especially regarding the transcription factor PeLBT1, which regulates lignin synthesis in moso bamboo. Therefore, it is necessary to develop a method that utilizes key candidate genes for moso bamboo lignin and employs modern biotechnology to improve the wood properties of plants (including bamboo). Summary of the Invention

[0003] The purpose of this invention is to provide a transcription factor PeLBT1 related to lignin synthesis in moso bamboo and its application. Overexpression of the transcription factor PeLBT1 or its encoding gene in rice can effectively reduce the lignin content in rice, providing a new gene resource for genetic engineering for improving plant properties.

[0004] To achieve the above objectives, the present invention provides a transcription factor PeLBT1 related to lignin synthesis in moso bamboo, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] The present invention also provides a gene PeLBT1 related to lignin synthesis in moso bamboo, the nucleotide sequence of which is shown in SEQ ID NO.2 and the amino acid sequence encoded by which is shown in SEQ ID NO.1.

[0006] The present invention also provides a biomaterial comprising the above-mentioned genes related to lignin synthesis in moso bamboo.

[0007] Furthermore, the biological material is a gene expression cassette, expression vector, cloning vector, or engineered bacteria.

[0008] The present invention also provides the application of the above-mentioned transcription factor PeLBT1 related to lignin synthesis in moso bamboo, or the above-mentioned gene PeLBT1 related to lignin synthesis in moso bamboo, or the above-mentioned biological material in regulating lignin content.

[0009] Furthermore, the application method is as follows: overexpress the transcription factor PeLBT1, which is related to the synthesis of lignin in moso bamboo, or the gene PeLBT1, which is related to the synthesis of lignin in moso bamboo, in rice plants to reduce the lignin content in rice.

[0010] The present invention also provides a method for reducing the lignin content in rice by overexpressing the transcription factor PeLBT1, whose amino acid sequence is shown in SEQ ID NO.1, or the gene shown in SEQ ID NO.2, in rice to reduce the lignin content in rice.

[0011] The advantages and positive effects of the bamboo lignin synthesis-related transcription factor PeLBT1 and its application described in this invention are as follows:

[0012] 1. This invention successfully isolated and identified the lignin synthesis-related transcription factor PeLBT1 and its encoding gene from moso bamboo, and then transferred it into wild-type rice through transgenic technology for functional verification. The lignin content of the transgenic rice was reduced by 13.4% and 30.5% compared with that of wild-type rice, respectively, indicating that the transcription factor PeLBT1 is related to lignin formation.

[0013] 2. This invention overexpresses the transcription factor PeLBT1 or its encoding gene in rice, a monocotyledonous model plant, which can effectively reduce the lignin content in rice, providing a theoretical basis and technical support for the subsequent use of genetic engineering technology to improve the quality of moso bamboo.

[0014] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0015] Figure 1 This is an agarose gel electrophoresis image of the CDS sequence amplification of PeLBT1 in Example 1 of the present invention, where lanes 1-6 are the amplification products under conditions of 55℃, 56.9℃, 58.8℃, 61.1℃, 63℃ and 65℃ respectively, and M is DL5000;

[0016] Figure 2 This is an electrophoresis diagram of the PeLBT1 cloning plasmid single enzyme digestion verification in Example 1 of the present invention, where lanes 1-4 are the enzyme digestion products of the four recombinant plasmids, and M is DL2000;

[0017] Figure 3 This is an electrophoresis diagram of double enzyme digestion verification of the PeLBT1 expression vector recombinant plasmid in Example 2 of the present invention. Lanes 1-4 are the double enzyme digestion products of the four recombinant plasmids, M1 is DL15000, and M2 is DL5000.

[0018] Figure 4The results of RT-PCR detection of PeLBT1 in rice plants in Example 3 of this invention are shown, where M: DNA molecular weight marker; 1-2: different transgenic rice plants; 3: pCAMBIA1300-Ubi-GFP-FLAG-PeLBT1 plasmid; 4: wild-type rice plant WT.

[0019] Figure 5 This is the verification result of the relative expression level of PeLBT1 transgenic rice in Example 4 of the present invention, where WT is wild-type rice, and OE-2 and OE-5 are transgenic rice lines;

[0020] Figure 6 The following are the expression results of lignin synthesis genes in PeLBT1 transgenic rice in Example 4 of this invention, where WT is wild-type rice, OE-2 and OE-5 are transgenic rice lines, A is the expression level of the key enzyme PAL1 gene for lignin synthesis, B is the expression level of the key enzyme PAL5 gene for lignin synthesis, C is the expression level of the key enzyme 4CL3 gene for lignin synthesis, D is the expression level of the key enzyme 4CL4 gene for lignin synthesis, E is the expression level of the key enzyme CAD2 gene for lignin synthesis, and F is the expression level of the key enzyme CAD8 gene for lignin synthesis.

[0021] Figure 7 The results of lignin content analysis in PeLBT1 transgenic rice in Example 5 of this invention are shown, where WT is wild-type rice, and OE-2 and OE-5 are transgenic rice lines. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0025] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0026] This invention provides a transcription factor PeLBT1 related to lignin synthesis in moso bamboo, the amino acid sequence of which is shown in SEQ ID NO.1. It should be noted that the moso bamboo transcription factor PeLBT1 can reduce the lignin content of the plant, and has broad application prospects in plant-oriented breeding, providing a new gene resource for plant material property improvement genetic engineering.

[0027] Furthermore, PeLBT1, a transcription factor related to lignin synthesis in bamboo, is an NAC transcription factor. NAC (NAM, ATAF1 / 2, CUC1 / 2) transcription factors are a plant-specific family of transcription factors and one of the largest families of transcription factors in plants. They are widely involved in all stages of plant growth and development, from seed germination and seedling growth to flowering, fruiting, and ripening, all of which rely on the regulation of NAC transcription factors. In terms of hormone regulation, NAC transcription factors can respond to plant hormones such as gibberellin, abscisic acid, and ethylene, influencing plant growth and development by regulating the expression of related genes. For example, in rice, OsNAC120 can transcribe and activate the gibberellin synthesis genes OsGA20ox1 and OsGA20ox3, promoting gibberellin biosynthesis and thus promoting rice plant growth. OsNAC120 inhibits abscisic acid biosynthesis by transcribe and suppressing the abscisic acid synthesis genes OsNCED3 and OsNCED4, thereby negatively regulating abscisic acid-mediated drought tolerance in rice. Overexpression of OsNAC2 increases the sensitivity of rice seedling roots to ethylene and can directly activate the expression of OsACO and OsACO3, enhancing ethylene synthesis and thus delaying seedling establishment. In addition, NAC transcription factors also play an important regulatory role in the face of biotic stress (such as pathogen infection) and abiotic stress (such as drought, high temperature, low temperature, salinity, etc.), helping plants resist the effects of adverse external environments and maintain their growth and survival. For example, in rice, OsNAC23, OsNAC95, OsNAC122, OsNAC131, and OsNAC6 play a positive regulatory role in rice blast resistance. They can activate the expression of defense-related genes in rice, enhancing its resistance to rice blast fungus. Overexpression of OsNAC75 and OsNAC6 can enhance rice's resistance to rice blast and drought stress, indicating that they play an important bridging role in rice's response to multiple stresses. Meanwhile, NACs participate in the synthesis of secondary cell walls. For instance, overexpression of OsNAC29 and OsNAC31 in rice affects the expression of secondary cell wall-related genes, leading to changes in the content and ratio of components such as cellulose and lignin in the stem. However, research on NACs as important transcription factors in bamboo remains lacking. Therefore, elucidating the mechanism of NAC-mediated lignin synthesis in moso bamboo and utilizing key candidate genes to develop methods for improving plant (including bamboo) properties using modern biotechnology is of great significance. The transcription factor PeLBT1, which is related to lignin synthesis in moso bamboo, is a key gene affecting lignin synthesis. When applied to plant cultivation, it can effectively reduce the lignin content in the plant.

[0028] This invention also provides a gene related to lignin synthesis in moso bamboo, which encodes the transcription factor PeLBT1, a lignin synthesis-related transcription factor in moso bamboo. It should be noted that the nucleic acid encoding PeLBT1 can be any nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO. 1. As described above, expression or overexpression of this nucleotide sequence in the plant can effectively reduce the lignin content in the plant.

[0029] In some specific embodiments, the nucleotide sequence of the above-mentioned bamboo lignin synthesis-related gene is shown in SEQ ID NO. 2. It should be noted that the nucleotide sequence shown in SEQ ID NO. 2 can encode the amino acid sequence shown in SEQ ID NO. 1. Furthermore, it should be understood that the nucleotide sequence in this invention can also be a sequence obtained by codon optimization of the sequence shown in SEQ ID NO. 2. As described above, expression or overexpression of this nucleotide sequence in the plant can effectively reduce the lignin content in the plant.

[0030] This invention also provides a biological material comprising the above-described nucleotide sequence. It should be noted that this biological material can be any biological vector that can be used to express the above-described nucleotide sequence, including but not limited to gene expression cassettes, expression vectors, cloning vectors, or engineered bacteria. Gene expression cassettes, expression vectors, and cloning vectors are well known in the art. Furthermore, the engineered bacteria are generally Agrobacterium, used to mediate the transfection of exogenous target genes into plants.

[0031] This invention also provides a method for reducing the lignin content in plants: introducing the nucleotide sequence shown in SEQ ID NO.2 into the plant, thereby reducing the lignin content. It should be noted that introducing the nucleotide sequence into the plant is a technique known in the art. In some specific embodiments, the above-mentioned nucleotide sequence can first be constructed into an expression vector, and then transgenic plants containing the above-mentioned nucleotide sequence can be obtained through Agrobacterium-mediated transformation, thereby reducing the lignin content of the plant.

[0032] In some specific embodiments, rice can be selected as the plant in the above-described method for reducing lignin content in plants. Rice and bamboo are both monocotyledonous plants, and rice plays an important role in botanical research, often being used as a model organism. Therefore, this invention uses rice plants for functional verification.

[0033] It should be noted that, in this invention, by introducing the above-mentioned nucleotide sequence into wild-type rice, the lignin content of the transgenic rice was reduced by 13.4% and 30.5% respectively compared with that of the wild-type rice, indicating that the nucleotide sequence in this invention can achieve significant results in reducing the lignin content of plants.

[0034] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0035] Example 1: Obtaining the coding region sequence of the PeLBT1 gene, a transcription factor related to lignin synthesis in moso bamboo.

[0036] Based on the open reading frame sequence of PeLBT1 in the bamboo genome database, primers were designed as follows: upstream primer: 5′-ATGGCCAAGACATCGCTTC-3′ (SEQ ID NO.3), downstream primer: 5′-TCAAGTAACATGATCATGAATCGTA-3′ (SEQ ID NO.4). RNA was extracted from bamboo leaves and reverse transcribed into cDNA as a template for amplification. PCR amplification was performed under different temperature conditions (55℃, 56.9℃, 58.8℃, 61.1℃, 63℃, and 65℃) to find the optimal amplification temperature. The reaction system (20 μL) consisted of: 10.0 μL of 2×Taq Master Mix (Dye Plus), 1.0 μL each of upstream and downstream primers, 1.5 μL of template, and 6.5 μL of ddH2O. The amplification program was: 95℃ for 5 min; 98℃ for 15 s; 55-65℃ for 30 s; 72℃ for 1 min 14 s, for 35 cycles. The PCR amplification products were detected by agarose gel electrophoresis. The results are shown below. Figure 1 As shown, the target band was excised and purified. The recovered DNA fragment was ligated into the pCE3 BluntVector vector, transformed into E. coli DH5α competent cells, and plasmids were extracted from positive clones and verified by enzyme digestion. Figure 2 Sequencing revealed that the inserted gene fragment (the recovered DNA fragment) was 1224 bp, as shown in SEQ ID NO.2.

[0037] Example 2: Construction of a plant expression vector carrying the PeLBT1 gene

[0038] Using moso bamboo cDNA as a template, primers were designed based on the sequence shown in SEQ ID NO.2, with BamHI and PmlⅠ restriction sites introduced at both ends of the primers, respectively. The primer sequences are as follows: Upstream primer: 5′-TGTT ACTTCTGCAGggatccATGGCCAAGACATCGCTTCC-3′ (SEQ ID NO.5) (BamHI site lowercase letters); Downstream primer: 5′-CTCACCATAGGCCTcacgtgAGTAACATGA TCATGAATCG-3′ (SEQ ID NO.6) (PmlⅠ site lowercase letters); PCR amplification of the deoxyribonucleotide sequence of the coding region of moso bamboo PeLBT1 yielded the amplification product; The reaction system (20 μL) was as follows: 10.0 μL of 2×Taq Master Mix (Dye Plus), 1.0 μL each of upstream and downstream primers, 1.5 μL of template, and ddH2O. 6.5 μL of amplification was performed using the following program: 95℃, 5 min; 98℃, 15 s; 55-65℃, 30 s; 72℃, 1 min 14 s, 35 cycles. The PCR amplification products were detected by agarose gel electrophoresis. The target band was excised and purified. The recovered DNA fragment was ligated into the pCAMBIA1300-Ubi-GFP-FLAG vector using a one-step cloning method under 50℃ water bath conditions for 10 min. This ligation was then transformed into *E. coli* DH5α competent cells. Kanamycin (50 μg / ml) was picked. -1 Single clones grown on resistant plates were used to extract plasmids, which were then identified by restriction enzyme mapping (e.g., ...). Figure 3 (as shown in the figure) and sequencing verification, the obtained recombinant expression vector was named pCAMBIA1300-Ubi-GFP-FLAG-PeLBT1.

[0039] Example 3: PeLBT1 transformation of rice and qPCR detection

[0040] Since rice and bamboo are both monocotyledonous grasses and are closely related, and using the recombinant expression vector obtained in Example 2, Agrobacterium was used to transform rice callus tissue. Through screening, two transgenic rice lines were obtained, and gene expression was detected. Total RNA was extracted from both transgenic and wild-type rice plants and reverse transcribed into cDNA. Using this cDNA as a template, PCR detection was performed using the primers from Example 1. Figure 4 As shown, the results indicate that the target gene expression was detected in all transgenic rice plants, but not in wild-type rice plants, proving that PeLBT1 was expressed in transgenic rice plants (OE-2 and OE-5 are transgenic rice plants, and WT is a wild-type rice plant).

[0041] Example 4: Detection of Lignin Synthesis Gene Expression in PeLBT1 Transgenic Rice

[0042] The expression level of PeLBT1 was determined using qPCR. Figure 5 It was found that the expression level of PeLBT1 in transgenic rice plants was significantly higher than that in wild-type rice plants, with the transgenic rice line OE-5 showing a 38-fold increase compared to the wild-type rice. Further analysis was conducted on the expression patterns of genes in the endogenous lignin biosynthesis pathway in transgenic Arabidopsis plants. Results Figure 6 As shown, compared with wild-type rice plants, the expression of key enzyme genes for lignin synthesis was downregulated in transgenic rice plants.

[0043] Example 5: Analysis of lignin content in PeLBT1 transgenic rice

[0044] The lignin content in the wild-type and transgenic water in Example 4 was determined by colorimetry, and the results are as follows: Figure 7 As shown, the results indicated that, compared to wild-type rice (WT), the lignin content in the transgenic PeLBT1-expressed rice line OE-2 was reduced by approximately 13.4%, and in the transgenic rice line OE-5, it was reduced by approximately 30.5%. In conclusion, overexpression of PeLBT1 can reduce the lignin content in rice.

[0045] Therefore, this invention utilizes the aforementioned transcription factor PeLBT1 related to lignin synthesis in moso bamboo and its application. Overexpression of the transcription factor PeLBT1 or its encoding gene in rice can effectively reduce the lignin content in rice, providing a new gene resource for genetic engineering for improving plant properties.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A transcription factor PeLBT1 related to lignin synthesis in moso bamboo, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene related to lignin synthesis in moso bamboo, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.2, and its encoded amino acid sequence is shown in SEQ ID NO.

1.

3. A biomaterial, characterized in that: The biomaterial contains the gene related to lignin synthesis in moso bamboo as described in claim 2, wherein the biomaterial is a gene expression cassette, expression vector, or engineered bacteria.

4. A method for reducing the lignin content in rice, characterized in that: Overexpression of transcription factor PeLBT1 (amino acid sequence shown in SEQ ID NO.1) or gene (SEQ ID NO.2) in rice reduced the lignin content in rice.

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