Moso bamboo lignin synthesis related transcription factor PeLBT1 and application thereof

Abstract

The invention relates to the technical field of gene engineering, and discloses a moso bamboo lignin synthesis related transcription factor PeLBT1 and application thereof. The amino acid sequence of the transcription factor PeLBT1 related to synthesis of moso bamboo lignin is as shown in SEQ ID NO. 1. The nucleotide sequence of the gene related to moso bamboo lignin synthesis is shown as SEQ ID NO.2, and the amino acid sequence coded by the gene is shown as SEQ ID NO.1. According to the moso bamboo lignin synthesis related transcription factor PeLBT1 and the application thereof, the transcription factor PeLBT1 or the coding gene thereof is overexpressed in rice, the lignin content in the rice can be effectively reduced, and a new gene resource is provided for plant wood property improvement gene engineering.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a transcription factor PeLBT1 related to lignin synthesis in Phyllostachys edulis and its application. Background Art

[0002] Phyllostachys edulis, as an important plant in the genus Phyllostachys of the subfamily Bambusoideae of the family Poaceae, occupies an important position in China's forestry resources. It has the characteristics of rapid growth, high yield, strong adaptability, etc., and is an excellent substitute for wood. Among the components of the cell wall of Phyllostachys edulis, lignin is a key factor affecting the wood properties of Phyllostachys edulis. Studying the regulatory mechanism of lignin synthesis in Phyllostachys edulis is of great significance for improving the wood properties of Phyllostachys edulis. However, in the industrial application of Phyllostachys edulis, the large amount of lignin brings many problems. In the papermaking process, a large amount of chemical agents and energy are required to remove lignin, which not only increases production costs but also generates a large amount of waste liquid polluting the environment. The research on the regulation of lignin synthesis by transcription factors is still in its infancy, especially the research on the transcription factor PeLBT1 regulating lignin synthesis in Phyllostachys edulis is still blank. Therefore, a method for improving the wood properties of plants (including bamboo) by using key candidate genes of lignin in Phyllostachys edulis and modern biotechnology means is necessary. Summary of the Invention

[0003] The object of the present invention is to provide a transcription factor PeLBT1 related to lignin synthesis in Phyllostachys edulis and its application. Overexpressing the transcription factor PeLBT1 or its coding gene in rice can effectively reduce the lignin content in rice, providing a new gene resource for genetic engineering of plant wood property improvement.

[0004] To achieve the above object, the present invention provides a transcription factor PeLBT1 related to lignin synthesis in Phyllostachys edulis, and its amino acid sequence is as shown in SEQ ID NO.1.

[0005] The present invention also provides a gene PeLBT1 related to lignin synthesis in Phyllostachys edulis, its nucleotide sequence is as shown in SEQ ID NO.2, and the amino acid sequence it encodes is as shown in SEQ ID NO.1.

[0006] The present invention also provides a biological material comprising the above-mentioned gene related to lignin synthesis in Phyllostachys edulis.

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

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

[0009] Furthermore, the application method is as follows: overexpress the transcription factor PeLBT1 related to moso bamboo lignin synthesis or the gene PeLBT1 related to moso bamboo lignin synthesis 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, which is to overexpress the transcription factor PeLBT1 with the amino acid sequence 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 transcription factor PeLBT1 related to moso bamboo lignin synthesis and its application according to the present invention are as follows:

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

[0013] 2. Overexpressing the transcription factor PeLBT1 or its encoding gene in the monocotyledonous model plant - rice can effectively reduce the lignin content in rice, providing a theoretical basis and technical support for subsequent improvement of moso bamboo quality using genetic engineering technology.

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

[0015] Figure 1 It is the agarose gel electrophoresis diagram of the CDS sequence amplification of PeLBT1 in Example 1 of the present invention. Among them, lanes 1-6 are the amplification products under the conditions of 55°C, 56.9°C, 58.8°C, 61.1°C, 63°C and 65°C respectively, and M is DL5000;

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

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

[0018] Figure 4RT-PCR detection results of PeLBT1 in rice plants in Example 3 of the present invention, 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 Verification results of the relative expression levels 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 Expression results of lignin synthesis genes in PeLBT1 transgenic rice in Example 4 of the present invention, where WT is wild-type rice, OE-2 and OE-5 are transgenic rice lines, A is the expression level of the key lignin synthesis enzyme PAL1 gene, B is the expression level of the key lignin synthesis enzyme PAL5 gene, C is the expression level of the key lignin synthesis enzyme 4CL3 gene, D is the expression level of the key lignin synthesis enzyme 4CL4 gene, E is the expression level of the key lignin synthesis enzyme CAD2 gene, and F is the expression level of the key lignin synthesis enzyme CAD8 gene;

[0021] Figure 7 Analysis results of lignin content in PeLBT1 transgenic rice in Example 5 of the present invention, where WT is wild-type rice, and OE-2 and OE-5 are transgenic rice lines; Detailed implementation manners

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

[0023] Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those of ordinary skill in the art to which the present invention belongs.

[0024] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually determined according to national standards. The experimental instruments, equipment and reagents not indicated the sources in the following examples are all commercially available raw materials.

[0025] Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0026] An embodiment of the present invention provides a transcription factor PeLBT1 related to lignin synthesis in Phyllostachys edulis, and its amino acid sequence is shown in SEQ ID NO.1. It should be noted that the Phyllostachys edulis transcription factor PeLBT1 can reduce the lignin content of plants, has broad application prospects in plant directional breeding, and provides new gene resources for genetic engineering of plant wood property improvement.

[0027] In addition, the transcription factor PeLBT1 related to lignin synthesis in Phyllostachys edulis is a NAC transcription factor. NAC (NAM, ATAF1 / 2, CUC1 / 2) transcription factors are a unique class of transcription factor families in plants and are also one of the largest transcription factor families in plants. They are widely involved in all stages of plant growth and development. From seed germination, seedling growth to plant flowering, fruiting and fruit ripening, they are all inseparable from the regulation of NAC transcription factors. In terms of hormone regulation, NAC transcription factors can respond to plant hormones such as gibberellin, abscisic acid, ethylene, etc., and affect the growth and development process of plants by regulating the expression of related genes. For example, OsNAC120 in rice can transcriptionally activate the gibberellin synthesis genes OsGA20ox1 and OsGA20ox3, promote the biosynthesis of gibberellin, and thus promote the growth of rice plants; OsNAC120 inhibits the biosynthesis of abscisic acid by transcriptionally suppressing the abscisic acid synthesis genes OsNCED3 and OsNCED4, thereby negatively regulating the drought tolerance of rice mediated by abscisic acid; Overexpression of OsNAC2 increases the sensitivity of roots to ethylene during the seedling stage of rice, and can directly activate the expression of OsACO and OsACO3, enhance ethylene synthesis, and thus delay seedling establishment. In addition, in the face of biotic stresses (such as pathogen infection) and abiotic stresses (such as drought, high temperature, low temperature, salinity, etc.), NAC transcription factors also play an important regulatory role, helping plants resist the influence of external adverse environments and maintaining their own growth and survival. For example, OsNAC23, OsNAC95, OsNAC122, OsNAC131 and OsNAC6 in rice have positive regulatory effects in the resistance to Magnaporthe oryzae. They can activate the expression of defense-related genes in rice and enhance the resistance of rice to Magnaporthe oryzae; After overexpression of OsNAC75 and OsNAC6, the resistance of rice to Magnaporthe oryzae and drought stress is enhanced, indicating that they play an important bridging role in rice's response to multiple stresses. At the same time, NAC is involved in the synthesis of secondary cell walls. For example, overexpression of OsNAC29 and OsNAC31 in rice affects the expression of secondary wall-related genes, resulting in changes in the content and proportion of components such as cellulose and lignin in the stem. However, the research on NAC as an important transcription factor in bamboo is still blank. Therefore, it is of great significance to analyze the mechanism of NAC in Phyllostachys edulis on lignin synthesis, use key candidate genes, and carry out methods for improving the wood properties of plants (including bamboo) with the help of modern biotechnology means. The transcription factor PeLBT1 related to lignin synthesis in Phyllostachys edulis in the present invention, as a key gene affecting lignin synthesis, can be effectively used to reduce the lignin content in plants when applied in plant cultivation.

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

[0029] In some specific embodiments, the nucleotide sequence of the above-mentioned gene related to moso bamboo lignin synthesis is as 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. In addition, it should be understood that the nucleotide sequence in the present invention can also be a sequence obtained by codon optimization of the sequence shown in SEQ ID NO.2. As described above, the expression or overexpression of this nucleotide sequence in plants can effectively reduce the lignin content in plants.

[0030] An embodiment of the present invention also provides a biological material containing the above nucleotide sequence. It should be noted that this biological material can be any biological vector that can be used for the expression of the above nucleotide sequence, including but not limited to gene expression cassettes, expression vectors, cloning vectors or engineering bacteria. Gene expression cassettes, expression vectors and cloning vectors are well known in the art. In addition, Agrobacterium is generally selected as the engineering bacterium to mediate the transfection of exogenous target genes into plants.

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

[0032] In some specific embodiments, in the above method for reducing the lignin content in plants, rice can be selected as the plant. Rice and moso bamboo are both monocotyledonous plants, and rice also has an important position in botanical research and is often used as a model organism. Therefore, the present invention conducts functional verification in rice plants.

[0033] It should be noted that in the present invention, by introducing the above nucleotide sequence into wild-type rice, the lignin content of the obtained transgenic rice is reduced by 13.4% and 30.5% respectively compared with that of the wild-type lignin, indicating that the nucleotide sequence in the present invention can achieve significant effects when applied to reduce the lignin content in plants.

[0034] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with specific examples, but the content of the present invention is not limited to the following examples only.

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

[0036] According to the open reading frame sequence of PeLBT1 in the moso bamboo genome database, primers were designed with the following sequences: forward primer: 5′-ATGGCCAAGACATCGCTTC-3′ (SEQ ID NO.3), reverse primer: 5′-TCAAGTAACATGATCATGAATCGTA-3′ (SEQ ID NO.4); Using moso bamboo leaves as materials, RNA was extracted and reverse transcribed into cDNA as the amplification template, and PCR amplification was carried out under different temperature conditions (55°C, 56.9°C, 58.8°C, 61.1°C, 63°C and 65°C) to find the optimal amplification temperature. The reaction system (20 μL) was as follows: 2×Taq Master Mix (Dye Plus) 10.0 μL, 1.0 μL each of the forward and reverse primers, 1.5 μL of the template, 6.5 μL of ddH2O. The amplification program was: 95°C, 5 min; 98°C, 15 s; 55 - 65°C, 30 s, 72°C, 1 min 14 s, 35 cycles. The PCR amplification products were respectively detected by agarose gel electrophoresis, and the detection results were as Figure 1 shown. The target band was cut out and purified and recovered. The recovered DNA fragment was ligated to the pCE3 BluntVector vector, and Escherichia coli DH5α competent cells were transformed. After extracting the plasmid from the positive clones and verifying by enzyme digestion ( Figure 2 shown), sequencing showed 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 according to the sequence shown in SEQ ID NO.2, and BamHⅠ and PmlⅠ restriction enzyme sites were introduced at both ends of the primers. The primer sequences are as follows: Forward primer: 5′-TGTT ACTTCTGCAGggatccATGGCCAAGACATCGCTTCC-3′ (SEQ ID NO.5) (the lowercase letters represent the BamHⅠ site); Reverse primer: 5′-CTCACCATAGGCCTcacgtgAGTAACATGA TCATGAATCG-3′ (SEQ ID NO.6) (the lowercase letters represent the PmlⅠ site); The deoxyribonucleotide sequence encoding the PeLBT1 of moso bamboo was amplified by PCR to obtain an amplification product; The reaction system (20 μL) is as follows: 2×Taq Master Mix (Dye Plus) 10.0 μL, 1.0 μL each of the forward and reverse primers, 1.5 μL of the template, 6.5 μL of ddH2O. The amplification program is as follows: 95 °C for 5 min; 98 °C for 15 s; 55 - 65 °C for 30 s, 72 °C for 1 min 14 s, for 35 cycles. The PCR amplification product was detected by agarose gel electrophoresis, the target band was cut and purified and recovered. The recovered DNA fragment was ligated to the pCAMBIA1300-Ubi-GFP-FLAG vector by one-step cloning method under the condition of 50 °C water bath for 10 min, and then transformed into Escherichia coli DH5α competent cells. Single colonies grown on the kanamycin (50 μg·ml -1 ) resistant plate were picked, plasmids were extracted, and identified by restriction enzyme mapping (as shown in Figure 3 ) and sequencing verification. The obtained recombinant expression vector was named pCAMBIA1300-Ubi-GFP-FLAG-PeLBT1.

[0039] Example 3 Transformation of PeLBT1 into Rice and qPCR Detection

[0040] Since both rice and bamboo are monocotyledonous gramineous plants and are very closely related, and using the recombinant expression vector obtained in Example 2, Agrobacterium was transformed to infect rice callus. Through screening, 2 transgenic rice lines were finally obtained and gene expression detection was carried out. Total RNA of transgenic rice plants and wild-type rice plants was extracted and reverse transcribed into cDNA respectively. Using this cDNA as a template, PCR detection was carried out with the primers in Example 1. As shown in Figure 4 , the results showed that the expression of the target gene was detected in 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, WT is wild-type rice plants).

[0041] Detection of the Expression of Lignin Synthesis Genes in PeLBT1 Transgenic Rice in Example 4

[0042] The expression level of PeLBT1 was determined by qPCR method. As Figure 5 can be seen, the expression level of PeLBT1 in transgenic rice plants was significantly higher than that in wild-type rice plants, and the transgenic rice line OE-5 was 38 times that of wild-type rice plants. Further analysis was carried out on the expression patterns of genes in the endogenous lignin biosynthesis pathway in transgenic Arabidopsis plants. The results Figure 6 showed that compared with wild-type rice plants, the expression of key enzyme genes for lignin synthesis in transgenic rice plants was down-regulated.

[0043] Example 5 Analysis of Lignin Content in PeLBT1 Transgenic Rice

[0044] The lignin content in the wild-type and transgenic rice in Example 4 was determined by colorimetry respectively. The determination results are as Figure 7 shown. The results showed that compared with wild-type rice (WT), the lignin content in the transgenic rice line OE-2 of transgenic PeLBT1 decreased by about 13.4%; the lignin content in the transgenic rice line OE-5 decreased by about 30.5%. In summary, overexpression of PeLBT1 can reduce the lignin content in rice.

[0045] Therefore, the present invention adopts the above-mentioned transcription factor PeLBT1 related to lignin synthesis in Phyllostachys edulis and its application. Overexpressing the transcription factor PeLBT1 or its coding gene in rice can effectively reduce the lignin content in rice, providing a new gene resource for genetic engineering of plant wood property improvement.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions 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 Phyllostachys edulis, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

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

1.

3. A biological material, characterized in that: It contains the gene related to moso bamboo lignin synthesis described in claim 2.

4. The biomaterial according to claim 3, wherein: The biological material is a gene expression cassette, an expression vector, a cloning vector or an engineered bacterium.

5. Use of the transcription factor PeLBT1 related to moso bamboo lignin synthesis described in claim 1, or the gene related to moso bamboo lignin synthesis described in claim 2, or the biological material described in claim 3 in regulating lignin content.

6. The application according to claim 5, characterized in that: The application method is: overexpressing the transcription factor PeLBT1 related to moso bamboo lignin synthesis or the gene PeLBT1 related to moso bamboo lignin synthesis in rice plants to reduce the lignin content in rice.

7. A method for reducing the lignin content in rice, characterized in that: Overexpressing the transcription factor PeLBT1 with the amino acid sequence shown in SEQ ID NO.1 or the gene shown in SEQ ID NO.2 in rice to reduce the lignin content in rice.

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