ABC transporter PeABCG1 of bamboo and its application

Abstract

The present invention belongs to the field of plant genetic engineering technology, and specifically relates to a bamboo ABC transporter protein, PeABCG1, and its application. The amino acid sequence of the transporter protein comprises the sequence shown in SEQ ID NO: 1. The bamboo ABC transporter encoding gene, PeABCG1, of the present invention is introduced into wild-type Arabidopsis thaliana through transgenic technology. The resulting transgenic Arabidopsis thaliana has an experimentally verified lignin content of at least 7% higher than that of the wild-type Arabidopsis thaliana. In other words, the bamboo ABC transporter protein or encoding gene provided by the present invention can be used in plants to effectively increase the lignin content of the plants, providing a new genetic resource for genetic engineering of plant wood properties improvement.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a bamboo ABC transporter protein PeABCG1 and an application thereof. Background Art

[0002] Lignin is an important component of plant cell walls, and its content and tissue specific mass are key factors affecting wood properties. Bamboo, one of the herbaceous plants containing lignin, belongs to the genus Phyllostachys in the subfamily Bambusoideae of the Poaceae family. It has the characteristics of fast growth and strong adaptability, high yield, and excellent physical properties, making it a good substitute for wood. Moso bamboo (Phyllostachysedulis) is the most representative timber bamboo species in my country. Studying the regulatory mechanisms of lignin synthesis, lignin monomer polymerization and deposition in moso bamboo is of great significance for bamboo wood property improvement and breeding. However, due to the biological characteristics of bamboo (Moso bamboo), such as the long time to reach flowering and low fruiting rate, the different flowering periods of different high-quality bamboo strains, and the difficulty of hybridization, bamboo breeding has made slow progress. Therefore, research on the regulatory mechanisms of lignin synthesis, lignin monomer polymerization and deposition in moso bamboo remains a blank.

[0003] Therefore, a method for improving the wood properties of plants (including bamboo) by utilizing key candidate genes of bamboo lignin and using modern biotechnology is necessary. Summary of the Invention

[0004] In response to the above problems, the present invention aims to provide a bamboo ABC transporter protein PeABCG1. By introducing the nucleic acid sequence encoding PeABCG1 into the plant through transgenic technology, the lignin content of the transgenic plant can be increased, thereby improving the wood properties of the plant.

[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 ABC transporter PeABCG1, whose 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 ABC transporter PeABCG1.

[0008] In another aspect, the present invention provides a biological material comprising the above-mentioned nucleic acid sequence.

[0009] In another aspect, the present invention provides a use of the above-mentioned bamboo ABC transporter PeABCG1 or the above-mentioned nucleic acid sequence or the above-mentioned biological material in increasing the lignin content of plants.

[0010] In another aspect, the present invention provides a method for increasing the lignin content in a plant, comprising: introducing the above-mentioned nucleic acid sequence or biological material into the plant to thereby increase the lignin content in the plant.

[0011] The beneficial effects of the present invention include: the gene encoding the bamboo ABC transporter PeABCG1 of the present invention is transferred into wild-type Arabidopsis thaliana through transgenic technology, and the lignin content of the obtained transgenic Arabidopsis thaliana is verified to be at least 7% higher than that of the wild-type Arabidopsis thaliana through experiments, that is, the bamboo ABC transporter PeABCG1 or the encoding gene provided by the present invention is applied to plants, which can effectively increase the lignin content of the plants, and provides a new gene resource for genetic engineering of plant wood property improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is an agarose gel electrophoresis image of the CDS sequence amplification of PeABCG1 in Example 1;

[0013] Figure 2 This is the electrophoresis diagram of double enzyme digestion verification of the recombinant plasmid of the PeABCG1 expression vector in Example 2;

[0014] Figure 3 Schematic diagram of the PeABCG1 expression vector pCAMBIA1300-3×FLAG-PeABCG1 constructed in Example 2;

[0015] Figure 4 This is the electrophoresis diagram of the PCR verification of the single clone colony of Agrobacterium transformed with PeABCG1 in Example 3;

[0016] Figure 5 PCR verification of PeABCG1 transgenic Arabidopsis thaliana in Example 4;

[0017] Figure 6 This is the analysis of lignin content in PeABCG1 transgenic Arabidopsis thaliana in Example 5;

[0018] Figure 7 The staining results of the PeABCG1 transgenic Arabidopsis thaliana flower stem sections in Example 5;

[0019] in, Figure 1 In the middle, lanes 1–6 are the amplification products under conditions of 56°C, 59°C, 62°C, 65°C, 68°C, and 71°C, respectively, and M1 is DL15000; Figure 2 In the middle, lanes 1-6 are 6 recombinant plasmid double-enzyme digestion products, M1 is DL15000; Figure 4 In the figure, lane 1 is the negative control (sterile water), lane 2 is the positive control (recombinant plasmid), lanes 3-6 are the colony PCR products of 4 single colonies, and M2 is DL5000; Figure 5In the figure, lane 1 is wild-type Arabidopsis, lane 2 is recombinant plasmid, lanes 3-7 are five different transgenic Arabidopsis lines, and M2 is DL5000; Figure 6 In the figure, WT is wild-type Arabidopsis, and OE is transgenic Arabidopsis; Figure 7 In the figure, A is wild-type Arabidopsis thaliana, and B is transgenic Arabidopsis thaliana. DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] The present invention provides a bamboo ABC transporter protein PeABCG1, whose amino acid sequence comprises the sequence shown in SEQ ID NO: 1. It should be noted that the bamboo ABC transporter protein PeABCG1 can increase the lignin content of plants, has broad application prospects in plant directed breeding, and provides a new gene resource for plant wood property improvement genetic engineering.

[0023] In addition, it should be noted that ATP-binding cassette transporters (ABC) are the largest family of transporters in organisms. This type of protein uses the energy generated by ATP hydrolysis to drive the transport of substances. The ABC transporter family includes 8 subfamilies, of which ABCG is the largest subfamily, which is further divided into white-brown complex (WBC) and pleiotropic drug resistance complex (PDR). WBC is a half-molecule ABCG transporter with one core unit, while PDR is a full-molecule ABCG transporter with two core units. ABCG has been widely studied in plants and plays an important role in plant hormone transport, epidermal cuticle formation, secondary metabolite secretion, and resistance to biotic and abiotic stresses. For example, AtABCG1 and AtABCG16 are involved in the transport of cuticle components in the epidermis related to floral organs; AtABCG36 and AtABCG40 can respectively participate in the transport of Cd 2+ and Pd 2+ Cell efflux; ABCG25, ABCG30, ABCG31 and ABCG40 in Arabidopsis are transporters of abscisic acid (ABA), which play the role of inputting or outputting ABA in different tissue parts of the plant respectively. At the same time, ABCG is involved in the transport of lignin monomers. For example, AtABCG29 in Arabidopsis is a transporter of H-type lignin monomer coumarin. However, the research on ABCG as an important transporter in bamboo is still blank. Therefore, it is of great significance to analyze the transport mechanism of lignin monomers by moso bamboo ABCG and to use key candidate genes to carry out molecular breeding methods for improving the wood properties of plants (including bamboo) with the help of modern biotechnology. The moso bamboo ABC transporter gene PeABCG1 in the present invention is a key gene affecting lignin synthesis. When used in the cultivation of plants, it can effectively increase the lignin content of the plants.

[0024] The present invention also provides a nucleic acid sequence encoding the aforementioned bamboo ABC transporter PeABCG1. It should be noted that the nucleic acid sequence encoding the aforementioned bamboo ABC transporter PeABCG1 can be any sequence encoding the sequence shown in SEQ ID NO: 1. As described above, expression or overexpression of this nucleic acid sequence in plants can effectively increase the lignin content in the plants.

[0025] In some specific embodiments, the nucleic acid sequence includes the sequence shown in SEQ ID NO: 2. It should be noted that the 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 nucleic acid sequence of the present invention can also be a codon-optimized sequence of the sequence shown in SEQ ID NO: 2. As described above, expression or overexpression of this nucleic acid sequence in a plant can effectively increase the lignin content in the plant.

[0026] The present invention also provides a biomaterial comprising the aforementioned nucleic acid sequence. It should be noted that the biomaterial can be any biological vector capable of expressing the aforementioned nucleic acid 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 typically used are Agrobacterium, which is used to mediate transfection of plants with exogenous target genes.

[0027] The embodiment of the present invention also provides the use of the above-mentioned bamboo ABC transporter PeABCG1 or the above-mentioned nucleic acid sequence or the above-mentioned biological material in increasing the lignin content of plants. It should be noted that the above-mentioned plants can be any plants that can be lignified, such as all woody plants or lignified herbaceous plants (such as bamboo); in addition, these plants can be wild plants, cultivated plants, or plants that have undergone gene editing.

[0028] The present invention also provides a method for increasing the lignin content in a plant, comprising: introducing the aforementioned nucleic acid sequence into the plant to increase the lignin content in the plant. It should be noted that introducing a nucleic acid sequence into a plant is a technique known in the art. In some specific embodiments, the aforementioned nucleic acid sequence can be first constructed into an expression vector, and then transgenic plants containing the nucleic acid sequence can be obtained through Agrobacterium-mediated transfection, thereby increasing the lignin content of the plant.

[0029] In some specific embodiments, the plant used in the above-mentioned method for increasing the lignin content in a plant can be Arabidopsis thaliana. It should be noted that in the present invention, by introducing the above-mentioned nucleic acid sequence into wild-type Arabidopsis thaliana, the lignin content of the transgenic Arabidopsis thaliana obtained was at least 7% higher than that of the wild-type, indicating that the application of the nucleic acid sequence of the present invention to increase the lignin content of a plant can achieve significant results.

[0030] 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.

[0031] Example 1 Obtaining the coding sequence of the bamboo ABC transporter gene PeABCG1

[0032] Based on the open reading frame sequence of PH02Gene09206 in the moso bamboo genome database, primers were designed with the following sequences: upstream primer: 5′-ATGGAGGGCGCGATGGAGAAGGT-3′, downstream primer: 5′-CTATCGTCGTTGGAAGTTCAACTTC-3′; using moso bamboo shoots as materials, RNA was extracted and reverse transcribed into cDNA as amplification template, and PCR amplification was performed under different temperature conditions (56°C, 59°C, 62°C, 65°C, 68°C and 71°C) to find the optimal amplification temperature. The reaction system (20 μL) was as follows: PrimeSTAR MaxPremix (2×) 10.0 μL, upstream and downstream primers 1.0 μL each, template 1.5 μL, ddH2O 6.5 μL, amplification program: 98°C, 10 s; 56-71°C, 15 s, 72°C, 2 min 40 s, 35 cycles, PCR amplification products were detected by agarose gel electrophoresis (detection results see Figure 1 ), the target band was excised and purified and recovered, the recovered DNA fragment was ligated into the pGEM-TEasy vector, and transformed into Escherichia coli DH5α competent cells. Blue-white screening was performed, and plasmids were extracted from positive clones, enzyme digestion verification, and sequencing were performed. The results showed that the inserted gene fragment (recovered DNA fragment) was 4479 bp, as shown in SEQ ID NO: 2.

[0033] BLAST comparison analysis with the moso bamboo genome database revealed that SEQ ID NO:2 was highly similar (99.88%) to the sequence of PH02Gene09206, with only 5 bases different, including 3 synonymous mutations and 2 non-synonymous mutations, none of which were located in the conserved domain. Further online comparative analysis using BLASTP software revealed that the amino acid sequence encoded by the gene (SEQ ID NO:1) had a high identity of 92% with OsABCG42 of rice (Oryza sativa); protein domain analysis showed that the protein had a typical ABC transporter (PF00005) conserved domain. Thus, the cloned gene encodes a member of the ABC family protein, and the gene was named PeABCG1 (as shown in SEQ ID NO:2).

[0034] Example 2 Construction of a plant expression vector carrying the PeABCG1 gene

[0035] Using bamboo cDNA as a template, primers were designed according to the sequence shown in SEQ ID NO:2, and KpnⅠ and BamHI restriction sites were introduced at both ends of the primers, respectively. The primer sequences are as follows: upstream primer: 5′-ACGGGGGACGAGCTCggtaccATGGAGGGCGCGATGGAG-3′ (KpnⅠ site in lowercase); downstream primer 5′-GTAGTCCATTCTAGAggatccTCGTCGTTGGAAGTTCAA-3′ (BamHI site in lowercase). PCR amplification of the deoxyribonucleotide sequence of the bamboo PeABCG1 coding region obtained the amplified product. The reaction system (20 μL) was as follows: PrimeSTAR MaxPremix (2×) 10.0 μL, upstream and downstream primers 1.0 μL each, template 1.5 μL, ddH2O 6.5 μL, and the amplification program was as follows: 98°C, 10 s; 65°C, 15 s, 72°C, 2 min The PCR amplification products were detected by agarose gel electrophoresis, and the target band was cut out for purification and recovery. The recovered DNA fragment was connected to the pCAMBIA1300-3×FLAG vector by one-step cloning method in a 50°C water bath for 10 min, and transformed into Escherichia coli DH5α competent cells. Kanamycin (50 μg ml -1 ) Single clones grown on the resistant plate were extracted and identified by enzyme digestion pattern (see Figure 2 ) and sequencing verification, the obtained recombinant expression vector was named pCAMBIA1300-3×FLAG-PeABCG1 (see Figure 3 ).

[0036] Example 3 Identification of monoclonal colonies containing the expression vector pCAMBIA1300-3×FLAG-PeABCG1

[0037] The expression vector pCAMBIA1300-3×FLAG-PeABCG1 constructed in Example 2 was transformed into Agrobacterium tumefaciens GV3101 competent strain by electroporation, and kanamycin (50 μg ml -1)-resistant plates were identified by PCR. Using a single colony formed after transformation with the PeABCG1 gene recombinant expression vector as a template, PCR amplification was performed using the following primers (upstream primer: 5′-ATGGAGGGCGCGATGGA-3′, downstream primer: 5′-TTCATCCAGCGAGGAGCATAGA-3′). The reaction system (20 μL) was as follows: 10.0 μL PrimeSTAR Max Premix (2×), 1.0 μL each of the upstream and downstream primers, 1.5 μL of template, and 6.5 μL of ddH2O. The amplification program was as follows: 98°C for 10 s, 62°C for 15 s, and 72°C for 1 min 30 s, for 35 cycles. The amplified fragment was 2231 bp from SEQ ID NO: 2. Sterile water was used as a negative control, and the recombinant plasmid was used as a positive control. The PCR electrophoresis diagram of Agrobacterium monoclonal colony transformed with expression vector pCAMBIA1300-3×FLAG-PeABCG1 plasmid is shown in the figure below. Figure 4 As shown, the results showed that the monoclonal colony contained the target gene fragment, and the Agrobacterium monoclonal bacterial liquid was obtained by shaking the bacteria, which can be used for infection and transformation experiments.

[0038] Example 4 PeABCG1 transformation into Arabidopsis thaliana and PCR detection

[0039] The bacterial solution obtained in Example 3 was used to transform wild-type Arabidopsis thaliana by the floral dipping method. -1 ) screening, and finally obtained 5 homozygous Arabidopsis transgenic lines, and gene expression detection was performed. Total RNA of transgenic Arabidopsis plants and wild-type Arabidopsis plants was extracted and reverse transcribed into cDNA, and the cDNA was used as a template and PCR detection (amplification and gel electrophoresis) was performed using the primers in Example 3; the reaction system (20 μL) was as follows: PrimeSTARMaxPremix (2×) 10.0 μL, upstream and downstream primers 1.0 μL each, template 1.5 μL, ddH2O 6.5 μL, amplification program: 98°C, 10 s; 62°C, 15 s, 72°C, 1 min 30 s, 35 cycles; the detection results are shown in FIG. Figure 5 As shown, the results showed that the target gene was detected in all five Arabidopsis transgenic lines, but not in wild-type Arabidopsis plants, demonstrating that PeABCG1 had been transferred into Arabidopsis plants and expressed therein.

[0040] Example 5 Analysis of lignin content in PeABCG1 transgenic Arabidopsis

[0041] The lignin content in the wild-type Arabidopsis thaliana flower stem and the lignin content in the transgenic Arabidopsis thaliana flower stem in Example 4 were measured using a colorimetric method. The results are shown in FIG. Figure 6As shown, the results showed that lignin in transgenic PeABCG1 Arabidopsis (OE) increased by about 7% compared with wild-type Arabidopsis (WT).

[0042] In addition, the flower stems of PeABCG1-overexpressing Arabidopsis and wild-type Arabidopsis were vibrated and sectioned, and stained with phloroglucinol (phloroglucinol is a commonly used lignin-specific dye that can react with lignin to produce red or purple-red). Figure 7 As shown ( Figure 7 In the figure, A (wild-type Arabidopsis thaliana) is red, and B (transgenic Arabidopsis thaliana) is purple. Figure 7 Compared with the wild type, the stems of transgenic Arabidopsis thaliana were stained darker. The staining was consistent with the results of lignin content determination, indicating that the lignin content of Arabidopsis thaliana increased after transgenic PeABCG1.

[0043] In summary, overexpression of PeABCG1 can promote the increase of lignin content in Arabidopsis thaliana.

[0044] 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. The ABC transporter PeABCG1 of moso bamboo is characterized by: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that It encodes the bamboo ABC transporter PeABCG1 described in claim 1.

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

2.

4. A biomaterial, characterized in that Comprising the nucleic acid molecule according to claim 2 or 3.

5. The biomaterial according to claim 4, characterized in that The biological material is a gene expression cassette, an expression vector, a cloning vector or an engineered bacterium.

6. Use of the bamboo ABC transporter PeABCG1 according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the biomaterial according to claim 4 or 5 in increasing the lignin content in Arabidopsis thaliana.

7. A method for increasing the lignin content in Arabidopsis thaliana, characterized in that: include: The nucleic acid molecule according to claim 2 or 3 or the biological material according to claim 4 or 5 is introduced into Arabidopsis thaliana to increase the lignin content in Arabidopsis thaliana.

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