A peony PlLAC15 gene, protein, and application and identification method thereof

Abstract

The present invention discloses a peony PlLAC15 gene, a protein, an application thereof and an identification method thereof, wherein the nucleotide sequence of the peony PlLAC15 gene is shown in SEQ ID NO. 1. The present invention promotes S-lignin accumulation, improves stalk strength, and creates a new tobacco germplasm with high stalk strength by transforming the constructed PlLAC15 gene overexpression vector into tobacco for expression.

Description

Technical Field

[0001] The invention relates to a peony PlLAC15 gene, a protein and an application and an identification method thereof, and belongs to the field of peony biotechnology. Background Art

[0002] Stem development is crucial for plant production. However, stem bending or lodging caused by insufficient stem strength can seriously reduce crop yield and ornamental quality of cut flowers (Chang YL, Cui HX, Wang YY, et al. Silicon spraying enhances wheat stem resistance to lodging under lightstress. Agronomy, 2023, 13: 2565; Tonooka M, Homma Y, Toyoizumi T, et al. Stem bending in cut gerbera under the condition of suppressing bacterial proliferation is associated with the weakening of the stem strength. Scientia Horticulturae, 2023, 319: 112153). Therefore, improving stem strength is an urgent problem to be solved in plant production. Plant stem strength is closely related to the secondary wall components of mechanical tissue. Among these components, cellulose is the main substance of the cell wall skeleton, and its deposition plays an important role in stabilizing the cell wall structure (Lee KJD, Marcus SE, Knox JP. Cell wall biology: perspectives from cell wall imaging. Molecular Plant, 2011, 4: 212-219).In barley (Guo BJ, Huang XY, Qi J, et al. Brittle culm 3, encoding a cellulose synthase subunit 5, is required for cell wall biosynthesis in barley (Hordeum vulgare L.). Frontiers in Plant Science, 2022, 13: 989406), soybean (Liu W, Deng Y, Hussain S, et al. Relationship between cellulose accumulation and lodging resistance in the stem of relay intercropped soybean [Glycine max (L.) Merr.]. Field Crops Research, 2016, 196: 261-267) and corn (Qi BQ, Hu J, Zhu LB, et al. Response of maizes talk to plant density on cellulose accumulation by modulating enzymes activities. Field Crops Research, 2023, 304: 109152), cellulose content is an important factor affecting stem strength. In addition, lignin in secondary cell walls provides structural support to plants.In wheat (Luo YL, ChangYL, Li CH, et al. Shading decreases lodging resistance of under wheat different planting densities by altering lignin monomer composition of stems. Frontiersin Plant Science, 2022, 13: 1056193), chrysanthemum (Zhao WQ, Ding L, Liu JY, et al. Regulation of lignin biosynthesis by an atypical bHLH protein CmHLB inchrysanthemum. Journal of Experimental Botany, 2022, 73: 2403-2419) and alfalfa (ZhangSM, Zhu HS, Wang L, et al. Effects of selenium on the lignin deposition pattern and stem mechanical properties of alfalfa (Medicago sativa L.). Journal of Agricultural and Food Chemistry, 2024, DOI: 10.1021 / acs.jafc.3c06684), lignin content was significantly correlated with stem strength.Some studies have found that compared with cellulose, lignin has a greater impact on stem strength (Hussain S, Iqbal N, Rahman T, et al. Shade effect on carbohydrates dynamics and stem strength of soybean genotypes. Environmental and Experimental Botany, 2019, 162, 374-382; Tian B, Liu L, Zhang L, et al. Characterization of culmmorphology, anatomy and chemical composition of foxtail millet cultivars differing in lodging resistance. Journal of Agricultural Science, 2015, 153, 1437-1448).

[0003] Lignin is a complex phenolic polymer widely found in plants. It can be divided into three types according to its composition structure: H-lignin, G-lignin and S-lignin. They are respectively polymerized from three monomers: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol (Boerjan W, Ralph J, Baucher M. Lignin biosynthesis. Annual Review of Plant Biology, 2003, 54: 519-546). Studies on model plants Arabidopsis thaliana and poplar have shown that laccase (LAC) and peroxidase (PER) can successively catalyze the polymerization of lignin monomers (Guo Yayu, Xu Huimin, Zhao Yuanyuan, et al. Research progress on plant lignification process and its regulation. Chinese Science: Life Sciences, 2020, 50 (2): 111-122.). Among them, LAC plays an important role in increasing the content of lignin. For example, overexpression of the sugarcane LAC gene in Arabidopsis can restore the lignin content of the Arabidopsis lac17 mutant (Cesarino I, Araújo P, Mayer JLS, et al. Expression of SofLAC, a new laccase in sugarcane, restores lignin content but not S:G ratio of Arabidopsis lac17 mutant. Journal of Experimental Botany, 2013, 64: 1769-1781); overexpression of the pear LAC1 gene leads to thickening of the cell walls of Arabidopsis xylem cells and intervascular fiber cells and a significant increase in lignin content (Cheng X, Li GH, Ma CH, et al. Comprehensive genome-wide analysis of the pear (Pyrus betschneideri) laccase gene (PbLAC) family and functional identification of PbLAC1 involved in lignin biosynthesis. Plos One, 2019, 14: e0210892).

[0004] Peony is a traditional famous flower in my country and a new high-end cut flower in recent years. However, the stem bending caused by insufficient stem strength seriously reduces the quality and commodity price of peony cut flowers. In the early stage, the inventors have found that lignin provides mechanical support for peony stems, and obtained candidate genes and transcription factors related to lignin biosynthesis (Zhao DQ, Luan YT, Xia X, et al. Lignin provides mechanical support to herbaceous peony (Paeonialactiflora Pall.) stems. Horticulture Research, 2020, 7: 213); and further identified three R2R3-MYB transcription factor family members to improve peony stem strength by regulating lignin biosynthesis and secondary wall thickening (Tang YH, Lu LL, Sheng ZP, et al. An R2R3-MYB network modulates stem strength by regulating lignin biosynthesis and secondary cell wall thickening in herbaceous peony. Plant Journal, 2023a, 113: 1237-1258). However, there has been no report on the genetic regulation of peony lignin monomer polymerization. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a peony PlLAC15 gene, protein and its application and identification method that can improve the stem strength.

[0006] Technical solution: To solve the above technical problems, the present invention provides a peony PlLAC15 gene, which is cloned by PCR amplification technology, and the nucleotide sequence of the peony PlLAC15 gene is shown in SEQ ID NO. 1. SEQ ID NO. 1 in the sequence consists of 1790 bases.

[0007] The present invention also provides a protein encoded by the peony PlLAC15 gene, the amino acid sequence of which is shown in SEQ ID NO. 2. Wherein, SEQ ID NO. 2 in the sequence table consists of 565 amino acids.

[0008] The present invention also provides an expression cassette, a recombinant vector, a recombinant bacterium or a cell, which contains the peony PlLAC15 gene.

[0009] Wherein, the recombinant vector is to introduce the peony PlLAC15 gene into the vector plasmid.

[0010] The recombinant bacteria are obtained by introducing the recombinant vector described in claim 4 into a host bacterium, and then selecting and identifying the positive strains and retaining them.

[0011] Wherein, the recombinant expression vector containing the PlLAC15 gene can be constructed using the existing plant binary expression vector pCAMBIA1301. The plant expression vector carrying the gene PlLAC15 of the present invention is transferred into Agrobacterium EHA105 cells, and then transformed into plant tissues by the leaf disc method, and the transformed host plant is tobacco. Further, the primers designed in the construction of the eukaryotic expression vector are SEQ ID NO.5 (5'-caggtcgactctagaggatccATGAAGTTTCCCATCGT CCAAT-3') and SEQ ID NO.6 (5'-ttcgagctcagatctggtaccACACGGTGGCATATCTGGTGG-3').

[0012] The present invention also provides the use of the peony PlLAC15 gene, peony PlLAC15 protein, expression cassette, recombinant vector, recombinant bacteria or cells in enhancing the strength of plant stems.

[0013] The present invention also provides a method for obtaining a plant with high stalk strength, comprising the following steps:

[0014] 1) making the plant contain the Paeonia lactiflora PlLAC15 gene; or

[0015] 2) The plant is made to express the protein encoded by the peony PlLAC15 gene.

[0016] Wherein, the method also includes transgenic, hybridization, backcrossing or asexual reproduction.

[0017] The present invention also provides a method for identifying plants with high stalk strength obtained by the method, comprising the following steps:

[0018] (1) determining whether the plant contains the Paeonia lactiflora PlILAC15 gene; or

[0019] (2) Determine whether the plant expresses the protein encoded by the Paeonia lactiflora PlLAC15 gene.

[0020] Among them, the transformed tobacco plants were screened by PCR and qRT-PCR verification. Further, PCR detection was carried out, and the primers designed were: upstream primer: 5'-AAATCCGCGTGCACGAGGT-3' (SEQ ID NO.7); downstream primer: 5'-TCGTTATGTTTATCGGCACTTTGCA-3' (SEQ ID NO.8); qRT-PCR detection was carried out, using tobacco NtActin (AB158612) as the internal reference gene, and the primers designed were: upstream primer NtActin-F: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO.9); downstream primer NtActin-R: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO.10); PlLAC15 gene primers were designed: upstream primer PlLAC15-F: 5'-GCTATTGTCATCTACCCC-3' (SEQ ID NO.11); downstream primer PlLAC15-R: 5'-GACCATTGATAAGATACGCA-3' (SEQ ID NO.12).

[0021] The present invention also provides a method for promoting the accumulation of S-lignin in plant stems, comprising the following steps:

[0022] 1) making the plant contain the Paeonia lactiflora PlLAC15 gene; or

[0023] 2) The plant is made to express the protein encoded by the peony PlLAC15 gene.

[0024] The plants include but are not limited to tobacco.

[0025] The present invention uses peony as material and provides an application of peony PlLAC15 gene in improving plant stem strength, which will provide new gene family members for creating high stem strength plant materials using LAC.

[0026] The invention constructs an overexpression vector of the peony PlLAC15 gene, transfers the pCAMBIA1301-PlLAC15 overexpression vector into tobacco by using the Agrobacterium-mediated leaf disc method, and measures the stalk strength when the plants grow to the full flowering stage. The stalk strength of the tobacco plants transferred with the PlLAC15 gene is increased by more than 50% compared with that of the wild-type plants, indicating that the overexpression of the peony PlLAC15 gene has the function of changing the stalk strength of the plants.

[0027] Among them, the cDNA full-length sequence of the peony PlLAC15 gene (SEQ ID NO.1)

[0028]

[0029] The amino acid sequence (SEQ ID NO. 2) was deduced from the cDNA sequence of the Paeonia lactiflora PlLAC15 gene.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention promotes S-lignin accumulation and improves stalk strength by transforming the constructed PlLAC15 gene overexpression vector into tobacco for expression, thereby creating a new tobacco germplasm with high stalk strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Nucleotide sequence of the peony PlLAC15 gene and its deduced amino acid sequence; the amino acid sequence is marked below the corresponding nucleotide sequence in the figure, and the rest is non-coding region; * indicates the stop codon;

[0032] Figure 2 : The amino acid sequence of the PlLAC15 gene of Paeonia lactiflora and the homologous phylogenetic tree of the LAC family of Arabidopsis thaliana;

[0033] Figure 3: Identification of transgenic tobacco plants with PlLAC15 gene based on PCR detection: 1: DL2000 DNA Marker; 2: wild-type plants; 3-4: transgenic peony PlLAC15 gene strains;

[0034] Figure 4 : Identification of transgenic tobacco plants with PlLAC15 gene based on qRT-PCR detection: ** indicates P < 0.01;

[0035] Figure 5 : Comparison of stalk strength between wild-type and transgenic PlLAC15 tobacco plants: ** indicates P < 0.01;

[0036] Figure 6 : Histochemical staining observation of the stems of wild-type and transgenic PlLAC15 tobacco plants: (A) stem diameter; (B) stem strength;

[0037] Figure 7 :Comparison of lignin content in stems of wild-type and transgenic PlLAC15 tobacco plants: (A) lignin content; (B) S-lignin content; (C) G-lignin content; (D) ratio of S-lignin to G-lignin content; where ** indicates P < 0.01. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0039] Example 1 Cloning of the full-length cDNA sequence of the peony PlLAC15 gene

[0040] Obtaining the full-length sequence of the PlLAC15 gene: Peony leaves were selected as materials, and the MiniBEST Plant RNAExtraction Kit (TaKaRa) was used to extract total RNA. The PrimeScript RT Reagent Kit (TaKaRa) was used to reverse transcribe and produce cDNA. The reverse transcription system was: 1μL RNA, 1μL Oligo (dT) 18, 1μL dNTP Mixture (10mM each), 2μL 5× PrimeScript Buffer, 0.25μL RNase Inhibitor, 0.25μL PrimeScript RTase, 4.5μL RNase Free ddH2O; reverse transcription program: 42℃ reaction for 60min, 70℃ extension for 15min. On this basis, PCR amplification was performed. The PCR amplification system was: 2μL cDNA, 2μL dNTP Mixture (10mM each), 2.5μL 10× Vazyme L Amp Buffer (Mg 2+ plus), 1μL Forward Primer (5'-TTTGCTCTGTTTACTTGTGACG-3' (SEQ ID NO.3)), 1μL Reverse Primer (5'-GCATCACTCTATTTCCACTACA-3' (SEQ ID NO.4)), 0.25μL VazymeLAmp DNA Polymerase (5U / μL), 16.3μL RNase Free ddH2O. The reaction conditions were: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 52℃ annealing for 30s, 72℃ extension for 60s, for a total of 35 cycles; 72℃ extension for 10min. The products were detected by 1% agarose gel electrophoresis and then separated and sequenced. The results are shown in Figure 1 As shown, the full length of the PlLAC15 gene cDNA is 1790 bp (the sequence is shown in SEQ ID NO.1), the open reading frame is 1698 bp, and a total of 565 amino acids are encoded (the sequence is shown in SEQ ID NO.2).

[0041] Example 2 Alignment of the amino acid sequence of the peony PlLAC15 gene with the Arabidopsis thaliana LAC family

[0042] The amino acid sequences of Arabidopsis LAC family were downloaded from The Arabidopsis Information Resource (TAIR) (https: / / www.arabidopsis.org / ) database. They and the amino acid sequence of PlLAC15 gene were expressed in FASTA format. Then, the evolutionary tree was constructed using MEGA software. The algorithm was NeighborJoining. After 1000 self-introductions, their most homologous amino acid sequences were observed. The results showed that it was clustered with Arabidopsis AtLAC15 ( Figure 2 ).

[0043] Example 3 Expression of Peony PlLAC15 Gene Overexpression Vector in Tobacco

[0044] Construction of overexpression vector of peony PlLAC15 gene: A pair of primers were designed to amplify the PlLAC15 sequence (upstream primer PlLAC15-F: 5'-caggtcgactctagaggatccATGAAGTTTCCCATCGTCCAAT-3' (SEQ ID NO.5), downstream primer PlLAC15-R: 5'-ttcgagctcagatctggtaccACACGGTGGCATATCTGGTGG-3' (SEQ ID NO.6)), which were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. PCR amplification system: 12.5μL 2×Phanta FlashMaster Mix (Vazyme), 1μL Forward Primer, 1μL Reverse Primer, 2μL DNA template, 8.5μL ddH2O. Reaction procedure: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 52℃ annealing for 5s, 72℃ extension for 10s, a total of 35 cycles; 72℃ extension for 1min. After the reaction, the PCR reaction solution was analyzed by agarose gel electrophoresis, and the TSP601-DNA gel recovery kit (Tsingke) was used to recover the large fragment of PlLAC15 containing the restriction site. The binary expression vector pCAMBIA1301 plasmid was double-digested with BamHI and KpnI. The enzyme digestion reaction system was: 2.0μL 10×CutSmart Buffer, 4μL pCAMBIA1301 plasmid, 0.4μL BamHI (20000U / mL), 0.4μL KpnI (20000U / mL), 13.2μL ddH2O; react at 37℃ for 1h. The double-digestion product was analyzed by agarose gel electrophoresis, and the TSP601-DNA gel recovery kit (Tsingke) was used to recover and purify the large fragment of the plasmid pCAMBIA1301. plus One step PCR Cloning Kit (Novoprotein) uses homologous recombination to connect two recovered products. The reaction system is: 1.0 μL 5× reaction buffer, 0.3 μL plus recombinase, 3.0 μL pCAMBIA1301 large fragment, 1.0 μL PlLAC15 large fragment; connect in a 50°C metal bath for 15 min and cool on ice, take 5 μL of the connection product and transform it into Trelief TM 5α competent cells (Tsingke), and then cultured on LB plates (containing Kan 50 mg / L) at 37°C overnight, picked positive single clones for expansion culture, extracted plasmid pCAMBIA1301-PlLAC15, and then double enzyme digestion and sequencing verification were performed until the pCAMBIA1301-LAC15 overexpression vector was successfully constructed.

[0045] Transformation of tobacco with the overexpression vector of the peony PlLAC15 gene: Take 5uL of the pCAMBIA1301-PlLAC15 overexpression vector plasmid and transform it into GV3101 (pSoup-p19) competent cells (TOLOBIO), and then culture it on a YEB plate (containing Rif 50mg / L and Kan 50mg / L) at 28℃ for 2d, pick the positive single clone and culture it in YEB liquid culture medium (containing Rif 50mg / L and Kan 50mg / L), 28℃, 200rpm overnight. Take 2mL of the shaken bacterial solution and add it to 50mL of liquid YEB (containing Rif 50mg / L and Kan 50mg / L) containing the same antibiotics, and culture it under the same conditions until OD 600=0.3-0.4. Pour the shaken bacteria into a 50mL centrifuge tube, centrifuge at room temperature at 5000rpm for 10min, and discard the supernatant for later use. First, add an appropriate amount of acetosyringone (20mg / mL) to a sterilized small triangular flask, add 5mL of MS0 (MS0 liquid basic culture medium, without agar and sucrose) to the centrifuge tube to dissolve the bacteria, beat it evenly with a gun, pour it into a small triangular flask with an appropriate amount of acetosyringone, and then add MS0 to 50mL. Add 50mL of MS0 (MS0 liquid basic culture medium, without agar and sucrose) to another sterilized small triangular flask for later use. Take the sterile seedling leaves of tobacco, cut them into small pieces (about 1cm×1cm), put them into a 50mL small triangular bottle with MS0, cut a total of 100 leaves and put them into the bottle, pour the leaves into a beaker covered with gauze, take the filtered leaves and add them into a small triangular bottle of 50mL MS0+100μL acetosyringone (100μmol / mL), infect for 8min, and shake gently during infection; after the infection is completed, filter out the bacterial solution, take out the leaves, use sterile filter paper to absorb the excess bacterial solution on the surface of the leaves, inoculate them in co-cultivation medium [MS medium + 3.0mg / L 6-benzylaminopurine (6-BA) + 0.1mg / L naphthaleneacetic acid (NAA) + 30g / L sucrose + 6.66% agar], and culture them in the dark for 3d; after the co-cultivation is completed, transfer to the resistance bud screening differentiation medium [MS medium + 3.0mg / L6-BA + 0.1mg / L NAA + 30g / L sucrose + 6.66% agar + 100mg / L carbenicillin (Cb) + 25mg / L hygromycin (Hyg)] for selection culture, subculture once every two weeks until differentiation and budding; when the adventitious buds reach more than 2cm, cut off the adventitious buds and transfer to rooting screening medium [1 / 2MS + 0.3mg / L indole butyric acid (IBA) + 30g / L sucrose + 6.66% agar + 50mg / L Cb + 8mg / L Hyg] for rooting screening. After 4-6 months of cultivation, transgenic tobacco with PlLAC15 gene can be obtained.

[0046] Example 4 Identification of tobacco plants transformed with the peony PlLAC15 gene

[0047] PCR identification: NuClean Plant Genomic DNA Kit (CWBIO) was used to extract tobacco leaf DNA. On this basis, specific primers of the PlLAC15 gene (Forward Primer: 5'-AAATCCGCGTGCACGAGGT-3' (SEQ ID NO.7), Reverse Primer: 5'-TCGTTATGTTTATCGGCACTTTGCA-3' (SEQ ID NO.8)) were designed for PCR amplification. Reaction system: 12.5μL 2×Rapid Taq Master Mix (Vazyme), 1μL Forward Primer, 1μL Reverse Primer, 2μL DNA template, 8.5μL ddH2O. Reaction procedure: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 52℃ annealing for 15s, 72℃ extension for 5s, a total of 35 cycles; 72℃ extension for 5min. After the reaction, the PCR reaction solution was detected by gel electrophoresis. Figure 3 It can be seen that a single bright band was detected in the transgenic tobacco with the peony PlLAC15 gene, but not in the wild-type tobacco.

[0048] qRT-PCR identification: MiniBEST Plant RNA Extraction Kit (TaKaRa) was used to extract total RNA from tobacco leaves, and HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme) was used to reverse transcribe total RNA into cDNA. The reaction system was: 1.0 μL RNA, 4.0 μL 4×gDNA wiper Mix, 11.0 μL RNase Free dH2O; the reaction conditions were: 42°C for 2 min. After the reaction was completed, 4.0 μL 5×HiScript III qRTSuperMix was added to the reaction solution; the reaction conditions were: 37°C for 15 min, 85°C for 5 s. The cDNA obtained by reverse transcription was used SYBR qPCR SuperMix Plus (Novoprotein) kit was used for qRT-PCR detection. On this basis, the tobacco NtActin (AB158612) gene was used as an internal reference (Forward Primer: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO.9), Reverse Primer: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO.10)), and specific primers for the PlLAC15 gene (Forward Primer: 5'-GCTATTGTCATCTACCCC-3' (SEQ ID NO.11), Reverse Primer: 5'-GACCATTGATAAGATACGCA-3' (SEQ ID NO.12)) were designed for qRT-PCR detection. Reaction system: 2 μL cDNA, 12.5 μL SYBR qPCR SuperMix Plus, 1μL Forward Primer, 1μL Reverse Primer, 8.5μL ddH2O. Reaction procedure: 95℃ pre-denaturation for 3min; 95℃ denaturation for 5s, 55℃ annealing for 30s, 72℃ extension for 30s, a total of 40 cycles; 72℃ extension for 10min. After the reaction, 2 -△△Ct Methods The relative expression level of genes was analyzed. The results of qRT-PCR identification showed that PlLAC15 had a significantly higher expression level in transgenic tobacco ( Figure 4 ).

[0049] Example 5 Phenotypic observation of tobacco plants transformed with the peony PlMYB103 gene

[0050] When the tobacco plants were cultured to flowering, the phenotypes of wild-type and transgenic peony PlLAC15 tobacco plants were compared. It was found that the upper stems of transgenic peony PlLAC15 tobacco plants were straighter than those of wild-type plants ( Figure 5 ).

[0051] Example 6 Determination of relevant morphological indexes of tobacco plants transformed with the peony PlLAC15 gene

[0052] The diameter and strength of the upper stems of the wild-type and transgenic peony PlLAC15 tobacco plants in Example 5 were measured using a vernier caliper and a stem strength tester (NK-2). The results showed that the diameter of the upper stems of the transgenic peony PlLAC15 tobacco plants was relatively higher than that of the wild-type, but the difference was not significant; while the stem strength was significantly improved, with an increase of more than 50% ( Figure 6), indicating that overexpression of the peony PlLAC15 gene can significantly increase the stem strength of tobacco plants.

[0053] Example 7 Determination of lignin content in the stems of tobacco plants transformed with the peony PlLAC15 gene

[0054] The lignin content was determined by gas chromatography-mass spectrometry (GC-MS). The whole stems of the wild-type and transgenic peony PlLAC15 tobacco plants in Example 5 were first dried at 80°C to constant weight, crushed, and passed through a 40-mesh sieve to obtain dry samples. 20 mg of dry samples were weighed in a reaction bottle, 1 mL of freshly prepared reaction solution (final concentration of 2.5% BF3 and 10% ethanethiol dissolved in dioxane) was added, and the reaction was placed in a 100°C constant temperature oven for 4 h, and the reaction bottle was shaken every 1 h. After that, the reaction bottle was placed at -20°C, the reaction was stopped for 5 min, 0.2 mL of internal standard tetracosane (dissolved in dichloromethane) with a concentration of 0.1 mg / mL was added, pH = 4 was adjusted with 0.4 mol / L NaHCO3, 2 mL of ultrapure water and 1 mL of CH2Cl2 were added, vortexed, and allowed to stand for stratification. The lower organic phase was aspirated, evaporated and redissolved in 0.4 mL CH2Cl2, 50 μL pyrimidine and 100 μL BSA were added, and the mixture was allowed to stand at 25 °C for 4 h. 2 μL was taken for analysis by GC-MS (Trace1310 ISQ, Thermo, USA). Chromatographic conditions: TG-5MS (30 m × 0.25 mm × 0.25 μm) column; the heating program was an initial temperature of 50 °C, which was increased to 220 °C at a rate of 35 °C / min, and then to 230 °C at a rate of 0.5 °C / min, and finally to 280 °C at a rate of 50 °C / min, and maintained for 7 min; the injection port temperature was 250 °C; the carrier gas flow rate was 1.2 mL / min; the split ratio was 20:1; and the injection volume was 2 μL. Mass spectrometry conditions: EI ion source, ion source temperature of 280°C, electron energy of 70 eV; transfer line temperature of 280°C; solvent delay time of 5 min; scanning range of 40 to 650 amu. Figure 7 It can be seen that compared with wild-type plants, the lignin content in the stems of transgenic peony PlLAC15 tobacco plants increased significantly, especially S-lignin increased by 15.39%, while G-lignin changed little, only increased by 3.8%, which led to a significant increase in S / G.

[0055] In summary, the present invention provides a full-length cDNA sequence of the peony PlLAC15 gene and its application in enhancing plant stem strength. By transforming the constructed PlLAC15 gene overexpression vector into tobacco for expression, the accumulation of S-lignin in the stem is promoted, the stem strength is improved, and a new tobacco germplasm with high stem strength is created.

Claims

1. A peony PlLAC15 A gene characterized by The peony PlLAC15 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The herbaceous peony according to claim 1 PlLAC15 The protein encoded by the gene is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. An expression cassette, a recombinant vector or a recombinant bacterium, characterized in that: Containing the peony according to claim 1 PlLAC15 Gene.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector is PlLAC15 Gene introduction vector plasmid.

5. The recombinant bacterium according to claim 3, characterized in that The recombinant bacteria are obtained by introducing the recombinant vector described in claim 4 into a host bacteria, and retaining positive strains after screening and identification.

6. The herbaceous peony according to claim 1 PlLAC15 Use of the gene, the expression cassette, the recombinant vector or the recombinant bacteria as described in claim 3 in enhancing the strength of tobacco stalks.

7. A method for obtaining tobacco with high stalk strength, characterized in that: The following steps are involved: 1) Making tobacco contain the nucleotide sequence of peony shown in SEQ ID NO.1 PlLAC15 Gene; or 2) Making tobacco express the peony root with the amino acid sequence shown in SEQ ID NO.2 PlLAC15 Protein encoded by a gene.

8. The method according to claim 7, characterized in that The methods also include transgenic, hybridizing, backcrossing or asexual propagation.

9. A method for identifying tobacco having high stalk strength obtained by the method of claim 7, characterized in that: The following steps are involved: (1) Determining whether the tobacco contains a peony root with a nucleotide sequence as shown in SEQ ID NO.1 PlLAC15 Gene; or (2) Determining whether the tobacco expresses the peony root with the amino acid sequence shown in SEQ ID NO.2 PlLAC15 Protein encoded by a gene.

10. A method for promoting the accumulation of S-lignin in tobacco stems, characterized in that: The following steps are involved: 1) Making tobacco contain the nucleotide sequence of peony shown in SEQ ID NO.1 PlLAC15 Gene; or 2) Making tobacco express the peony root with the amino acid sequence shown in SEQ ID NO.2 PlLAC15 Protein encoded by a gene.

Images