Paeonia lactiflora PlLHL1 gene and protein thereof, and application of PlLHL1 gene and protein thereof in regulation and control of plant stalk strength

Abstract

The invention discloses a Paeonia lactiflora PlLHL1 gene, a protein of the Paeonia lactiflora PlLHL1 gene and application of the Paeonia lactiflora PlLHL1 According to the invention, the constructed PlLHL1 gene overexpression vector is transformed into tobacco for expression, so that the lignin accumulation is reduced, the stalk strength is reduced, and a new tobacco germplasm with low stalk strength is created.

Description

Technical Field

[0001] The invention belongs to the field of peony biotechnology, and specifically relates to a peony PlLHL1 gene and a protein thereof and applications thereof in regulating plant stem strength. Background Art

[0002] Peony (Paeonia lactiflora Pall.) is a traditional famous flower in my country and is known as the "May Flower God". In the fresh cut flower market, peony is sought after by consumers for its full flower shape and bright color, becoming a new high-end cut flower with great development potential. Although my country is the main origin and planting country of peony, with rich variety resources, most of them cannot become excellent cut flower materials due to insufficient stem strength, resulting in bent stems and drooping flowers. This has led to 90% of peony cut flowers on the market coming from foreign varieties. Therefore, it is urgent to solve the problem of insufficient stem strength of peony in my country, so as to promote the healthy development of my country's peony fresh cut flower industry.

[0003] The strength of plant stems is closely related to the development of secondary walls of mechanical tissues. Earlier, we have found that lignin can provide mechanical support for peony stems (Zhao DQ, Luan YT, Xia X, et al. Lignin provides mechanical support to herbaceous peony (Paeonia lactiflora Pall.) stems. Horticulture Research, 2020, 7: 213).Subsequently, we identified multiple lignin biosynthesis genes that regulate peony stem strength through transgenic studies, including the hydroxycinnamoyl-CoA shikimate / quinate hydroxycinnamoyltransferase gene PlHCT1 (Zhao DQ, Xu C, Luan YT, et al. Silicon enhances stem strength by promoting lignin accumulation in herbaceous peony (Paeonia lactiflora Pall.). International Journal of Biological Macromolecules, 2021, 190: 769-779), caffeic acid-O-methyltransferase PlCOMT1 (Zhao DQ, Luan YT, Shi WB, et al. Melatonin enhances stem strength by increasing lignin content and secondary cell wall thickness in herbaceous peony. Journal of Experimental Botany, 2022, 73: 5974-5991), and laccase genes PlLAC4 and PlLAC15 (Tang YH, Lu LL, Sheng ZP, et al. An R2R3-MYB network modulatesstem strength by regulating lignin biosynthesis and secondary cell wallthickening in herbaceous peony.Plant Journal,2023,113:1237-1258;Yin YH,ZuoSQ,Zhao MH,et al.PlLAC15facilitates syringyl lignin deposition to enhancestem strength in herbaceous peony.Agriculture,2024,14:1609).On this basis, we further identified three R2R3-MYB transcription factor family members that improve peony stem strength by regulating lignin biosynthesis and secondary cell wall thickening in herbaceous peony. Plant Journal, 2023, 113: 1237-1258); peony WRKY transcription factor family member PlWRKY41 enhances stem strength by promoting secondary cell wall thickening (Tang YH, Lu LL, Huang XQ, et al. The herbaceous peony transcription factor WRKY41 apromotes secondary cellwall thickening to enhance stem strength. Plant Physiology, 2023, 191: 428-445); in addition, it was found that peony SPL transcription factor family member PlSPL2 negatively regulates stem development (Tang YH, Xu HJ, Yu RK, et al. The SBP-box transcription factor PlSPL2 negatively regulates stem development in herbaceous peony. Plant Cell Reports, 2024, 43: 275). However, there have been no reports on the role of other transcription factors in regulating peony stem strength.

[0004] The bHLH family is the second largest family of eukaryotic transcription factors after MYB and is widely present in plants. Its name comes from its highly conserved basic / helix-loop-helix special domain, which consists of two parts, one is the basic amino acid region and the other is the helix-loop-helix region (HLH). Its basic domain is located at the N-terminus of bHLH and contains about 15 amino acids and 6 basic residues. It is mainly involved in the binding of DNA to the E-box (CANNTG) or G-box (CACGTG) motif in the target gene. bHLH transcription factors are widely involved in plant growth and development, morphological construction and stress response (Zhu Lulu, Zhou Bo. Regulation of bHLH proteins in plant development and abiotic stress. Molecular Plant Breeding, 2022, 20(20): 6750-6760). Although it has been found that bHLH transcription factors may be involved in regulating lignin accumulation (Gao ZY, Sun WJ, Wang J, et al. GhbHLH18 negatively regulates fiber strength and length by enhancing lignin biosynthesis in cotton fibers. Plant Science, 2019, 286: 7-16; Onohata T, Gomi K. Overexpression of jasmonate-responsive OsbHLH034 in rice results in the induction of bacterial blight resistance via an increase inlignin biosynthesis. Plant Cell Reports, 2020, 39: 1175-1184), there has been no report on its function in regulating stem strength. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a new peony PlLHL1 gene.

[0006] The technical problem that the present invention needs to solve is to provide the protein PlLHL1 encoded by the peony PlLHL1 gene.

[0007] The technical problem that the present invention also aims to solve is to provide an expression cassette, a recombinant vector or a recombinant strain containing the peony PlLHL1 gene.

[0008] The technical problem that the present invention also aims to solve is to provide the application of the peony PlLHL1 gene, the protein PlLHL1, the expression cassette, the recombinant vector or the recombinant strain in regulating the strength of plant stems.

[0009] The final technical problem to be solved by the present invention is to provide a method for obtaining plants with low stalk strength.

[0010] Technical solution: In order to solve the above technical problems, the present invention provides a peony PlLHL1 gene, the full-length cDNA sequence of the peony PlLHL1 gene is shown in SEQ ID NO. 1. SEQ ID NO. 1 in the sequence table consists of 2328 bases.

[0011] The present invention also includes the protein PlLHL1 encoded by the peony PlLHL1 gene, and its amino acid sequence is shown in SEQ ID NO. 2. Wherein, SEQ ID NO. 2 in the sequence table consists of 678 amino acids.

[0012] The present invention also includes an expression cassette, a recombinant vector or a recombinant strain, which contains the peony PlLHL1 gene.

[0013] Wherein, the recombinant vector is a eukaryotic expression vector containing the peony PlLHL1 gene. Preferably, the eukaryotic expression vector includes the intermediate plant expression vector pCAMBIA1301.

[0014] The present invention can use the existing plant binary expression vector pCAMBIA1301 to construct a recombinant expression vector containing the PlLHL1 gene. The plant expression vector carrying the PlLHL1 gene of the present invention is transferred into Agrobacterium GV3101 cells and then transformed into plant tissues by leaf disc method. The transformed host plant is tobacco.

[0015] Furthermore, the primers designed for constructing the eukaryotic expression vector are SEQ ID NO.5 (5'-caggtcgactctagaggatccATGGGTCCTCACTTGCAGCA-3') and SEQ ID NO.6 (5'-ttcgagctcagatctggtaccCGCAGCCTTGGGTTGCAA-3').

[0016] Furthermore, transformed tobacco plants were screened by PCR and qRT-PCR verification.

[0017] Further, PCR detection was carried out, and the primers for the PlLHL1 gene were designed as follows: upstream primer: 5'-AAACTCAAACATCGTGCTC-3' (SEQ ID NO.7); downstream primer: 5'-ACCTGTCCAACAATCCCT-3' (SEQ ID NO.8);

[0018] qRT-PCR detection was carried out, and tobacco NtActin (AB158612) was used as the internal reference gene. 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); the primers designed for the PlLHL1 gene were: upstream primer: 5'-AAACTCAAACATCGTGCTC-3' (SEQ ID NO.7); downstream primer: 5'-ACCTGTCCAACAATCCCT-3' (SEQ ID NO.8).

[0019] Wherein, the recombinant strain is obtained by introducing the recombinant vector into the host strain.

[0020] The present invention also includes the application of the peony PlLHL1 gene, the protein PlLHL1, the expression cassette, the recombinant vector or the recombinant strain in regulating the strength of plant stems.

[0021] Wherein, the regulating plant stem strength includes reducing the plant stem strength or increasing the plant stem strength. Preferably, reducing the plant stem strength includes overexpressing the Peony PlLHL1 gene.

[0022] The present invention also includes a method for obtaining a plant with low stem strength, which comprises introducing the peony PlLHL1 gene into the plant.

[0023] The plants include but are not limited to tobacco.

[0024] The method comprises introducing the peony PlLHL1 gene into an overexpression vector, and then transferring the overexpression vector into tobacco.

[0025] The invention constructs an overexpression vector of the peony PlLHL1 gene, transfers the pCAMBIA1301-PlLHL1 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 PlLHL1 gene is significantly reduced compared with that of the wild-type plants, indicating that the overexpression of the peony PlLHL1 gene has the function of changing the stalk strength of the plants.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the present invention reduces lignin accumulation and stalk strength by transforming the constructed PlLHL1 gene overexpression vector into tobacco for expression, thereby creating a new tobacco germplasm with low stalk strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The nucleotide sequence of the PlLHL1 gene of Paeonia lactiflora and its deduced amino acid sequence are shown in the figure. The deduced amino acid sequence is marked below the corresponding nucleotide sequence, and the rest is a non-coding region; * indicates a stop codon.

[0028] Figure 2 The amino acid sequence of the PlLHL1 gene of peony and the homologous phylogenetic tree and sequence alignment of the bHLH family of Arabidopsis thaliana: A: homologous phylogenetic tree of PlLHL1 and the bHLH family of Arabidopsis thaliana; B: sequence alignment of PlLHL1 with AtLHL1 and AtLHL2.

[0029] Figure 3 This is the subcellular localization map of peony PlLHL1.

[0030] Figure 4 This is the identification diagram of transgenic tobacco plants with the PlLHL1 gene based on PCR detection: 1 and 7: DL2000 DNA Marker; 2 and 6: wild-type plants; 3-5: transgenic peony PlLHL1 gene strains.

[0031] Figure 5 This is the identification diagram of transgenic PlLHL1 gene tobacco plants based on qRT-PCR detection: where ** indicates P<0.01.

[0032] Figure 6 The figure is a phenotypic comparison of wild-type and transgenic PlLHL1 gene tobacco plants.

[0033] Figure 7 Comparison of stem diameters between wild-type and transgenic PlLHL1 tobacco plants: * indicates P<0.01, ** indicates P<0.01.

[0034] Figure 8 Comparison of stem strength between wild-type and transgenic PlLHL1 tobacco plants: ** indicates P < 0.01.

[0035] Fig. 9 Comparison of lignin content in the stems of wild-type and transgenic PlLHL1 tobacco plants: ** indicates P < 0.01. DETAILED DESCRIPTION

[0036] The technical scheme of the present invention is further described in detail below through specific examples, but it is necessary to point out that the following examples are only used to describe the content of the invention and do not constitute a limitation on the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are all carried out according to conventional steps, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0037] Example 1 Cloning of the full-length cDNA sequence of the peony PlLHL1 gene

[0038] Obtaining the full-length sequence of the PlLHL1 gene: Peony leaves were selected as materials, and the MiniBEST Plant RNA Extraction Kit (TaKaRa) was used to extract total RNA. II 1st Strand cDNA Synthesis Kit (Vazyme) was used to reverse transcribe cDNA. The first step was to use a reverse transcription system of 1 μL RNA, 1 μL Oligo(dT) 23 VN (50μM), 6μL RNase Free ddH2O; reverse transcription procedure: 65℃ reaction for 5min, quickly put on ice to cool, and stand on ice for 2min. Second step reaction: add 10μL 2×RT Mix and 2μL HiScript II Enzyme Mix to the mixture in the previous step; reverse transcription procedure: 50℃ reaction for 45min, 85℃ reaction for 2min. On this basis, PCR amplification was performed. The PCR amplification system is: 2μL cDNA, 10μL 2×Phanta Max Master Mix (Vazyme), 1μL Forward Primer (5'-TGTGGATTGATTGAGTTAGG-3' (SEQ ID NO.3)), 1μL Reverse Primer (5'-CTCACAAATCACTCGCAATA-3' (SEQ ID NO.4)), 6μL RNase Free ddH2O. The reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 48℃ annealing for 15 s, 72℃ extension for 60 s, for a total of 35 cycles; 72℃ extension for 10 min. The products were detected by 1% agarose gel electrophoresis and then separated and sequenced. Figure 1 As shown, the full length of the PlLHL1 gene cDNA is 2328 bp, the open reading frame is 2037 bp, and it encodes a total of 678 amino acids.

[0039] The full-length cDNA sequence of the PlLHL1 gene of Paeonia lactiflora (SEQ ID NO.1)

[0040]

[0041] The amino acid sequence inferred from the cDNA sequence of the peony PlLHL1 gene (SEQ ID NO.2)

[0042] MGPHLQQILRSLCLNTEWKYAVFWKLKHRARMVLTLEDAYYDNHEIHERKRSSKTLVNLCDGQYLHDPLGLAVAKMSYHVYSLGEGIVGQVAITGKHQWIFADKPVTKSCSSFEYCDGWQTQFSAGIRTIVVVAVVPHGVVQLGSLHKVVEDIKLVNRIREVFFALQNS LVGYIDSPIQCNLKSNLKPPDISARSSCSEVVQDYLHHLDETVNRGSSDACSPVFPCLDKYSNGTYAIPLPGIHQKELVEVVNKDERGVESSMPRSKLLEPNSEIFNFEYQKEKQRKLFDERKCEEESSFWRDVGMVSDHNVTLHAEHNLVFPYLPPDLIQSWDGVKFDH QQDSSLHIEPPSDMQLEKDSDMKLDFQFSAGCELHEALGPAFLKQSHINYEEKTEVGTSIELPEGMTCSQLTTDSVSENLLEAVVAKVCHSSSDTKSEKSMCKSLLITENLPEPSSHTKDTICSASGQPSLIDENSPEISPGGISSTCLSTYSEQLERSGKGNKKRARP GENTRPRPRDRQLIQDRIKELRELIPSGSKCSIDSLLERTIKHMLFLQSTTKHADKLKKCAESKAKEKGYDQGSSWAVEVGSHLKVCTIIVENLNMNGQMLVEMLCEECSHFLEIAEAIRSLGLMILKGVTEARGDKTWICFVVEGQNNRSMHRMDILWSLVQILQPKAA

[0043] Example 2: Alignment of amino acid sequence of peony PlLHL1 gene with homologous phylogenetic tree and sequence of Arabidopsis thaliana bHLH family

[0044] 171 Arabidopsis bHLH family amino acid sequences were downloaded from the database of The Arabidopsis Information Resource (TAIR) (https: / / www.arabidopsis.org / ), and they and the amino acid sequence of the PlLHL1 gene were expressed in FASTA format. Then, the evolutionary tree was constructed using MEGA7.0 software, with the NeighborJoining algorithm and 1000 self-introductions. Their most homologous amino acid sequences were observed, and the results showed that it was clustered with Arabidopsis AtLHL1 ( Figure 2 A).

[0045] The amino acid sequences of peony PlLHL1, Arabidopsis thaliana AtLHL1 and AtLHL2 were further compared using DNAMAN 9.0 software, which showed that PlLHL1 had a highly conserved bHLH domain ( Figure 2 B).

[0046] Example 3 Subcellular localization of Peony PlLHL1

[0047] Gene-specific primers containing Sal I restriction sites were designed (PlLHL1-F1: 5'-cggggatcctctagagtcgacATG GGTCCTCACTTGCAGCA-3' (SEQ ID NO.5); PlLHL1-R1: 5'-caccatggtactagtgtcgacCGCAGCCTTGGGTTGCAA-3' (SEQ ID NO.6)). The plasmid returned by Nanjing Qingke Biotechnology Co., Ltd. that was successfully cloned and sequenced was used as a template for PCR amplification. The PCR amplification system was: 10μL 2×PhantaMax Master Mix (Vazyme), 2μL plasmid, 2μL primer, 6μL RNase Free H2O. The reaction procedure was: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 50℃ annealing for 15s, 72℃ extension for 60s, 35 cycles; 72℃ extension for 5min. After detection by 1% agarose gel electrophoresis, the amplified product was recovered by gel. The pCAMBIA2300 vector was further linearized by single enzyme digestion using Sal I. The enzyme digestion system was as follows: 0.4 μL Sal I (20000 U / mL), 2 μL Cutsmart Buffer, 1 μL pCAMBIA2300, 16.6 μL RNase Free H2O. The digestion was performed in a 37°C water bath for 1 h. The digestion product was detected by 1% agarose gel electrophoresis and recovered from the gel. Plus recombinase was used to construct the vector pCAMBIA2300-PlLHL1 from the two gel recovery products and then transformed into Agrobacterium. Next, the positive monoclonal clones containing the recombinant vector plasmid pCAMBIA2300-PlLHL1 and the empty vector plasmid pCAMBIA2300 on the YEB solid medium (5 g / L beef extract (Solarbio, Product No. B8530) + 1 g / L yeast extract (Oxoid, Product No. LP0021B) + 5 g / L peptone (Solarbio, Product No. P8450) + 5 g / L sucrose (Sangon, Product No. A502792-0500) + 0.5 g / L magnesium sulfate heptahydrate (Shanghai Test, Product No. 10013018) + 1.5% agar powder (Solarbio Cat#A8190)) were picked and transferred to YEB liquid medium (50 mg / L Kana (kalamycin) and 20 mg / L Rif (rifampicin), and the other components were the same as the YEB solid medium except that agar powder was not added), and cultured overnight at 28°C and 200 rpm until the OD 600 = 0.7, collect the cells after centrifugation, resuspend the cells in YEB liquid medium without antibiotics to wash out the antibiotics, and add an equal volume of resuspension solution to suspend Agrobacterium to OD 600 =0.7, and let stand at room temperature for 1 hour to rejuvenate the bacteria, and then inject them into 35-40 day old tobacco leaves through the lower epidermis, and culture them in weak light for 36-48 hours. The fluorescence signal in the epidermal cells of the leaf dorsal surface was observed using an ultra-high resolution laser confocal microscope (TCSSP8 STED, Leica, Germany) to determine the subcellular localization of the PlLHL1 protein. The excitation light for green fluorescent protein (GFP) was 488nm, and the excitation light for nuclear co-localization protein (NLS-mCherry) was 561nm. Figure 3 It can be seen that the green fluorescent protein fused to P1LHL1 and the red fluorescent protein expressed in the nucleus are localized in the same position in tobacco leaves, indicating that P1LHL1 is localized in the cell nucleus.

[0048] Example 4 Expression of Peony PlLHL1 Gene Overexpression Vector in Tobacco

[0049] Construction of overexpression vector of peony PlLHL1 gene: A pair of primers were designed to amplify the PlLHL1 sequence (PlLHL1-F1: 5'-caggtcgactctagaggatccATGGGTCCTCACTTGCAGCA-3' (SEQ ID NO.5); PlLHL1-R1: 5'-ttcgagctcagatctggtaccCGCAGCCTTGGGTTGCAA-3' (SEQ ID NO.6)). PCR amplification system: 12.5μL 2×Phanta Flash Master 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 10s, 72℃ extension for 130s, a total of 35 cycles; 72℃ extension for 1min. After the reaction, the PCR reaction solution was analyzed by agarose gel electrophoresis, and the TSP602-DNA gel recovery kit (Tsingke) was used to recover the large fragment of PlLHL1 containing the restriction site (SEQ ID NO.11). The binary expression vector pCAMBIA1301 plasmid was double-digested with BamH I and Kpn I. The reaction system was: 2.0 μL 10×CutSmart Buffer, 4 μL pCAMBIA1301 plasmid, 0.4 μL BamH I (20000U / mL), 0.4 μL Kpn I (20000U / mL), 13.2 μL ddH2O; react at 37°C for 1 hour. The double-digested product was analyzed by agarose gel electrophoresis, and the TSP602-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 (Cat. No. NR005-018), 3.0 μL pCAMBIA1301 large fragment, 1.0 μL PlLHL1 large fragment; connect in a 50°C metal bath for 15 min and cool on ice, take 5 μL of the ligation product to transform Trelief TM5α 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-PlLHL1, and then double enzyme digestion and sequencing verification were performed until the pCAMBIA1301-PlLHL1 overexpression vector was successfully constructed.

[0050] Large fragment of PlLHL1 from Paeonia lactiflora containing restriction site (SEQ ID NO.11)

[0051]

[0052] Transformation of tobacco with peony PlLHL1 gene overexpression vector: Take 5 μL of pCAMBIA1301-PlLHL1 overexpression vector plasmid to transform GV3101 (pSoup-p19) competent cells (TOLOBIO), and then culture on YEB solid culture medium plates (containing Rif 50 mg / L and Kan 50 mg / L) at 28°C for 2 days, pick positive single clones in YEB liquid culture medium (containing Rif 50 mg / L and Kan 50 mg / L), and culture overnight at 28°C and 200 rpm. Take 2 mL of the shaken bacterial solution and add it to 50 mL of liquid YEB containing the same antibiotics (containing Rif 50 mg / L and Kan 50 mg / L), and culture 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 the sterilized small triangular flask, add 5mL MS0 (MS0 liquid basic culture medium, without agar and sucrose (Haibo Biological, Product No. HB8469-5)) 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 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 100-150 leaves in total 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+3.0mg / L 6-BA+0.1mg / L 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+3.0mg / L 6-BA+0.1mg / L NAA+30g / L sucrose+6.66% agar+100mg / L Cb+25mg / L Hyg], 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 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 PlLHL1 gene can be obtained.

[0053] Example 5 Identification of tobacco plants transformed with the peony P1LHL1 gene

[0054] PCR identification: NuClean Plant Genomic DNA Kit (CWBIO) was used to extract tobacco leaf DNA. On this basis, specific primers of the PlLHL1 gene (upstream primer: 5'-AAACTCAAACATCGTGCTC-3' (SEQ ID NO.7), downstream primer: 5'-ACCTGTCCAACAATCCCT-3' (SEQ ID NO.8)) were designed for PCR amplification. Reaction system: 12.5μL2×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 subjected to gel electrophoresis. Figure 4 It can be seen that a single bright band was detected in the transgenic tobacco with the peony PlLHL1 gene, but not in the wild-type tobacco.

[0055] qRT-PCR identification: Total RNA was extracted using the MiniBEST Plant RNAExtraction Kit (TaKaRa), and the HiScript III RT SuperMix for qPCR (+gDNAwiper) (Vazyme) kit was used to reverse transcribe the total RNA into cDNA. The reaction system was: 1.0 μL RNA, 4.0 μL 4×gDNAwiper Mix, 11.0 μL RNase FreedH2O; the reaction conditions were: 42°C for 2 min. After the reaction was completed, 4.0 μL 5×HiScript III qRT SuperMix was added to the reaction solution of the first step; 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 the specific primers of the PlLHL1 gene (upstream primer: 5'-AAACTCAAACATCGTGCTC-3' (SEQ ID NO.7), downstream primer: 5'-ACCTGTCCAACAATCCCT-3' (SEQ ID NO.8)) were used for qRT-PCR detection. Reaction system: 2μL cDNA, 12.5μL 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 PlLHL1 had a significantly higher expression level in transgenic tobacco ( Figure 5 ).

[0056] Example 6 Phenotypic observation of tobacco plants transformed with the peony P1LHL1 gene

[0057] When the tobacco plants were cultured to flowering, the phenotypes of wild-type and transgenic peony PlLHL1 tobacco plants were compared. It was found that the upper stems of transgenic peony PlLHL1 tobacco plants were significantly bent compared with the wild-type ( Figure 6 ).

[0058] Example 7 Determination of morphological indexes of tobacco plants transformed with the peony P1LHL1 gene

[0059] The diameter and strength of the upper stems of wild-type and transgenic peony PlLHL1 tobacco plants were measured using a vernier caliper and a stem strength meter (NK-2). The results showed that the diameter of the upper stems of transgenic peony PlLHL1 tobacco plants was relatively lower than that of the wild-type ( Figure 7 ), the stem strength decreased significantly ( Figure 8 ), indicating that overexpression of the peony PlLHL1 gene could significantly reduce the stem strength of tobacco plants.

[0060] Example 8 Determination of lignin content in the stems of tobacco plants transformed with the peony PlLHL1 gene

[0061] The lignin content of tobacco plant stems was determined using a lignin content detection kit (Beijing Solebow Technology Co., Ltd., catalog number BC4200). The specific steps are as follows: the sample was dried at 80°C to constant weight, crushed, and passed through a 40-mesh sieve, then 5 mg was weighed into a 1.5 mL centrifuge tube; the spectrophotometer was preheated for 30 min, zeroed with glacial acetic acid, and the wavelength was set to 280 nm; 500 μL of reagent one was added to the sample tube, reacted in a 65°C water bath for 30 min, centrifuged at room temperature and 8000 g for 5 min, the supernatant was removed, and the sediment was retained at the bottom; then 500 μL of reagent two was added, vortexed for 5 min, centrifuged at room temperature and 8000 g for 5 min, the supernatant was removed, and the sediment was retained at the bottom; then 500 μL of reagent three was added, vortexed for 5 min, centrifuged at room temperature and 8000 g for 5 min, the supernatant was removed, and the sediment was retained at the bottom; then 500 μL of reagent four and 20 μL of reagent five were added in sequence. Add 500μL of reagent 4 and 20μL of reagent 5 to the blank tube in sequence. Mix both thoroughly, place in an 80℃ water bath for 40min (wrap with sealing film) for acetylation, shake for 30S every 10min, and then cool naturally to room temperature. Add 500μL of reagent 6 respectively, mix thoroughly, centrifuge at room temperature and 8000g for 10min, and take the supernatant for testing. Pipette 20μL of supernatant and mix with 980μL of glacial acetic acid, pipette the reaction solution into a 1mL quartz cuvette, and measure the absorbance A at 280nm; calculation formula: ΔA=A measurement tube-A blank tube, lignin content (mg / g)=2.184×ΔA÷W. From Fig. 9 It can be seen that compared with the wild-type plants, the lignin content of the transgenic peony PlLHL1 gene tobacco plants was significantly lower, indicating that overexpression of the peony PlLHL1 gene can significantly reduce the accumulation of lignin in the stems of tobacco plants.

[0062] In summary, the present invention provides a full-length cDNA sequence of the peony PlLHL1 gene and its protein and its application in regulating the plant stem strength. By transforming the constructed PlLHL1 gene overexpression vector into tobacco for expression, the accumulation of stem lignin is reduced, the stem strength is reduced, and a new tobacco germplasm with low stem strength is created.

Claims

1. A peony PlLHL1 A gene characterized by The peony PlLHL1 The full-length cDNA sequence of the gene is shown in SEQ ID NO.

1.

2. The herbaceous peony according to claim 1 PlLHL1 The protein PlLHL1 encoded by the gene is characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. An expression cassette, a recombinant vector or a recombinant strain, characterized in that: It contains the peony root described in claim 1 PlLHL1 Gene.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector contains the peony PlLHL1 The eukaryotic expression vector of the gene, preferably, the eukaryotic expression vector includes the intermediate plant expression vector pCAMBIA1301.

5. The recombinant strain according to claim 3, characterized in that The recombinant strain is obtained by introducing the recombinant vector into the host strain.

6. The herbaceous peony of claim 1 PlLHL1 Use of the gene, the protein PlLHL1 described in claim 2, the expression cassette, the recombinant vector or the recombinant strain described in claim 3 in regulating the strength of plant stems.

7. The use according to claim 6, characterized in that: The regulating plant stem strength comprises reducing the plant stem strength or increasing the plant stem strength. Preferably, reducing the plant stem strength comprises overexpressing the peony described in claim 1 PlLHL1 Gene.

8. A method for obtaining plants with low stalk strength, characterized in that The method comprises the steps of: PlLHL1 The gene is introduced into the plant.

9. The method for obtaining a plant with low stalk strength according to claim 8, characterized in that: The plants include tobacco.

10. The method for obtaining a plant with low stalk strength according to claim 8, characterized in that: The method comprises the steps of: PlLHL1 The gene is introduced into an overexpression vector, and then the overexpression vector is transferred into tobacco.

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