PsLTP gene, vector, recombinant bacteria and application for delaying peony flower senescence

Abstract

The present invention relates to the field of genes, and specifically to a PsLTP gene, vector, recombinant bacteria and application for delaying peony flower senescence. In order to solve the technical problem of delaying petal senescence and extending the flowering period, the present invention provides a PsLTP1 gene for delaying peony flower senescence and extending the flowering period of peony flowers, and its nucleotide sequence is shown in SEQ ID NO: 1. The present invention verifies that overexpression of PsLTP1 in peony petal discs delays petal senescence, while petal senescence is accelerated after VIGS silencing, and the content of hydrogen peroxide and superoxide anion increases, indicating that PsLTP1 is involved in the senescence process of peony petals. The present invention also provides a vector, a recombinant bacteria and application thereof containing the PsLTP1 gene.

Description

Technical Field

[0001] The present invention relates to the field of genes, and in particular to a method for delaying the senescence of peony flowers. PsLTP Genes, vectors, recombinant bacteria and their applications. Background Art

[0002] peony( Paeonia section Moutan DC.) is native to China and has high ornamental, medicinal and oil value (Wang et al., 2019). There are more than 2,000 cultivars of peony, which are known for their large flowers, diverse colors, rich fragrance and symbol of wealth and prosperity. They are known as the "king of flowers". Peonies are often used in courtyards, potted plants and special peony gardens, attracting tourists from all over the world and have great ornamental and economic value. However, the natural flowering period of peony is short, and the life span of a single flower is only 3 to 5 days, which seriously restricts its direct and indirect economic benefits.

[0003] However, peony is a perennial woody plant, and the efficiency of conventional hybrid breeding is very low; the genetic basis and molecular regulatory mechanism of peony flowering are lacking, and it is difficult to solve the problem of short flowering time of peony through breeding and cultivation technology (Guo et al., 2022; Zhang et al., 2019a). Therefore, delaying petal senescence and extending the flowering period have become the key to breaking through the restrictions on the economic benefits of peony; exploring the genes related to petal senescence and revealing the regulatory mechanism of petal senescence are of great guiding significance for accelerating the selection and breeding of new peony varieties with long flowering periods, and have become one of the focuses of the ornamental horticulture industry.

[0004] Nonspecific lipid transfer proteins (nsLTPs) are a family of small basic proteins that are widely distributed in plants. They are able to bind and transfer various lipids between cell membranes or between plants and the environment (Missaoui et al., 2022; Salminen et al., 2016). nsLTP proteins contain eight conserved cysteine ​​motifs (8CM), and their general form is C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C (Yang et al., 2022; Zhang et al., 2019b). Edstam et al. (2011) divided nsLTPs into LTP1 (about 9KD), LTP2 (about 7KD) and ck types based on intron position, GPI anchoring, eight conserved cysteine ​​motifs and sequence similarity. LTP1-type lipid transporter genes in Arabidopsis are all predicted to encode pathogenesis-related proteins; f, h, g, and k exist in non-flowering plants, d and g types are mainly found in early land plants, while some other types are limited to a single species, such as h type, which is only found in Selaginella chinensis (Edstam et al., 2011).

[0005] nsLTPs are involved in a variety of biological processes, including lipid transport, lipid metabolism, cuticle formation, cell wall extension, signal transduction, plant stress response, and plant growth and development (Ahmed et al., 2017; Chae et al., 2010; Jung et al., 2009; Liu et al., 2015; Zhang et al., 2010; Zhou et al., 2019). nsLTP Overexpression in Arabidopsis and rice can significantly increase seed oil content (Song et al., 2021; Wang et al., 2015). nsLTP Overexpression of these genes can improve the resistance of Arabidopsis, tobacco, potato and rice to pathogen infection (Jung et al., 2005; Patkar and Chattoo, 2006; Wang et al., 2021). nsLTP Overexpression of can also improve salt tolerance and cold tolerance in tomato and rice (Gangadhar et al., 2016; Zhao et al., 2020); some nsLTP It has been shown to be involved in the regulation of reactive oxygen species (ROS) scavenging, which is essential for plant survival under stress conditions (Wang et al., 2014). At3g51600 The gain-of-function mutant ltp-1 (LTP1 type) showed defects in sexual reproduction, as well as dwarfing of primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and multiple branches, indicating that LTP Involved in a variety of plant growth and development processes (Chae et al., 2010). However, another LTP1 type in Arabidopsis nsLTP ( At5g59320 ) can increase the content of abscisic acid (ABA), thereby making the pathogens susceptible (Gao et al., 2016).

[0006] In previous studies, the inventors determined the gene expression profile during peony petal aging and found that PsLTP1 The gene is involved in the senescence process of peony petals and has application prospects in delaying peony flower senescence and prolonging flowering period, and the present invention is particularly proposed. Summary of the invention

[0007] In order to solve the technical problem of delaying petal senescence and extending the flowering period, the present invention provides a method for delaying peony flower senescence and extending the flowering period of peony flowers. PsLTP1The gene has a nucleotide sequence as shown in SEQ ID NO: 1.

[0008] ATGGCTGGTTCAGGATTCCTCAAGCTCGCTTGTGTGGCATTTGCCTTGATGATGGTGGCTGCACCTTGTGCAGAAGCCATAACATGTGGTCAGGTGACGAGCAGCCTGACTCCATGCATTGGTTACCTGAGAAGCGGTGGAGCAGTCCCACCGGCCTGCTGCAGTGGCGTCAAGTCCCT CAACGGTGCTGCTAAGACCACACCGGACCGCCAGACTGCCTGCAACTGCCTTAAATCAGCAGCTGGTAGCATCTCTGGCATCAATTTGAGTTTTGCTAGTGGCCTTCCTGGCAAGTGTGGCGTCAACATTCCTTACAAGATTAGCCCCTCCACTGACTGCAGCACATTAAAATAA (SEQ ID NO: 1)

[0009] Furthermore, the PsLTP1 The amino acid sequence of the PsLTP1 protein encoded by the gene is shown in SEQ ID NO:2.

[0010] MAGSGFLKLACVAFALMMVAAPCAEAITCGQVTSSLTPCIGYLRSGGAVPPACCSGVKSLNGAAKTTPDRQTACNCLKSAAGSISGINLSFASGLPGKCGVNIPYKISPSTDCSTLK (SEQ ID NO: 2).

[0011] The present invention also provides a PsLTP1 protein, whose amino acid sequence is shown in SEQ ID NO:2.

[0012] The present invention also provides a vector, which is a plasmid vector comprising the PsLTP1 Gene.

[0013] Furthermore, the vector is a pSuper 1300-GFP vector or a pBI121 vector and PsLTP1 Plasmids obtained by gene recombination.

[0014] The present invention also provides a recombinant bacterium comprising the vector.

[0015] Furthermore, the recombinant bacteria is Escherichia coli or Agrobacterium, preferably Escherichia coli (E. coli) strain DH5ɑ, Agrobacterium strain GV3101, or Agrobacterium strain EHA105.

[0016] The present invention also provides the PsLTP1 Application of the gene, PsLTP1 protein, the vector or the recombinant bacteria in delaying peony petal senescence and / or extending the flowering period of peony flowers.

[0017] Furthermore, the application may include the following methods:

[0018] ① Improve the quality of peony PsLTP1 Gene expression, such as gene modification or introduction into the vector;

[0019] ② Use PsLTP1 The gene, the vector or the recombinant bacteria express the PsLTP1 protein, and then the solution containing the PsLTP1 protein is used to infect the peony flowers;

[0020] ③ Cultivate and amplify the recombinant bacteria, and use the suspension containing the recombinant bacteria to infect peony flowers.

[0021] The present invention also provides a method for delaying peony flower senescence, comprising:

[0022] (1) Design primer pairs PsLTP1 The open reading frame of pBI21- PsLTP1 Overexpression vector and transform Agrobacterium EHA105;

[0023] (2) Resuspend the bacteria in infection solution until the OD value reaches 1.0-1.2, and immerse the peony buds in the infection solution for infection.

[0024] Furthermore, the infection solution contains: 2.132 g / L MES, 0.9522 g / L MgCL2, and 0.5 mol / L AS.

[0025] Compared with the prior art, the beneficial effects of the present invention include at least:

[0026] 1. The present invention is the first to discover PsLTP1 gene, and proved PsLTP1 Genes involved in the aging process of peony petals.

[0027] 2. The present invention successfully amplified PsLTP1 Gene and explored its regulatory mechanism in peony flowers.

[0028] 3. The present invention constructs PsLTP1 The gene vector and recombinant bacteria have verified its application prospects in delaying the aging of peony flowers and prolonging the flowering period. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 for PsLTP1 Phylogenetic analysis of genes.

[0030] Figure 2 The amino acid sequence alignment of PsLTP1 protein and AtLTP1.12 protein.

[0031] Figure 3 for PsLTP1 The spatiotemporal expression patterns of genes in 'Xue Ying Tao Hua'; Figure 3 A in PsLTP1 Tissue expression patterns; Figure 3 B is PsLTP1 Expression patterns during petal development.

[0032] Figure 4 The subcellular localization results of GFP-PsLTP1, where NLS-BFP represents the nuclear localization signal; GFP represents the excitation fluorescence; Bright represents the result observed under bright field; Merge represents the composite result of overlapping NLS-BFP, GFP and bright field.

[0033] Figure 5 For pBI121- PsLTP1 Vector construction results, where M represents DL 2000 Marker; P represents positive; N represents negative; Figure 5 A in pBI121- PsLTP1 ORF amplification results; Figure 5 B is pBI121- PsLTP1 E. coli bacterial solution PCR results; Figure 5 C in pBI121- PsLTP1 PCR results of Agrobacterium culture.

[0034] Figure 6 The results of functional identification of overexpressed PsLTP1 in peony petal senescence; Figure 6 A in PsLTP1 Phenotypic observation of overexpression of 'Xue Ying Tao Hua'; Figure 6 Middle B is qRT-PCR analysis PsLTP1 Expression levels in overexpressing petals; Figure 6 C is PsLTP1 Detection of hydrogen peroxide (DAB) and superoxide anion (NBT) in petals of overexpressed 'Xueyingtaohua'; Figure 6 D in the figure is qRT-PCR detection of senescence-related genes in overexpressed petals PsSAG113 expression level.

[0035] Figure 7 For TRV2- PsLTP1 Vector construction results; M represents DL 2000 Marker; P represents positive; N represents negative; Figure 7 A in the middle is TRV2- PsLTP1 ORF amplification results; Figure 7 B in the middle is TRV2- PsLTP1 E. coli bacterial solution PCR results; Figure 7 C in the middle is TRV2- PsLTP1 PCR results of Agrobacterium culture.

[0036] Figure 8 The results of functional identification of silencing PsLTP1 in peony petal senescence; Figure 8 A in PsLTP1 Observation of the phenotype of silent 'Xue Ying Tao Hua'; Figure 8 Middle B is qRT-PCR analysis PsLTP1 expression levels in silent petals; Figure 8 C is PsLTP1 Silence detection of hydrogen peroxide (DAB) and superoxide anion (NBT) in the petals of 'Xue Ying Tao Hua'; Figure 8 D in the figure is qRT-PCR detection of senescence-related genes in silenced petals PsSAG113 expression level. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0038] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0039] The materials, instruments, etc. involved in the embodiments of the present invention are as follows:

[0040] Material Handling:

[0041] In mid-to-late October, four-year-old peony 'Xue Ying Tao Hua' was moved into pots by individual plants. After growing in a 20℃ greenhouse for 2 weeks, it was transferred to a 2~4℃ cold storage for 28 days, and then moved to a 20℃ greenhouse (13 h light / 11 h dark). When the flower buds were soft and the sepals were loosely turned outward, they were cut off together with the pedicels and immediately placed in water. The petals were taken at 0, 24, 48, 60 and 72 hours, respectively, and quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use.

[0042] Commonly used vectors and strains:

[0043] Escherichia coli ( E. coli ) strain DH5ɑ, Agrobacterium strain GV3101, Agrobacterium strain EHA105, pBI121 vector, and pSuper 1300-GFP vector.

[0044] Main enzymes and reagents:

[0045] QuickCut restriction endonuclease, 5×ABscript Ⅲ RT Mix (purchased from ABdonal), 2×FlashMaster Mix (purchased from Vazyme), 2×Rapid Taq Master Mix (purchased from Vazyme), SteadyPure Plant RNA Extraction Kit (Accurate Biology, AG21019, China) (purchased from Accurate), EVO M-MLV Mix Kit with gDNA Clean for qPCR (AccurateBiotechnology (Hunan) Co., Ltd., China) (purchased from Accurate), qPCRSYBR GreenPremix Pro Taq HS qPCR Kit Ⅱ (Accurate Biology, AG11701, China) (purchased from Accurate Biology).

[0046] LB Broth, LB Broth with Agar, Ampicillin, Kanamycin, Rifampicin, Acetosyringone, Dimethyl Sulfoxide, MES.

[0047] Preparation of antibiotics:

[0048] Kan (Kanamycin): Weigh 1 g of powder and add ddH2O to dissolve and dilute to 20 mL. Filter and dispense at a concentration of 50 mg / mL.

[0049] Amp (ampicillin): weigh 1 g of powder and add ddH2O to dissolve and dilute to 20 mL. Filter and dispense at a concentration of 50 mg / mL.

[0050] Rif (Rifampicin): Weigh 500 mg of powder and dissolve in DMSO to make up to 25 mL. Filter with an organic filter membrane and dispense at a concentration of 50 mg / mL.

[0051] Preparation of culture medium:

[0052] LB liquid medium: weigh 25 g LB broth powder and add 1 L ddH2O, sterilize at 121℃ for 20 min, add antibiotics as needed, and store at 4℃.

[0053] LB solid medium: weigh 40 g LB broth with Agar powder, add 1 L ddH2O, sterilize at 121℃ for 20 min, add corresponding antibiotics as needed, pour into plates, seal, and store at 4℃.

[0054] Infection solution: weigh 2.132 g MES, 0.9522 g MgCL2, and 0.5 M AS, add ddH2O to make up to 1 L, and use immediately.

[0055] Main instruments:

[0056] UV clean bench, UV spectrophotometer, high-speed refrigerated centrifuge (Heal Force), pipette (Eppendorf), fluorescence real-time quantitative PCR instrument, pH meter, Leica laser confocal microscope (Leica TCS-SP2), constant temperature and light incubator.

[0057] Embodiment 1: PsLTP1 Gene cloning and analysis

[0058] From the model plant Arabidopsis thaliana website TAIR AtLTP1.12 (AT5G59320.1), Arabidopsis thaliana AtLTP The sequence is a seed sequence, and the local 'Xueyingtaohua' flower development transcriptome database Blast query PsLTP Gene, get 'Snow Reflects Peach Blossoms' PsLTP Specific primers were designed, and cDNA samples of petals of 'Xue Ying Tao Hua' at different stages were used as templates. According to the instructions of EVO M-MLV Mix Kit with gDNA Clean for qPCR, a PsLTP The gene is 354 bp in length and its nucleotide sequence is shown in SEQ ID NO: 1. AtLTPs Multiple sequence alignment was performed and phylogenetic trees were constructed (e.g. Figure 1 ), PsLTP and AtLTP1.12 The amino acid sequences were translated by DNAMAN, and the sequence was shown in SEQ ID NO: 2. PsLTP and AtLTP1.12 Amino acid sequence data were compared (e.g. Figure 2 ), named PsLTP1 .

[0059] Embodiment 2: PsLTP1 Analysis of spatiotemporal expression of genes in 'Snow Reflecting Peach Blossoms'

[0060] Real-time fluorescence quantitative PCR was used to detect the expression level of PsLTP1 gene in roots, stems, leaves, sepals, bracts, stamens and petals.

[0061] Using actin gene as internal reference, real-time quantitative primers were designed online using NCBI (Table 1) and synthesized by Bioengineering (Shanghai).

[0062] Table 1: PsLTP1 Gene real-time quantitative primers

[0063]

[0064] According to qPCRSYBR Green Premix Pro Taq HS qPCR Kit Ⅱ, prepare 20 μL real-time quantitative fluorescence PCR reaction system, the reaction system is shown in Table 2, set three sample replicates and three technical replicates. The reaction was performed in AppliedBiosystems Quant Studio TM 5 Fluorescence quantitative PCR system operation, using 2 -ΔΔ The CT method was used to analyze the relative expression of the data.

[0065] Table 2: PCR amplification system

[0066]

[0067] Results PsLTP1 The gene was expressed at a higher level in the petals of 'Xue Ying Tao Hua' ( Figure 3 Then, the petals of 'Xue Ying Tao Hua' at different developmental stages were detected. PsLTP1 expression, found PsLTP1 The expression level gradually increased during S1-S5 ( Figure 3 Middle B).

[0068] Embodiment 3: PsLTP1 Subcellular localization of genes

[0069] 3.1 GFP- PsLTP1 Construction of fusion expression vector

[0070] 3.1.1 Primer design

[0071] Designed using CE Design V1.04 PsLTP1 Specific primers for gene recombination with pSuper1300 (GFP) vector, primer sequences are shown in Table 3.

[0072] Table 3: PsLTP1 GFP primer

[0073]

[0074] 3.1.2 Amplification and recovery of target genes

[0075] The cDNA of the petals of 'Xue Ying Tao Hua' at different stages was used as amplification template and amplified with 2× FlashMaster Mix. PsLTP1 The open reading frame of the gene was amplified according to the reagent instructions, and the amplified product was detected by gel electrophoresis. The detection band was consistent with the size of the target gene product.

[0076] Cut the above strips according to Fast Pure Ⓡ The product was purified according to the instructions of Gel DNA Extraction Mini Kit (Vazyme, Nanjing).

[0077] 3.1.3 Enzyme digestion and ligation

[0078] use XB I. Kpn pSuper-1300 (GFP) was double-digested at the I restriction site, and the mixture was placed in a PCR instrument at 37°C for 30 min and reacted at 50°C for 20 min.

[0079] The target gene and the above enzyme digestion solution were recombined using 5×ABscript Ⅲ RT Mix, and the reaction system was shown in Table 4. The reaction conditions were 50°C for 15 min.

[0080] Table 4: Recombination reaction system

[0081]

[0082] 3.1.4 Transformation into competent E. coli DH5ɑ

[0083] Take out the DH5ɑ competent medium from the -80℃ refrigerator and thaw it on ice. Transfer 10 μL of the recombinant plasmid to the DH5ɑ competent medium with a pipette. Ice bath for 30 min, heat shock in a 42℃ water bath for 45s, and ice bath for 2 minutes. Add 800 μL of LB liquid medium without antibiotics. Place the transformed bacterial solution in a 37℃ shaker and set the speed to 200 rpm for 60 min. Centrifuge the above bacterial solution at 5000 rpm for 30s, keep 50 μL of the supernatant, resuspend it, and spread it on LB solid medium containing Kan for overnight culture.

[0084] 3.1.5 Bacterial liquid PCR and plasmid extraction

[0085] Single clones were selected for culture, turbid strains were identified by PCR, and the strains corresponding to the positive bands were sent for sequencing and sequence comparison. PsLTP1 The strains corresponding to the sequences with consistent sequence alignment were expanded overnight and the recombinant plasmids were extracted.

[0086] 3.1.6 Transformation into competent Agrobacterium GV3101

[0087] Take out the GV3101 competent medium from the -80℃ refrigerator and place it on ice to thaw. Pipette 1 μg of the above plasmid and transfer it to the GV3101 competent medium. Ice bath for 5 min, liquid nitrogen for 5 min, 37℃ water bath for 5 min, ice bath for 5 min. Add 800 μL of LB liquid medium without antibiotics, put the transformed bacterial solution into a 28℃ shaker, set the speed to 200 rpm, and recover for 2-3 h. Centrifuge at 5 000 rpm for 30 s, keep 50 μL of supernatant, resuspend it, and spread it on LB solid medium containing Kan+Rif, and invert it for 48-72 h. Pick a single colony for bacterial solution PCR, detect the amplified product by gel electrophoresis, and select the bacterial solution with the same size as the target band for amplification and preservation.

[0088] 3.2 Preparation of Nicotiana benthamiana

[0089] In a sterilized clean bench, add 100 μL of sodium hypochlorite and 900 μL of Tween to the EP tube containing tobacco seeds. Place in a 37°C shaker at 200 rpm for 1 h. Add a mixed solution of sodium hypochlorite and Tween to the EP tube, shake by hand, and wash 6 times, 2 min each time. Add ddH2O to blow the seeds onto the filter paper, and after drying, sprinkle the seeds on the sterilized culture medium and seal. Cultivate at 4°C for 3 days, and light culture for about a week to obtain seedlings. Cover the seedlings with plastic wrap after transplanting, remove the film after one week, and culture for about 3 weeks before subsequent genetic transformation experiments.

[0090] 3.3 Transient expression in Nicotiana benthamiana

[0091] Agrobacterium containing GFP-PsLTP1 recombinant plasmid, protective bacteria P19 and strains containing nuclear localization signal NLS-BFP were propagated and shaken, and the OD value was measured to 0.6-0.8. After centrifugation at 4 000 rpm for 5 min, they were resuspended in infection solution and the OD value was measured to 0.6-0.8. The above three bacteria were mixed in a ratio of 1:1:1 and left to stand in the dark for 3 h. Three-week-old tobacco was genetically transformed and injected into the lower epidermis of tobacco with a sterile syringe. The treatment was dark for 1 d and light for 3 d.

[0092] 3.4 Laser confocal microscopy observation

[0093] Place a drop of water in the center of the slide, tear off the lower epidermis of the tobacco and spread it in the water, cover it with a cover glass from one side, and use filter paper to absorb excess water from one side to make a temporary slide. Observe the slide with a specific excitation wavelength.

[0094] 3.5 Experimental Results

[0095] NLS-BFP is a nuclear localization signal used to observe the location, shape and size of the cell nucleus. PsLTP1 The subcellular localization results of the fusion protein showed that, consistent with the control group, green fluorescence (GFP- PsLTP1 ) overlaps with the blue fluorescence (NLS-BFP), and the green fluorescence fills the positions in the cytoplasm and nucleus, indicating that PsLTP1 Located in the cytoplasm and nucleus (e.g. Figure 4 as shown).

[0096] Example 4: Overexpression of peony petals PsLTP1 Gene

[0097] 4.1 Construction of pBI121- PsLTP1 Overexpression vector

[0098] Design of pBI21- PsLTP1 Seamless cloning primers (Table 5) PsLTP1 The open reading frame of was amplified, and the vector construction and transformation steps in Example 3 subcellular localization were used to construct pBI21- PsLTP1 The overexpression vector was used to transform Agrobacterium EHA105.

[0099] Table 5: PsLTP1 Primers required for ligation to pBI121 vector

[0100]

[0101] Amplified PsLTP1 The sequence was tested by gel electrophoresis, and the electrophoresis results showed that the size of the target band was consistent with the expectation ( Figure 5 A in the figure). According to the seamless cloning method, the target band was connected to the vector and transformed into the DH5ɑ E. coli strain. The transformed DH5ɑ was then spread on a plate containing antibiotics and a monoclonal strain with good growth was selected. To avoid the emergence of strains with empty vector self-ligation, PCR identification was performed to select the target band with the same size as the vector. PsLTP1 The matching strains were sent to the company for sequencing ( Figure 5 B), the sequencing results were compared with the target sequence to confirm that the clone was correct. Then, the recombinant plasmid was extracted and transformed into the Agrobacterium EHA105 strain, the strain was spread on the culture medium containing Kan and Rif, and the single clone strain was selected for PCR identification to screen the positive strain ( Figure 5Middle C).

[0102] 4.2 Agrobacterium preparation

[0103] Take pBI21 and pBI21- PsLTP1 200 μL of Agrobacterium were inoculated into 50 mL of LB+Kan+Rif medium and cultured overnight for activation. The overnight culture solution was inoculated into LB+Kan+Rif liquid medium at a ratio of 1:210 and cultured at 28°C with a shaker at 210 rpm for 8-12 h until the OD value reached 1.0-1.2. The bacteria were collected by centrifugation at 4000 rpm for 8 min. The supernatant was discarded and the bacteria were resuspended in the infection solution until the OD value reached 1.0-1.2. The pBI21 resuspension was used as the control group, and the pBI21- PsLTP1 The resuspension was used as the experimental group and placed in a large beaker for later use.

[0104] 4.3 Infection of peony buds

[0105] Take peony buds according to the experimental material treatment method, and poke a few needle holes at the top of the buds in advance. Place the treated peony buds upside down in a beaker filled with infection solution. Soak the entire bud in the infection solution, place the beaker in a vacuum pump to evacuate, evacuate at 0.1 atmosphere for 20 minutes, slowly release the air, and repeat 3 times. Rinse with tap water twice and rinse with bacterial water once. Place in 8℃ dark conditions for 2-3 days and then transfer to normal culture (16 h day / 8 h night) for 2-3 days.

[0106] 4.4 PsLTP1 and aging-related genes PsSAG113 Expression detection

[0107] The peony buds that had been treated in darkness at 8°C for 3 days were taken out and cultured under normal light. The normal light time was recorded as day 0. On the second day, RNA was extracted from the peony petals and cDNA was obtained by reverse transcription. Then, qRT-PCR technology was used to detect the expression of 5-mercaptopropionate in peony petals. PsLTP1 Extraction of RNA from peony flowers: Total RNA extraction from peony flowers at different stages SteadyPure PlantRNA Extraction Kit instructions. cDNA synthesis: refer to EVO M-MLV Mix Kit with gDNAClean for qPCR kit instructions, according to the two-step method, remove genomic DNA and reverse transcription reaction.

[0108] Select PsLTP1 The flower buds with higher expression levels than the control were used as the overexpression successful flower buds for subsequent experiments. qRT-PCR technology was used to detect senescence-related genes in the control and overexpression successful peony flower buds PsSAG113The expression amount.

[0109] The results show that PsLTP1 The expression level of α was significantly increased in the overexpressed peony buds, indicating that the α was successfully overexpressed in peony. PsLTP1 Gene( Figure 6 (B) Overexpression PsLTP1 Phenotypic observation of peony buds revealed that overexpression PsLTP1 In the peony flowers of the treated group, the petals began to fall on the 4th day and were almost completely shed on the 8th day, while the petals of the peony flowers of the control group began to fall on the 3rd day and were almost completely shed on the 6th day ( Figure 6 Middle A), indicating overexpression PsLTP1 It can delay the falling of peony petals. PsSAG113 It is a maker gene related to aging. qRT-PCR detection of overexpression in peony buds PsSAG113 The gene expression level was found PsSAG113 The expression level of Figure 6 D), so it is inferred PsLTP1 It can delay the shedding and aging process of peony petals.

[0110] 4.5 DAB and NBT staining

[0111] To explore PsLTP1 To determine whether overexpression treatment would affect the accumulation of reactive oxygen species and superoxide anions, DAB and NBT staining was performed on the peony petals in the control and overexpression groups. Holes were punched at the same position of the peony petals in the control and overexpression groups to obtain petal discs, which were then placed in 50 mL centrifuge tubes. DAB and NBT dye solutions (Coolbo, Beijing) were added, respectively, and the vacuum pump was used to evacuate for 30 min, followed by slow release. The staining was performed overnight at room temperature, and the stained petals were decolorized with 95% ethanol in an 80°C water bath until clear the next day.

[0112] The results showed that the petals of the control group were stained darker, while the overexpression PsLTP1 The petals are lightly dyed, indicating PsLTP1 The accumulation of reactive oxygen species and superoxide anions was reduced after overexpression treatment ( Figure 6 Middle C).

[0113] Example 5: Silence in Peony Petals PsLTP1 Gene

[0114] 5.1 TRV2- PsLTP1 Vector construction

[0115] According to the SGN VIGS Tool, gene-specific sequences were retrieved and specific primers were designed (Table 6). PsLTP1The open reading frame of TRV2- PsLTP1 vector and transformed into Agrobacterium EHA105.

[0116] Table 6: PsLTP1 Primers required for ligation of TRV2 vector

[0117]

[0118] Amplification using specific primers PsLTP1 The sequence was sequenced and tested by gel electrophoresis. The electrophoresis results showed that the size of the target band was consistent with the expected value ( Figure 7 A in the figure). According to the seamless cloning method, the target band was connected to the vector and transformed into the DH5ɑ E. coli strain. The transformed DH5ɑ was then spread on a plate containing antibiotics and a monoclonal strain with good growth was selected. To avoid the emergence of strains with empty vector self-ligation, PCR identification was performed to select the target band with the same size as the vector. PsLTP1 The matching strains were sent to the company for sequencing ( Figure 7 B), the sequencing results were compared with the target sequence to confirm that the clone was correct. Then, the recombinant plasmid was extracted and transformed into the Agrobacterium EHA105 strain, the strain was spread on the culture medium containing Kan and Rif, and the single clone strain was selected for PCR identification to screen the positive strain ( Figure 7 Middle C).

[0119] 5.2 Agrobacterium preparation

[0120] Take TRV1, TRV2 and TRV2- PsLTP1 200 μL of Agrobacterium was inoculated into 50 mL of LB+Kan+Rif medium and cultured overnight for activation. Take the overnight cultured bacterial solution and inoculate it into LB+Kan+Rif liquid medium at a ratio of 1:210, and culture it in a shaking incubator at 210 rpm at 28°C for 8-12 h until the OD value reaches 1.0-1.2. Centrifuge at 4000 rpm for 8 min to collect the bacteria. Pour off the supernatant and resuspend the bacteria with the infection solution until the OD value reaches 1.0-1.2. Mix equal volumes of TRV1 and TRV2 infection solutions as the control group, and mix TRV1 and TRV2- PsLTP1 Mix equal volumes as the experimental group and place in a large beaker in the dark for 3-5 hours for later use.

[0121] 5.3 Infection of peony buds

[0122] The peony buds were infected according to the method of Example 4.

[0123] 5.4 PsLTP1 and aging-related genes PsSAG113 Expression detection

[0124] The peony buds that had been treated in darkness at 8°C for 3 days were taken out and cultured under normal light. The normal light time was recorded as day 0. On the second day, RNA was extracted from the peony petals and cDNA was obtained by reverse transcription. Then, qRT-PCR technology was used to detect the expression of 5-mercaptopropionate in peony petals. PsLTP1 The expression level of PsLTP1 The flower buds with expression levels lower than the control were used as the successful silencing flower buds for subsequent experiments. qRT-PCR technology was used to detect senescence-related genes in the control and successfully silenced peony flower buds PsSAG113 The amount of expression.

[0125] In the peony bud PsLTP1 After silencing treatment, qRT-PCR was used to detect PsLTP1 The results showed that PsLTP1 The expression level of α was significantly reduced in the silenced peony buds, indicating that α was successfully silenced in peony. PsLTP1 Gene( Figure 8 Middle B).

[0126] To silence PsLTP1 Phenotypic observation of peony buds revealed that PsLTP1 In the peony flowers of the treated group, the petals began to fall on the 3rd day and were almost completely detached on the 6th day, while the petals of the peony flowers of the control group began to fall on the 4th day and were almost completely detached on the 8th day ( Figure 8 Middle A), indicating silence PsLTP1 It can cause peony petals to fall prematurely. qRT-PCR detection of successfully silenced peony buds PsSAG113 The gene expression level was found PsSAG113 The expression level of Figure 8 D), further explanation PsLTP1 It can delay the shedding and aging process of peony petals.

[0127] 5.5 DAB and NBT staining

[0128] According to the method of Example 4, the peony petals of the control group and the silent group were stained and decolorized to clear.

[0129] The results showed that the petals of the control group were lightly stained, while the petals of the PsLTP1-silenced plants were darker stained, indicating that the accumulation of reactive oxygen species and superoxide anions increased after PsLTP1 silencing treatment ( Figure 8 Middle C).

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for delaying peony flower senescence and extending peony flower flowering period PsLTP1 A gene characterized by Said PsLTP1 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The method according to claim 1 PsLTP1 A gene characterized by Said PsLTP1 The amino acid sequence of the PsLTP1 protein encoded by the gene is shown in SEQ ID NO:

2.

3. A PsLTP1 protein for delaying peony flower senescence and extending peony flower flowering period, characterized in that: The amino acid sequence of the PsLTP1 protein is shown in SEQ ID NO:

2.

4. A carrier, characterized in that The vector is a plasmid vector comprising the PsLTP1 Gene.

5. The vector according to claim 4, characterized in that The vector is pSuper 1300-GFP vector or pBI121 vector and PsLTP1 Plasmids obtained by gene recombination.

6. A recombinant bacterium, characterized in that: The recombinant bacterium comprises the vector according to claim 4 or 5.

7. The recombinant bacterium according to claim 6, characterized in that The recombinant bacteria is Escherichia coli or Agrobacterium.

8. The recombinant bacterium according to claim 7, characterized in that The recombinant bacteria is Escherichia coli (E. coli) strain DH5ɑ, Agrobacterium strain GV3101, or Agrobacterium strain EHA105.

9. The method according to claim 1 or 2 PsLTP1 The invention relates to an application of the gene, the PsLTP1 protein described in claim 3, the vector described in claim 4 or 5, or the recombinant bacteria described in any one of claims 6 to 8 in delaying the senescence of peony petals and / or extending the flowering period of peony flowers.

10. The use according to claim 9, characterized in that The application includes any of the following methods: ① Improve the quality of peony PsLTP1 Gene expression; ② Use PsLTP1 The gene, the vector or the recombinant bacteria express the PsLTP1 protein, and then the solution containing the PsLTP1 protein is used to infect the peony flowers; ③ Cultivate and amplify the recombinant bacteria, and use the suspension containing the recombinant bacteria to infect peony flowers.

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