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

Abstract

The present invention relates to the field of genes, and in particular to a method for delaying the senescence of peony flowers. PsILR In order to solve the technical problem of delaying petal senescence and extending the flowering period, the present invention provides a gene for delaying peony flower senescence and extending the flowering period of peony flowers. PsILR The present invention verifies that the overexpression of the gene in peony petals is PsILR The senescence of petals was delayed, while the senescence of petals was accelerated after VIGS silencing, and the contents of hydrogen peroxide and superoxide anion increased, indicating that PsILR Participate in the aging process of peony petals. The present invention also provides a PsILR Gene vectors, recombinant bacteria and their applications.

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. PsI 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.

[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] The petal senescence process is closely related to the content of free IAA, and petal senescence is usually accompanied by petal wilting, fading and falling off (Miao Meimei, 2016). Among all plant hormones, auxin plays an important role in regulating the process of petal shedding. Increased auxin levels in plants can inhibit petal shedding, while decreased auxin content can promote petal shedding (Gao Qiang et al., 2018).

[0005] The auxin amidohydrolase gene was first discovered in Arabidopsis (Bartel, 1995). It is a metalloenzyme involved in the homeostasis of the plant hormone auxin and belongs to the amidohydrolase superfamily, the peptidase family M20, and the M20D subfamily. Structurally, auxin amidohydrolase has two vertical domains. The larger catalytic domain carries a binuclear metal center, and the smaller "satellite" domain usually serves as a dimerization interface and participates in the stabilization and regulation of enzyme activity.

[0006] Previous studies have shown that AtILR1, AtIAR3 and AtILL2 genes in Arabidopsis participate in regulating the extension of hypocotyls, the occurrence of lateral roots and the extension of root hairs under light conditions by regulating the content of free IAA. Among them, AtIAR3 participates in regulating the response of Arabidopsis to drought stress by hydrolyzing IAA-Ala. In recent years, there has been a certain research foundation on the regulatory mechanism of auxin on the occurrence of peony petal abscission, but the role and regulatory mechanism of auxin amidohydrolase in peony petal senescence are still unclear.

[0007] In previous studies, the inventors determined the gene expression profile during peony petal aging and found that PsI 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

[0008] 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. PsI The gene has a nucleotide sequence as shown in SEQ ID NO: 1.

[0009]

[0010] Furthermore, the PsI The amino acid sequence of the PsILR protein encoded by the gene is shown in SEQ ID NO:2.

[0011] MEWPFYLCICLLMLSTSSCSGLDLSSLTRELLESAREQHFFDWLKRVRRDMHEYPELAFEEHKTSQLIRSELDSLGVEYVWPVAKTGLVASIGSGTQPWFALRADMD ALPIQELVEWEHKSRIDGKMHACGHDAHVTMLLGAAKLLHNKRNELKGTVKLVFQPGEEGRGGAYHMLKEGALDNFQAIFGLHVLPEMPTGTIGSRPGPMLAGAGRF IATIQGRGGHAASPHVNRDPILAASFTIVALQQIVSRETDPLEARVVTVGFIEGGQAVNVIPETVRFGGTFRSMTTEGLFYVQQRIKEIVEKQAAVHRCNASVDFME ETLRPYPATVNDETMYKHGKRIGEVLLGKPNVQLIPMTMGAEDFSFYSQKLAAAFFMIGAKNDTLKSDIRVHSPHFVIDEEVLAIGAALHAAVAISYLDGHVVD (SEQ ID NO: 2).

[0012] The present invention also provides a PsILR protein, whose amino acid sequence is shown in SEQ ID NO:2.

[0013] The present invention also provides a vector, which is a plasmid vector comprising the PsI Gene.

[0014] Furthermore, the vector is a pSuper 1300-GFP vector or a pBI121 vector and PsI Plasmids obtained by gene recombination.

[0015] The present invention also provides a recombinant bacterium comprising the vector.

[0016] Furthermore, the recombinant bacteria is Escherichia coli or Agrobacterium, preferably Escherichia coli (E. coli) strain DH5ɑ, Agrobacterium strain GV3101, or Agrobacterium strain EHA105.

[0017] The present invention also provides the PsI Application of the gene, PsILR protein, the vector or the recombinant bacteria in delaying senescence of peony petals and / or extending the flowering period of peony flowers.

[0018] Furthermore, the application may include the following methods:

[0019] ① Improve the quality of peony PsI Gene expression, such as gene modification or introduction into the vector;

[0020] ② Use PsI The gene, the vector or the recombinant bacteria express the PsILR protein, and then the solution containing the PsILR protein is used to infect the peony flowers;

[0021] ③ Cultivate and amplify the recombinant bacteria, and use the suspension containing the recombinant bacteria to infect peony flowers.

[0022] The present invention also provides a method for delaying peony flower senescence, comprising:

[0023] (1) Design primer pairs PsI The open reading frame of pBI21- PsI Overexpression vector and transform Agrobacterium EHA105;

[0024] (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.

[0025] Furthermore, the infection solution contains: 2.132 g / L MES, 0.9522 g / L MgCL2, and 0.5 mol / L AS.

[0026] Compared with the prior art, the beneficial effects of the present invention include at least:

[0027] 1. The present invention is the first to discover PsI genes, and proved PsI Genes involved in the aging process of peony petals.

[0028] 2. The present invention successfully amplified PsI Gene and explored its regulatory mechanism in peony flowers.

[0029] 3. The present invention constructs PsI 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

[0030] Figure 1 The secondary structure prediction of PsILR protein; A: α-helix; B: β-sheet; C: β-turn; D: random coil.

[0031] Figure 2 for PsI Phylogenetic analysis of genes.

[0032] Figure 3 for PsI The spatiotemporal expression patterns of genes in 'Xue Ying Tao Hua'; Figure 3 Middle A shows the states of flowers at different stages S1-S5; Figure 3 B is PsI Tissue expression patterns; Figure 3 C is PsI Expression patterns during petal development.

[0033] Figure 4 For pBI121- PsI Vector construction results, where M represents DL 2000 Marker; P represents positive; N represents negative; Figure 4 A in pBI121- PsI ORF amplification results; Figure 4 B is pBI121- PsI E. coli bacterial solution PCR results; Figure 4 C in pBI121- PsI PCR results of Agrobacterium culture.

[0034] Figure 5 For overexpression PsI Functional identification results in peony petal senescence; Figure 5 A in PsI Phenotypic observation of overexpression of 'Xue Ying Tao Hua'; Figure 5 Middle B is qRT-PCR analysis PsI Expression levels in overexpressing petals; Figure 5 C is PsI Detection of hydrogen peroxide (DAB) and superoxide anion (NBT) in petals of overexpressed 'Xueyingtaohua'; Figure 5 D in the figure is qRT-PCR detection of senescence-related genes in overexpressed petals PsSAG13 expression level.

[0035] Figure 6 For TRV2- PsI Vector construction results; M represents DL 2000 Marker; P represents positive; N represents negative; Figure 6 A in the middle is TRV2- PsI ORF amplification results; Figure 6 B in the middle is TRV2- PsI E. coli bacterial solution PCR results; Figure 6 C in the middle is TRV2- PsI PCR results of Agrobacterium culture.

[0036] Figure 7 For silence PsI Functional identification results in peony petal senescence; Figure 7 A in PsI Observation of the phenotype of silent 'Xue Ying Tao Hua'; Figure 7 Middle B is qRT-PCR analysis PsI expression levels in silent petals; Figure 7 C is PsI Silence detection of hydrogen peroxide (DAB) and superoxide anion (NBT) in the petals of 'Xue Ying Tao Hua'; Figure 7 D in the figure is qRT-PCR detection of senescence-related genes in silenced petals PsSAG13 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 S1, S2, S3, S4 and S5 (i.e., according to the state of the flowers: initial opening S1, half-opening S2, full bloom S3, initial decay S4, decay S5), quick-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 of MES (4-morpholineethanesulfonic acid), 0.9522 g of MgCl2, and 0.5 M of AS (acetosyringone), add ddH2O to make up to 1 L, and use it freshly prepared.

[0055] Main instruments:

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

[0057] Example 1: PsI Cloning and analysis of genes

[0058] According to the EST sequences of genes obtained from the proteome related to peony flower senescence and suppression subtractive hybridization (SSH) library, blast in the local transcriptome database to obtain the PsI sequence of 'Snow Reflecting Peach Blossom'. PsI The open reading frame (ORF) of the gene is 1278 bp, encoding a polypeptide of 425 amino acids, and its nucleotide sequence is shown in SEQ ID NO: 1. PsI Using DANMAN software to translate the obtained gene into a protein sequence, the encoded PsILR protein has the molecular formula C

[0059] H 2101 H 3297 N 577 O 607 S 20 , with a molecular weight of 46992.99 D, a theoretical isoelectric point pI of 5.98; an instability index of 40.56, suggesting that the protein is unstable; a liposolubility index of 90.66; the total number of negatively charged residues (Asp + Glu): 51; the total number of positively charged residues (Arg + Lys): 41; the total average hydrophilicity index is -0.058, indicating that it is a hydrophilic protein. Predicting its secondary structure includes 39.53% α-helix, 6.59% β-turn, 16.94% extended region, and 36.94% random coil (as Figure 1 shown). It can be seen from the results that in the amino acid sequence deduced from the PsILR gene, leucine (Leu) has the largest proportion, accounting for 9.9%; tryptophan (Trp) and cysteine (Cys) have the smallest proportion, accounting for 1.2%.

[0060] Perform blastp on the PsILR protein sequence on the NCBI website to find homologous proteins, and use MEGA 5.0 software to construct a phylogenetic tree (as Figure 2 As shown in the figure, PsILR has the highest homology with Populus trichocarpa, followed by Salix babylonica and Psoralea corylifolia.

[0061] Embodiment 2: PsI Analysis of spatiotemporal expression of genes in 'Xue Ying Tao Hua'

[0062] Real-time fluorescence quantitative PCR was used to detect the expression level of PsILR gene in roots, stems, leaves, sepals, bracts, stamens and petals.

[0063] Using actin gene as internal reference, real-time quantitative primers were designed online using NCBI (Table 1) and synthesized by Bioengineering (Shanghai).

[0064] Table 1: PsI Gene real-time quantitative primers

[0065]

[0066] 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.

[0067] Table 2: PCR amplification system

[0068]

[0069] The status of peony flowers in different stages from S1 to S5 Figure 3 As shown in A, the expression level in the calyx of 'Xue Ying Tao Hua' is high, and the expression level in the petals is moderate ( Figure 3 Then, the petals of 'Xue Ying Tao Hua' at different developmental stages were tested. PsI expression, found PsI The expression level gradually increased during S1-S4 and began to decrease during S5 ( Figure 3 (B).

[0070] Example 3: Overexpression of peony petals PsI Gene

[0071] 3.1 Construction of pBI121- PsI Overexpression vector

[0072] 3.1.1 Primer design

[0073] Designed using CE Design V1.04 PsI Specific primers for gene recombination with pBI121 vector, primer sequences are shown in Table 3.

[0074] Table 3: PsI Primers required for ligation to pBI121 vector

[0075]

[0076] 3.1.2 Amplification and recovery of target genes

[0077] The cDNA of the petals of 'Xue Ying Tao Hua' at different stages was used as amplification template and amplified with 2× FlashMaster Mix. PsI 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 ( Figure 4 Middle A).

[0078] Cut the above strips according to Fast Pure Ⓡ The product was purified according to the instructions of Gel DNA Extraction Mini Kit (Vazyme, Nanjing).

[0079] 3.1.3 Enzyme digestion and ligation

[0080] use XB I. Sma pBI121 was double-digested with I restriction enzyme site, and the mixture was placed in a PCR instrument at 37°C for 30 min and reacted at 50°C for 20 min.

[0081] 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.

[0082] Table 4: Recombination reaction system

[0083]

[0084] 3.1.4 Transformation into competent E. coli DH5ɑ

[0085] 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.

[0086] 3.1.5 Bacterial liquid PCR and plasmid extraction

[0087] 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. PsI The strains corresponding to the sequences with consistent sequence alignment were cultured overnight, and the recombinant plasmids were extracted ( Figure 4 Middle B).

[0088] 3.1.6 Transformation into competent Agrobacterium EHA105

[0089] Take out the EHA105 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 EHA105 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 culture 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 culture medium containing Kan+Rif, and invert and culture for 48-72 h. Pick a single colony for bacterial solution PCR, detect the amplified product by gel electrophoresis, screen the bacterial solution of the positive strain, amplify and shake, and preserve the bacteria ( Figure 4 Middle C).

[0090] 3.2 Agrobacterium preparation

[0091] Take pBI21 and pBI21- PsI 200 μL of Agrobacterium were 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 at 28℃ with a shaker at 210 rpm 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 mentioned above until the OD value reaches 1.0-1.2. Use the pBI21 resuspension as the control group and the pBI21- PsIThe resuspension was used as the experimental group and placed in a large beaker for later use.

[0092] 3.3 Infection of peony buds

[0093] Take peony buds according to the experimental material treatment method, punch holes with a hole puncher to form petal discs. Place the treated peony petal discs in a beaker filled with infection solution. Soak the petal discs in the infection solution, place the beaker in a vacuum pump to evacuate air at 0.1 atmosphere for 20 minutes, slowly release air, and repeat 3 times. Rinse with tap water twice and with bacterial water once. Place in 8℃ dark conditions for 1 day and then transfer to normal culture (16 h day / 8 h night) for 2-3 days.

[0094] 3.4 PsI and aging-related genes PsSAG13 Expression detection

[0095] The peony petal discs treated in darkness at 8℃ for 1 day were taken out for normal light culture. The normal light time was recorded as day 0. On the second day, RNA was extracted from the peony discs, and cDNA was obtained by reverse transcription. Then, the expression level of PsILR in peony was detected by qRT-PCR. The discs with higher PsILR expression than the control were selected as the overexpression buds for subsequent experiments. The expression level of the senescence-related gene PsSAG13 in the control and overexpression peony discs was detected by qRT-PCR.

[0096] The results show that PsI The expression level of α was significantly increased in the overexpressed peony petal disc, indicating that the α was successfully overexpressed in peony. PsI Gene( Figure 5 (B) Overexpression PsI Phenotypic observation of peony petal discs revealed that overexpression PsI In the peony petal disk of the treated plant, a small part of the petal disk began to curl on the 3rd day, and partly rotted and browned on the 6th day, while the petals of the peony in the control group mostly curled on the 3rd day, and almost completely rotted and browned on the 6th day ( Figure 5 Middle A), indicating overexpression PsI It can delay the decay of peony petals. PsSAG13 It is a maker gene related to aging. qRT-PCR detection of overexpression in peony buds PsSAG13 The gene expression level was found PsSAG13 The expression level of Figure 5 D), so it is inferred PsI It can delay the shedding and aging process of peony petals.

[0097] 3.5 DAB and NBT staining

[0098] To explore PsI 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. The vacuum pump was used to evacuate for 30 min, and then the air was slowly released. The staining was carried out overnight at room temperature, and the stained petals were decolorized with 95% ethanol in an 80°C water bath until clear the next day. The results showed that the petals in the control group were stained darker, while those in the overexpression group were darker. PsI The petals are lightly dyed, indicating PsI The accumulation of reactive oxygen species and superoxide anions was reduced after overexpression treatment ( Figure 5 Middle C).

[0099] Example 4: Silence in Peony Petals PsI Gene

[0100] 4.1 TRV2- PsI Vector construction

[0101] According to the SGN VIGS Tool, gene-specific sequences were retrieved and specific primers were designed (Table 5). PsI The open reading frame of was amplified, and TRV2- PsI vector and transformed into Agrobacterium EHA105.

[0102] Table 5: PsI Primers required for ligation of TRV2 vector

[0103]

[0104] Amplification using specific primers PsI 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 6 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. PsI The matching strains were sent to the company for sequencing ( Figure 6 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 6 Middle C).

[0105] 4.2 Agrobacterium preparation

[0106] Take TRV1, TRV2 and TRV2- PsI 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- PsI Mix equal volumes as the experimental group and place in a large beaker in the dark for 3-5 hours for later use.

[0107] 4.3 Infection of peony buds

[0108] The peony buds were infected according to the method of Example 3.

[0109] 4.4 PsI and aging-related genes PsSAG13 Expression detection

[0110] 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. PsI The expression level of PsI 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 PsSAG13 The amount of expression.

[0111] In the peony bud PsI After silencing treatment, qRT-PCR was used to detect PsI The results showed that PsI The expression level of α was significantly reduced in the silenced peony buds, indicating that α was successfully silenced in peony. PsI Gene( Figure 7 Middle B).

[0112] To silence PsI Phenotypic observation of peony buds revealed that PsI 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 in the control group began to fall on the 4th day and were almost completely detached on the 8th day ( Figure 7 Middle A), indicating silence PsIIt can cause peony petals to fall prematurely. qRT-PCR detection of successfully silenced peony buds PsSAG13 The gene expression level was found PsSAG13 The expression level of Figure 7 D), further explanation PsI It can delay the shedding and aging process of peony petals.

[0113] 4.5 DAB and NBT staining

[0114] According to the method of Example 3, the peony petals of the control group and the silent group were stained and decolorized to clear.

[0115] The results showed that the petals of the control group were lighter in color, while the petals of the silent PsI The petals are darker, indicating PsI The accumulation of reactive oxygen species and superoxide anions increased after silencing ( Figure 7 Middle C).

[0116] 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 PsI A gene characterized by Said PsI The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

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

2.

3. A carrier, characterized in that The vector is a plasmid vector comprising the PsI Gene.

4. The vector according to claim 3, wherein The vector is pSuper 1300-GFP vector or pBI121 vector and PsI Plasmids obtained by gene recombination.

5. A recombinant bacterium, characterized in that: The recombinant bacterium comprises the vector according to claim 3 or 4.

6. The recombinant bacterium according to claim 5, characterized in that The recombinant bacteria is Escherichia coli or Agrobacterium.

7. The recombinant bacterium according to claim 6, characterized in that The recombinant bacteria is Escherichia coli (E. coli) strain DH5ɑ, Agrobacterium strain GV3101, or Agrobacterium strain EHA105.

8. The method according to claim 1 PsI The invention relates to an application of the gene, the PsILR protein described in claim 2, the vector described in claim 3 or 4, or the recombinant bacteria described in any one of claims 5 to 7 in delaying the senescence of peony petals and / or extending the flowering period of peony flowers.

9. The use according to claim 8, characterized in that The application includes any of the following methods: ① Improve the quality of peony PsI Gene expression; ② Use PsI The gene, the vector or the recombinant bacteria express the PsILR protein, and then the solution containing the PsILR 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.

Images