A gene of ERF84 associated with petal senescence in Osmanthus fragrans and its application

Abstract

This invention discloses an *Osmanthus fragrans* petal senescence-related gene, ofERF84, and its applications, belonging to the field of plant molecular biology. Based on previous research and bioinformatics analysis software, the function of this gene was preliminarily predicted. The full-length gene sequence was obtained through cloning, and an overexpression vector was constructed and genetically transformed into *Nicotiana macrophylla*. The results showed that compared with wild-type plants, the petals of transgenic plants withered 2 days later, indicating that the ofERF84 gene plays an important regulatory role in petal senescence. As an important transcription factor regulating the petal senescence process, the ofERF84 gene can be used in genetic engineering for some plants with short flowering periods, and has practical application value.

Description

[0001] This invention is a divisional application, with the original application number being 2022116311221, the application date being December 19, 2022, and the invention title being "A gene related to the aging of Osmanthus fragrans petals and its application". Technical Field

[0002] This invention belongs to the field of plant molecular biology, and in particular relates to a senescence-related gene in the petals of Osmanthus fragranscv. 'rixianggui' and its application. Background Technology

[0003] Osmanthus fragrans, belonging to the Oleaceae family and the Osmanthus genus, is a beautiful tree with a rich fragrance when in bloom, and is widely planted in gardens, courtyards, and scenic spots. In recent years, the rise of molecular biology has provided novel approaches to regulating plant flowering time, such as transgenic technology. Studies have shown that the development and senescence of plant flowers are regulated by many transcription factors. ERF transcription factors are a class of transcription factors unique to plants and have significant biological importance in plant development. Related research indicates that ERF transcription factors can regulate plant flowering time, thereby promoting or delaying petal senescence. No reports on this aspect have been found in osmanthus-related research; therefore, functional studies of ERF transcription factors are of great significance. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a senescence-related gene of ERF84 in Osmanthus fragrans that can significantly slow down petal senescence; another purpose of this invention is to provide an application of the senescence-related gene of ERF84 in Osmanthus fragrans in slowing down petal senescence in plants.

[0005] Technical solution: The present invention relates to a senescence-related gene of ERF84 in Osmanthus fragrans petals, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0006] The amino acid sequence of the ofERF84 gene expression protein, which is related to the senescence of Osmanthus fragrans petals, is shown in SEQ ID NO.5.

[0007] The nucleotide sequence of the ofERF84 gene is shown in SEQ ID NO.2:

[0008] ATGAGCAACTCAGACAAATCTGAGAAGAAGTACAAAGGCATTCGATGTCGAAAATGGGGCAAATGGGTATCTGAAATTCGAGTTCCGGGCACGCGAGATCGACTGTGGTTAGGATCATACTCCACGCCGGAGGCTGCAGCAGTAGCTCATGACACAGCCTTGTATTGTCTCCGAGGAGAGTCGTGCTCACTGGAAAATTTCA ACTTCCCTCGATGTTACCGGCTAGTGTCGAATAGGAATGTCACCAAGGTCTGTGCAAAGGGTCTCGTCGGATGCTGGCATGGCCATTGATGCACAATTTTTAGCCTCAAGGAGTTCTGATAATGTGGAGGAAGTTGACCAAAATGGGATGGGTTTTGGGCTAGATGATCAGGAACAATTGAGCATTTCCGTTGAAGATTA TCTCAGATGGCCTTCTATATGA

[0009] The nucleotide sequence of the protein expressed by the ofERF84 gene is shown in SEQ ID NO.5:

[0010] MSNSDKSEKKYKGIRCRKWGKWVSEIRVPGTRDRLWLGSYSTPEAAAV AHDTALYCLRGESCSLENFNFPSMLPASARIGMSPRSVQRVSSDAGMAIDAQF LASRSSDNVEEVDQNGMGFGLDDQEQLSISVEDYLRWPSI-

[0011] Vectors or host bacteria containing the above-mentioned Osmanthus fragrans petal senescence-related gene ofERF84.

[0012] As a further improvement to the above scheme, the vector is a plant recombinant expression vector.

[0013] As a further improvement to the above scheme, the plant recombinant expression vector is 1305-GFP-ofERF84.

[0014] This invention provides the application of the senescence-related gene ofERF84 in Osmanthus fragrans petals in slowing down the senescence of plant petals.

[0015] Preferably, the plant is cinnamon or tobacco.

[0016] This invention provides a method for slowing down the senescence of plant petals, the method being:

[0017] 1) Construct the vector ofERF84 containing the senescence-related genes of the Osmanthus fragrans petals;

[0018] 2) Transform the constructed vector into plants or plant cells;

[0019] 3) Cultivate transgenic plantlets; cultivate the above-mentioned plants for production application.

[0020] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: Based on previous research and bioinformatics analysis software, the function of this gene was preliminarily predicted. The full-length gene sequence was obtained through cloning, and an overexpression vector was constructed and genetically transformed into Nicotiana macrophylla. The results showed that, compared with wild-type plants, the petals of the transgenic plants withered 2 days later, indicating that the gene of this invention plays an important regulatory role in petal senescence. As an important transcription factor gene regulating the petal senescence process, it can be used in genetic engineering for some plants with short flowering periods, and has practical application value. Attached Figure Description

[0021] Figure 1 A picture of Osmanthus fragrans in full bloom;

[0022] Figure 2 Agarose gel electrophoresis image of the amplified product of the target gene of ERF24; M: DL2,000 DNA Marker; Lanes 1-2: of ERF24;

[0023] Figure 3 PCR of E. coli with 1305-GFP vector of ERF24, M: DL2,000 DNA Marker; lanes 1-5: PCR of E. coli with 1305-GFP-ofERF24 vector;

[0024] Figure 4 Agrobacterium 1305-GFP vector ofERF24 for PCR of ERF24, M: DL2,000 DNA Marker; lanes 1-5: Agrobacterium 1305-GFP-ofERF24 vector for PCR of ERF24.

[0025] Figure 5 For the detection of positive seedlings of ERF24, M: DL2,000 DNA Marker; lanes 1-9: PCR of 1305-GFP-ofERF24 transgenic plants;

[0026] Figure 6 For observation of the flowering period of positive seedlings of ERF24, S1: initial flowering period; S2: 2 days after initial flowering period; S3: 4 days after initial flowering period; S4: 6 days after initial flowering period.

[0027] Figure 7 Agarose gel electrophoresis image of the target gene amplification product of ofERF84, M: DL2,000 DNA Marker; Lane 1: ofERF84;

[0028] Figure 8 PCR of E. coli with 1305-GFP vector of ERF84, M: DL2,000 DNA Marker; lanes 1-5: PCR of E. coli with 1305-GFP-ofERF84 vector;

[0029] Figure 9 Agrobacterium 1305-GFP vector ofERF84: PCR of Agrobacterium 1305-GFP vector; M: DL2,000 DNA Marker; Lanes 1-5: PCR of Agrobacterium 1305-GFP-ofERF84 vector.

[0030] Figure 10 For the detection of positive seedlings of ERF84, M: DL2,000 DNA Marker; lanes 1-15: PCR of 1305-GFP-ofERF84 transgenic plants;

[0031] Figure 11 For observation of the flowering period of positive seedlings of ERF84, S1: initial flowering period; S2: 2 days after initial flowering period; S3: 4 days after initial flowering period; S4: 6 days after initial flowering period.

[0032] Figure 12 Agarose gel electrophoresis image of the target gene amplification product of ofERF109, M: DL2,000 DNA Marker; lanes 1-2: ofERF109;

[0033] Figure 13 PCR of E. coli with 1305-GFP vector of ERF109, M: DL2,000 DNA Marker; lanes 1-5: PCR of E. coli with 1305-GFP-ofERF109 vector;

[0034] Figure 14 Agrobacterium 1305-GFP vector of ERF109: PCR of Agrobacterium 1305-GFP vector; M: DL2,000 DNA Marker; Lanes 1-5: PCR of Agrobacterium 1305-GFP-ofERF109 vector.

[0035] Figure 15 For the detection of positive seedlings of ERF109, M: DL2,000 DNA Marker; lanes 1-11: PCR of 1305-GFP-ofERF109 transgenic plants;

[0036] Figure 16 For observation of the flowering period of positive seedlings of ERF109, S1: initial flowering period; S2: 2 days after initial flowering period; S3: 4 days after initial flowering period; S4: 6 days after initial flowering period. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments. In the following embodiments, operations not described in detail are routine biological experimental procedures, which can be performed with reference to molecular biology experimental manuals and existing publicly available journal articles, or according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0038] The materials used in this application are flowers that bloomed during the peak flowering period of mature Osmanthus fragrans trees. Figure 1 Images of Osmanthus fragrans during its peak bloom period, such as... Figure 1 As shown, flowers of Osmanthus fragrans during its peak blooming period were collected from the Osmanthus germplasm resource bank of Jiangsu Vocational College of Agriculture and Forestry in October 2020. They were immediately flash-frozen in liquid nitrogen and then stored at -80℃. The large-flowered tobacco seedlings used were provided by the research group of Wang Lianggui at Nanjing Forestry University.

[0039] In this embodiment, total RNA was extracted from plants using the TIANGEN Plant RNA Extraction Kit (DP432). TaKaRa PrimeScript was used. TM The RT Master Mix (Perfect Real Time) reverse transcription kit reverse transcribes the extracted RNA into cDNA. The resulting cDNA is then diluted 10 times with water and stored at -20°C.

[0040] Example 1: Construction of an overexpression vector for the ofERF24 gene of *Cinnamomum camphora*

[0041] (1) Obtaining the target gene

[0042] Based on the Osmanthus whole genome database previously published by the research group, one gene sequence was screened and compared with the sequence of the model plant Arabidopsis thaliana, and named ofERF24.

[0043] The nucleotide sequence of the ofERF24 gene is shown in SEQ ID NO.1:

[0044] ATGGATTCCTCTGTTTTTCACTCCATGAATTCTGATAATTTTTCCTCTGATTCATCCTTTGGATCACCAGAATCTTTTCCATGTTGGGATGTAAAATTCTTGGAGGAAAATTCACTTCCTTTCAATGAAAATGATTCTGAAGAAATGCTTCTGTATGGTGTGATAGCTCAAGCTGCACAAGAAGAAAATTCTGAATCAAATTCATCTGATCATGAAACAAAAGAAGATGAAGTAAATTCAGTACCAGACAAAAAACCCACCAAGGAAAAGTCTTACAGGGGTGTAAGAAAAAGGCCATGGGGGAAATTTGCAGCTAAAATAAGGGATTCTACAAGGAATGGAATAAGGGTTTGGTTAGGAACATTTGATACTGCAGAAGCTGCTGCTTTAGCTTACGATCAAGCGGCGTTTTCGATGCGTGGCCAGGCTGCGATTATGAATTTTCCTGTGGAAAGGGTGAGGGAATCACTGTGTGAAATGAAGTGTATAACTGAGGAAGATTGTTCACCTGTTATTGCTTTAAAAAGGAAACATTCGATGAGGAGGAGAAATGGGAACAAGAAAAGCAAGGGGAGGAAAATTAAGGAGGAAAATGTGGCGGTGTTTGAGGATTTGGGTGCTGATTATTTGGAACAACTTCTGAGTACAAGTTGA

[0045] The nucleotide sequence of the protein expressed by the ofERF24 gene is shown in SEQ ID NO.4:

[0046] MDSSVFHSMNSDNFSSDSSFGSPESFPCWDVKFLEENSLPFNENDSEEMLLYGVIAQAAQEENSESNSSDHETKEDEVNSVPDKKPTKEKSYRGVRKRPWGKFAAKIRDSTRNGIRVWLGTFDTAEAAALAYDQAAFSMRGQAAIMNFPVERVRESLCEMKCITEEDCSPVIALKRKHSMRRRNGNKKSKGRKIKEENVAVFEDLGADYLEQLLSTS-

[0047] (2) Design primers

[0048] The full-length nucleotide sequence of the gene was analyzed using BioXM software to determine restriction enzyme sites, and SpeⅠ and SmaⅠ enzymes were selected as the two restriction endonucleases. Primers were designed using Premier Primer 5.0 software. The required information was filled in, including the sequence near the restriction sites on the vector, the full length of the target gene, and the two restriction sites (5′ and 3′ ends) in sequence. The designed sequences were synthesized by Jereh Biotech.

[0049] F1: 5′-AGGACAGCCCAGATCACTAGT-3′,

[0050] R1: 5′-GCTCACCATGGATCCCCCGGG-3′.

[0051] (3) Gene cloning

[0052] Using cDNA diluted 10-fold as a template, the full-length gene sequence was cloned using NEB's Q5 high-fidelity enzyme system. The PCR reaction mixture consisted of: 10 μL 5×Q5 Reaction Buffer, 10 μL 5×Q5 High GC Enhancer, 4 μL 2.5 mM dNTPs, 2.5 μL 10 μM Primer-F, 2.5 μL 10 μM Primer-R, 0.5 μL cDNA, 0.5 μL Q5 High-Fidelity DNA Polymerase, and up to 50 μL Nuclease-Free Water. The PCR conditions were: 98℃ for 30 s; 98℃ for 10 s, 60℃ for 30 s, 72℃ for 45 s, 30 cycles; 72℃ for 5 min; 10℃ for infinity.

[0053] Add 5 μL of 10× Loading Buffer to the PCR product and spot it into the lanes of a 1% agarose gel. Run agarose gel electrophoresis at 120V and 200mA for 20 min. Observe, acquire images, and cut the target bands using a gel imaging system. Figure 2 The image shows the agarose gel electrophoresis result of the amplified product of the target gene of ERF24.

[0054] (4) T-vector bacterial culture PCR

[0055] The T vector was activated and cultured beforehand by removing it from the -80℃ ultra-low temperature freezer. A 10 μL reaction mixture was used: 0.5 μL Forward Primer, 0.5 μL Reverse Primer, 5 μL 2×PCR Mix, 1 μL bacterial culture, and 3 μL ddH2O. The reaction conditions were: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min; and termination of the reaction at 25℃. The amplified products were then subjected to agarose gel electrophoresis.

[0056] (5) PCR of E. coli culture with 1305-GFP vector

[0057] The reaction mixture consisted of 0.5 μL forward primer, 0.5 μL reverse primer, 5 μL 2×PCR Mix, 1 μL E. coli culture, and 3 μL ddH2O. The reaction conditions were: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, for 35 cycles; 72℃ final extension for 10 min; and termination at 25℃. The amplified products were then subjected to agarose gel electrophoresis. Figure 3 PCR of E. coli with the 1305-GFP vector of ofERF24.

[0058] Example 2: Construction of an overexpression vector for the ofERF84 gene of *Cinnamomum camphora*

[0059] (1) Obtaining the target gene

[0060] Based on the Osmanthus whole genome database previously published by the research group, one gene sequence was screened and compared with the sequence of the model plant Arabidopsis thaliana, and named ofERF84.

[0061] The nucleotide sequence of the ofERF84 gene is shown in SEQ ID NO.2:

[0062] ATGAGCAACTCAGACAAATCTGAGAAGAAGTACAAAGGCATTCGATG TCGAAAATGGGGCAAATGGGTATCTGAAATTCGAGTTCCGGGCACGCGAGATCGACTGTGGTTAGGATCATACTCCACGCCGGAGGCTGCAGCAGTAGCTCATGACACAGCCTTGTATTGTCTCCGAGGAGAGTCGTGCTCACTGGAAAATTTCAACTTCCCTTCGATGTTACCGGCTAGTGCTCGAAT AGGAATGTCACCAAGGTCTGTGCAAAGGGTCTCGTCGGATGCTGGCATGGCCATTGATGCACAATTTTTAGCCTCAAGGAGTTCTGATAATGTGGAGGAAGTTGACCAAAATGGGATGGGTTTTGGGCTAGATGATCAGGAACAATTGAGCATTTCCGTTGAAGATTATCTCAGATGGCCTTCTATATGA

[0063] The nucleotide sequence of the protein expressed by the ofERF84 gene is shown in SEQ ID NO.5:

[0064] MSNSDKSEKKYKGIRCRKWGKWVSEIRVPGTRDRLWLGSYSTPEAAAV AHDTALYCLRGESCSLENFNFPSMLPASARIGMSPRSVQRVSSDAGMAIDAQF LASRSSDNVEEVDQNGMGFGLDDQEQLSISVEDYLRWPSI-

[0065] (2) Design primers

[0066] The full-length nucleotide sequence of the gene was analyzed using BioXM software to determine restriction enzyme sites, and SpeⅠ and SmaⅠ enzymes were selected as the two restriction endonucleases. Primers were designed using Premier Primer 5.0 software. The required information was filled in, including the sequence near the restriction sites on the vector, the full length of the target gene, and the two restriction sites (5′ and 3′ ends) in sequence. The designed sequences were synthesized by Jereh Biotech.

[0067] F2: AGGACAGCCCAGATCACTAGTATGAGCAACTCAGACAAATCTGAG R2: GCTACCATGGATCCCCCGGGTATAGAAGGCCATCTGAGATAATCT

[0068] (3) Gene cloning

[0069] Using cDNA diluted 10-fold as a template, the full-length gene sequence was cloned using NEB's Q5 high-fidelity enzyme system. The PCR reaction mixture consisted of: 10 μL 5×Q5 Reaction Buffer, 10 μL 5×Q5 High GC Enhancer, 4 μL 2.5 mM dNTPs, 2.5 μL 10 μM Primer-F, 2.5 μL 10 μM Primer-R, 0.5 μL cDNA, 0.5 μL Q5 High-Fidelity DNA Polymerase, and up to 50 μL Nuclease-Free Water. The PCR conditions were: 98℃ for 30 s; 98℃ for 10 s, 60℃ for 30 s, 72℃ for 45 s, 30 cycles; 72℃ for 5 min; 10℃ for infinity.

[0070] Add 5 μL of 10× Loading Buffer to the PCR product and spot it into the lanes of a 1% agarose gel. Run agarose gel electrophoresis at 120V and 200mA for 20 min. Observe, acquire images, and cut the target bands using a gel imaging system. Figure 7 The image shows the agarose gel electrophoresis result of the amplified product of the target gene of ofERF84.

[0071] (4) T-vector bacterial culture PCR

[0072] The T vector was activated and cultured beforehand by removing it from the -80℃ ultra-low temperature freezer. A 10 μL reaction mixture was used: 0.5 μL Forward Primer, 0.5 μL Reverse Primer, 5 μL 2×PCR Mix, 1 μL bacterial culture, and 3 μL ddH2O. The reaction conditions were: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min; and termination of the reaction at 25℃. The amplified products were then subjected to agarose gel electrophoresis.

[0073] (5) PCR of E. coli culture with 1305-GFP vector

[0074] The reaction mixture consisted of 0.5 μL forward primer, 0.5 μL reverse primer, 5 μL 2×PCR Mix, 1 μL E. coli culture, and 3 μL ddH2O. The reaction conditions were: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, for 35 cycles; 72℃ final extension for 10 min; and termination at 25℃. The amplified products were then subjected to agarose gel electrophoresis. Figure 8 PCR of E. coli with the 1305-GFP vector of ofERF84.

[0075] Example 3: Construction of an overexpression vector for the cinnamon ofERF109 gene

[0076] (1) Obtaining the target gene

[0077] Based on the Osmanthus whole genome database previously published by the research group, one gene sequence was screened and compared with the sequence of the model plant Arabidopsis thaliana, and named ofERF109.

[0078] The nucleotide sequence of the ofERF109 gene is shown in SEQ ID NO.3:

[0079]

[0080] The nucleotide sequence of the protein expressed by the ofERF109 gene is shown in SEQ ID NO. 6:

[0081] MANPGDTGEFIRFPVVPGNSETTTTTAQPHQPTQSWERIQQPDFLHSGEPMFFGFDQAREMPAMVPTIAAPGQTYGEWNFRPDPSGTGGSGNIHYPPSSPYYSSSSGSWLAGQKRRRGQDDSVTQFPEQVPRILGGFGESSFSVKTEVAEAGGTSVAPPPTTTVEHQPP PPETSTEEQGERRKYRGVRQRPWGKWAAEIRDPHKAARVWLGTFETAEAAARAYDEAALRFRGNKAKLNFPENVNLPPPGPPPPLPPAFLQNQPFQNTADFQHQKPTSMLEQMLYASSMAAAASSYPLQFPSQQQTINSQTQGNQTQENTPYYPAPPWTSSTHCPSSPS-

[0082] (2) Design primers

[0083] The full-length nucleotide sequence of the gene was analyzed using BioXM software to determine restriction enzyme sites, and SpeⅠ and SmaⅠ enzymes were selected as the two restriction endonucleases. Primers were designed using Premier Primer 5.0 software. The required information was filled in, including the sequence near the restriction sites on the vector, the full length of the target gene, and the two restriction sites (5′ and 3′ ends) in sequence. The designed sequences were synthesized by Jereh Biotech.

[0084] F3:AGGACAGCCCAGATCACTAGTATGGCGAATCCAGGAGACAC

[0085] R3:GCTCACCATGGATCCCCCGGGACTAGGAGATGAAGGACAGTGGGT

[0086] (3) Gene cloning

[0087] Using cDNA diluted 10-fold as a template, the full-length gene sequence was cloned using NEB's Q5 high-fidelity enzyme system. The PCR reaction mixture consisted of: 10 μL 5×Q5 Reaction Buffer, 10 μL 5×Q5 High GC Enhancer, 4 μL 2.5 mM dNTPs, 2.5 μL 10 μM Primer-F, 2.5 μL 10 μM Primer-R, 0.5 μL cDNA, 0.5 μL Q5 High-Fidelity DNA Polymerase, and up to 50 μL Nuclease-Free Water. The PCR conditions were: 98℃ for 30 s; 98℃ for 10 s, 60℃ for 30 s, 72℃ for 45 s, 30 cycles; 72℃ for 5 min; 10℃ for infinity.

[0088] Add 5 μL of 10× Loading Buffer to the PCR product and spot it into the lanes of a 1% agarose gel. Run agarose gel electrophoresis at 120V and 200mA for 20 min. Observe, acquire images, and cut the target bands using a gel imaging system. Figure 12 Agarose gel electrophoresis image of the amplified product of the target gene of ERF109.

[0089] (4) T-vector bacterial culture PCR

[0090] The T vector was activated and cultured beforehand by removing it from the -80℃ ultra-low temperature freezer. A 10 μL reaction mixture was used: 0.5 μL Forward Primer, 0.5 μL Reverse Primer, 5 μL 2×PCR Mix, 1 μL bacterial culture, and 3 μL ddH2O. The reaction conditions were: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min; and termination of the reaction at 25℃. The amplified products were then subjected to agarose gel electrophoresis.

[0091] (5) PCR of E. coli culture with 1305-GFP vector

[0092] The reaction mixture consisted of 0.5 μL forward primer, 0.5 μL reverse primer, 5 μL 2×PCR Mix, 1 μL E. coli culture, and 3 μL ddH2O. The reaction conditions were: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, for 35 cycles; 72℃ final extension for 10 min; and termination at 25℃. The amplified products were then subjected to agarose gel electrophoresis. Figure 13PCR of E. coli using the 1305-GFP vector of ofERF109.

[0093] Example 4: Transformation of Agrobacterium tumefaciens EHA105

[0094] (1) Take out Agrobacterium EHA105 stored in a -80℃ ultra-low temperature freezer and thaw it on ice. Add 5-10 μL of plasmid each time, and then freeze it in ice bath for 5 min, liquid ammonia for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min in sequence.

[0095] (2) Add 800 μL of antibiotic-free LB liquid medium and incubate at 28°C and 200 rpm on a shaker for 2-3 hours;

[0096] (3) After the culture is completed, centrifuge the bacterial solution at 6000r for 1min, discard part of the supernatant, and leave 100μL to spread evenly on LB solid medium (containing 50mg / L Kana), seal with sealing film, and invert in an incubator at 28℃ for 40-48h.

[0097] (4) Bacterial detection and backup: If the target band in the bacterial detection is correct and the brightness is consistent, pick the corresponding colonies in the backup plate into LB liquid medium (containing 50 mg / L Kana) and shake them. Then, preserve the bacterial solution and 50% glycerol at a volume ratio of 3:7. After quick freezing in liquid nitrogen, store in an ultra-low temperature freezer at -80℃.

[0098] Figure 4 Agrobacterium tumefaciens culture PCR using the 1305-GFP vector of ofERF24; Figure 9 Agrobacterium tumefaciens culture PCR of ofERF84 1305-GFP vector; Figure 14 Agrobacterium tumefaciens culture PCR using the 1305-GFP vector of ofERF109.

[0099] Example 5: Infecting large-flowered tobacco and screening to obtain resistant plants

[0100] (1) Explant disinfection: After harvesting the tender leaves of Nicotiana macrophylla, the surface of the leaves was cleaned with detergent to remove the dust layer. After rinsing with running water for 30 minutes, the leaves were transferred to a clean bench for disinfection. First, 75% ethanol was poured into a beaker and shaken to ensure that the ethanol fully contacts the surface of the tender leaves for 30 seconds. The leaves were then rinsed three times with sterile water. Next, the leaves were soaked in 5% NaClO for 10 minutes and rinsed four times with sterile water. The moisture on the surface of the leaves was then absorbed with sterile filter paper. After disinfection, the leaf edges and veins were removed with a sterile scalpel. The remaining leaves were then cut into small pieces of 0.5 × 0.5 cm for infection.

[0101] (2) Shaking culture: The 1305-GFP empty vector and the vectors containing the target genes prepared in Examples 1-3 were taken out and thawed on ice. The bacterial culture was added to 20 mL of LB liquid medium (containing 50 mg / L Kana) using a pipette and incubated in the dark on a shaker at 28°C and 200 rpm until the bacterial culture OD was reached. 600 = Between 0.4 and 0.45;

[0102] (3) Infection: Use sterile tweezers to transfer the cut tobacco leaves into the infection solution and infect for 10 minutes, shaking the conical flask once every 2 minutes;

[0103] (4) Co-culture: After the infected leaves are taken out, they are laid flat on sterile filter paper. After the bacterial solution dries slightly, the leaves are laid flat on the symbiotic culture medium (Ms+B5+1mg / L 6-BA+0.1mg / L NAA+30g / L Sucrose+8g / L Agro, pH=5.7+200um / L AS) and incubated in the dark at 25℃ for 3 days;

[0104] (5) Screening culture: After co-culturing for 3 days, the leaves were transferred to the screening medium (Ms+B5+1mg / L 6-BA+0.1mg / L NAA+30g / L Sucrose+8g / L Agro, pH=5.7+300mg / L timentin+5mg / Bialaphos) and cultured. The medium was changed every 15 days until resistant callus and resistant buds grew.

[0105] (6) Rooting medium: When the resistant shoots grow to about 2cm, they are transferred to rooting medium (1 / 2MS + 30g / L Sucrose + 8g / L Agro, pH = 5.8 + 200mg / L timentin + 5mg / L Bialaphos + 20mg / L hygremycin) for rooting culture.

[0106] (7) Hardening off seedlings: After the tissue culture seedlings have taken root, harden them off. Wash off the culture medium attached to the roots with pure water, transfer the seedlings to sterilized substrate, place them in an acclimatization room, and maintain them normally.

[0107] Example 6: Observation of the senescence period of transgenic plants

[0108] (1) Detection of positive tobacco seedlings: Total RNA was extracted from tobacco leaves using the TIANGEN Plant RNA Extraction Kit (DP432). TaKaRa PrimeScript was used for further analysis. TMThe extracted RNA was reverse transcribed into cDNA using the RT Master Mix (Perfect Real Time) reverse transcription kit. The full-length gene sequence was cloned using the NEB Q5 high-fidelity enzyme system. A 10 μL PCR reaction mixture was used for detection: 5 μL 2×PCR Mix, 0.5 μL Primer F, 0.5 μL Primer R, 1 μL bacterial culture, and 3 μL ddH2O. The PCR reaction conditions were: 94℃, 10 min; 94℃, 30 s; 58℃, 30 s; 72℃, 1 min 30 s; 72℃, 1 min; 25℃, infinity. The PCR products were then mixed with 5 μL of 10×Loading Buffer and detected by agarose gel electrophoresis. Plants matching the target band were considered positive. Figure 5 For positive test results of ofERF24; Figure 10 For positive test results of ofERF84; Figure 15 For positive test results of ERF109.

[0109] (2) Flowering period observation: After routine maintenance and management, the transgenic tobacco plants began to flower. Figure 6 , Figure 11 , Figure 16 It can be seen that wild-type plants enter the senescence stage 4 days after the initial flowering stage. However, the process from the initial flowering stage to the senescence stage in transgenic positive plants can last 6 days, which is 2 days longer than that of wild-type plants.

Claims

1. A gene associated with petal senescence in Osmanthus fragrans. ofERF84 Its expressed protein contains genes ofERF84 The application of vectors or host bacteria in slowing down the senescence of plant petals, characterized by, Gene ofERF84 Overexpression in plants; genes ofERF84 The nucleotide sequence is shown in SEQ ID NO.2, gene. ofERF84 The amino acid sequence of the expressed protein is shown in SEQ ID NO.5; the plant is Nicotiana macrocarpa.

2. The application according to claim 1, characterized in that, Used for gene amplification ofERF84 The primer pair is F2: AGGACAGCCCAGATCACTAGTATGAGCAACTCAGACAAATCTGAG R2: GCTCACCATGGATCCCCCGGGTATAGAAGGCCATCTGAGATAATCT.

3. The application according to claim 1, characterized in that, The vector is a plant recombinant expression vector.

4. The application according to claim 3, characterized in that, The plant recombinant expression vector is 1305-GFP-ofERF84.

5. A method for slowing down the senescence of plant petals, characterized in that, The method is as follows: 1) Constructing senescence-related genes for Osmanthus fragrans petals. ofERF84 Overexpression vectors; genes ofERF84 The nucleotide sequence is shown in SEQ ID NO.2, gene. ofERF84 The amino acid sequence of the expressed protein is shown in SEQ ID NO.5; 2) The constructed vector was transformed into a plant or plant cell; the plant was Nicotiana macrocarpa; 3) Cultivate transgenic plantlets; cultivate the above-mentioned plants for production application.

Images