Peony plerf15 protein, encoding gene thereof and application thereof in plant high temperature tolerance

Abstract

The present application discloses a peony PlERF15 protein, an encoding gene thereof and application thereof in plant high-temperature tolerance. PlERF15 The present application is constructed by transforming a gene super-expression vector into tobacco for expression, reduces active oxygen accumulation of plants, especially tobacco, reduces relative conductivity, improves chlorophyll fluorescence parameters Fv / Fm , and creates new tobacco germplasm with strong high-temperature tolerance.

Description

Technical Field

[0001] This invention belongs to the field of peony cultivation technology, specifically involving the peony PlERF15 protein and its encoding gene and its application in plant heat resistance. Background Technology

[0002] In recent years, high-temperature stress has had an increasingly significant impact on the growth and development of horticultural plants, weakening their growth vigor and leading to reduced yield and deteriorated quality, thus attracting considerable attention from researchers. Breeding heat-resistant varieties and implementing corresponding technical measures are particularly important for mitigating high-temperature stress.

[0003] Peony (Paeonia lactiflora Pall.) is a traditional famous flower in my country. It is widely popular for its colorful flowers, rich flower shapes and colors. It is widely used in courtyard cultivation, cut flower production and special garden construction. Peony prefers cool climate, but the high temperature in summer in the middle and lower reaches of the Yangtze River and south of it has a certain inhibitory effect on the growth and development of peony. It will cause peony to enter a semi-dormant state earlier, and at the same time, it will cause serious pests and diseases, reducing its ornamental value (Zhao Daqiu, Han Chenxia, ​​Tao Jun. Identification of heat resistance of different peony varieties. Journal of Yangzhou University (Agriculture and Life Sciences), 2015, 36(4):105-109; Yang Y, Sun M, Li SS, et al. Germplasm resources and genetic breeding of Paeonia: a systematic review. Horticulture Research, 2020, 7:107).

[0004] Previous studies have shown that peony undergoes significant physiological, biochemical, and cellular structural changes to resist the damage caused by high-temperature stress, thereby establishing a new metabolically stable equilibrium to adapt to high-temperature stress (Zhang Jiaping, Li Danqing, Nie Jingjing, et al. Physiological and biochemical responses and heat tolerance evaluation of peony under high-temperature stress. Journal of Nuclear Agricultural Sciences, 2016, 30: 1848-1856; Wu YQ, Zhao DQ, Han CX, et al. Biochemical and molecular responses of herbaceous peony to high temperature stress. Canadian Journal of Plant Science, 2016, 96: 474-484). In recent years, with the rapid development of molecular biology, a large number of studies have focused on the heat signal transduction pathways and transcriptional regulatory networks of plants in response to high-temperature stress. However, due to the lack of genomic information on peony, research on its heat tolerance at the molecular level has been relatively slow. Currently, only the heat shock protein gene PlHSP70, tryptophan decarboxylase gene PlTDC, and mitogen-activated protein kinase gene PlMAPK1 have been reported to enhance the high-temperature tolerance of peony plants (Zhao DQ, Xia X, Su JH, et al. Overexpression of herbaceous peony HSP70 confers high temperature tolerance. BMC Genomics, 2019, 20:70; Zhang TT, Tang YH, Luan YT, et al. Herbaceous peony AP2 / ERF transcription factor binds the promoter of the tryptophan and ecarboxylase gene to enhance high-temperature stress tolerance. Plant, Celland Environment, 2022, 45:2729-2743; Qian Y, Cheng ZY, Meng JS, et al. PlMAPK1 facilitates growth and photosynthesis of herbaceous peony (Paeonia lactiflora Pall.) under high-temperature stress. Scientia Horticulturae, 2023, 310:111701).Transcriptional regulation directly affects gene expression in eukaryotes, and transcription factors, as key molecular switches, play a crucial role in plant responses to heat stress (Zhang HM, Zhu JH, Gong ZZ, et al. Abiotic stress responses in plants. Nature Reviews Genetics, 2022, 23:104-119). However, there are few reports on transcription factors related to the heat tolerance of peony.

[0005] The ERF transcription factor family is one of the key regulatory factors in plant responses to abiotic stress. These transcription factors can be activated or inhibited under abiotic stress conditions, thereby initiating or inhibiting the transcription of genes related to stress responses. For example, ERF transcription factors play a crucial role in plant responses to abiotic stresses such as drought, high salinity, and low temperature (Mizoi J, Shinozaki K, Yamaguchi-Shinozaki. AP2 / ERF family transcription factors in plantabiotic stress responses. Biochimica Et Biophysica Acta-gene Regulatory Mechanisms, 2012, 1819: 86-96). Tobacco NtERF1 binds to the DRE cis-element in the promoter of the ABA synthesis gene NtSDR, activating the expression of ABA synthesis-related genes and increasing the ABA content in the plant, thereby enhancing its tolerance to abiotic stresses such as low temperature (Wu LJ, Chen XL, Ren HY, et al. ERF protein JERF1 that transcriptionally modulates the expression of abscisic acid biosynthesis-related gene enhances the tolerance under salinity and cold intobacco. Planta, 2007, 226:815-825). However, to date, research on the role of ERF transcription factors in plant tolerance to high temperature stress is relatively limited. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a peony PlERF15 protein with high temperature resistance and the peony PlERF15 gene encoding the protein.

[0007] Another technical problem that this invention aims to solve is to provide expression cassettes, recombinant vectors, recombinant cells or recombinant strains and their construction methods.

[0008] The technical problem to be solved by this invention is to provide the application of peony PlERF15 protein, peony PlERF15 gene, expression cassette, recombinant vector, recombinant cell or recombinant strain in plant heat resistance.

[0009] The final technical problem to be solved by the present invention is to provide a method for obtaining plants with heat resistance and a method for identifying whether the plants have heat resistance.

[0010] Technical Solution: To solve the above-mentioned technical problems, the present invention provides peony PlERF15 protein, the amino acid sequence of which is shown in SEQ ID NO.2. SEQ ID NO.2 in the sequence listing consists of 220 amino acids.

[0011] The present invention also provides a peony PlERF15 gene encoding the aforementioned protein, the sequence of which is shown in SEQ ID NO.1. SEQ ID NO.1 in the sequence listing consists of 882 bases.

[0012] The present invention also provides expression cassettes, recombinant vectors, recombinant cells or recombinant strains containing the peony PlERF15 gene described above.

[0013] The recombinant vector is obtained by introducing the peony PlERF15 gene into a plant expression vector.

[0014] The plant expression vector of the present invention includes the existing plant binary expression vector pCAMBIA1301.

[0015] The present invention also provides a method for constructing the recombinant vector, comprising the following steps: amplifying the peony PlERF15 gene and ligating it with an expression vector.

[0016] Furthermore, the primers designed for constructing the eukaryotic expression vector were SEQ ID NO.5 (5'-caggtcgactctagaggatccATGGATTACTCTTTTTCACACCACC-3') and SEQ ID NO.6 (5'-ttcgagctcagatctggtaccCCAAGGAGTAGCACTTTCTGATAAGT-3').

[0017] The present invention also provides a method for transforming a plant expression vector carrying the gene PlERF15 of the present invention into Agrobacterium EHA105 cells, and then transforming it into plant tissues by leaf disc method. The host plant being transformed is tobacco.

[0018] Transformed tobacco plants were screened using PCR and qRT-PCR verification. Further, PCR and qRT-PCR detection were performed, using tobacco NtActin (AB158612) as an internal reference gene. Primers were designed as follows: upstream primer NtActin-F: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO.7); downstream primer NtActin-R: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO.8); primers for the PlERF15 gene were designed as follows: upstream primer PlERF15-F: 5'-GAGTCATCGGAAACAGCA-3' (SEQ ID NO.9); downstream primer PlERF15-R: 5'-ACGCCTCTGTAGGACTTTT-3' (SEQ ID NO.10).

[0019] This invention also provides the application of the peony PlERF15 protein, the peony PlERF15 gene, the expression cassette, the recombinant vector, the recombinant cell or recombinant strain in plant heat resistance.

[0020] The present invention also provides a method for obtaining plants with heat resistance, comprising the following steps:

[0021] 1) To make the plant contain the peony PlERF15 protein described above; or

[0022] 2) To induce the plant to express the peony PlERF15 gene.

[0023] This includes steps such as genetic modification, hybridization, backcrossing, or asexual reproduction.

[0024] The present invention also provides a method for identifying plants with heat resistance obtained by the method, comprising the following steps:

[0025] 1) To identify whether the plant contains the peony PlERF15 protein;

[0026] 2) Identify whether the plant contains the peony PlERF15 gene.

[0027] The plants mentioned include, but are not limited to, tobacco.

[0028] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention is the first to obtain the peony PlERF15 gene and PlERF15 protein with plant heat resistance. By constructing an overexpression vector for the peony PlERF15 gene, and using Agrobacterium-mediated leaf disc method to transfer the pCAMBIA1301-PlERF15 overexpression vector into tobacco, after two months of cultivation, the tobacco plants transformed with the PlERF15 gene grew normally, while the leaves of wild-type tobacco wilted, indicating that overexpression of the peony PlERF15 gene has the function of altering the plant's heat resistance. This invention, by transforming the constructed PlERF15 gene overexpression vector into tobacco for expression, reduces the accumulation of reactive oxygen species in plants, especially tobacco, lowers the relative conductivity, and increases the chlorophyll fluorescence parameter Fv / Fm, creating a new tobacco germplasm with strong heat resistance. Attached Figure Description

[0029] Figure 1 The results of PCR amplification of the full-length cDNA of the peony PlERF15 gene were detected; where M: DL 2000 marker; 1: PCR amplification product.

[0030] Figure 2 The amino acid sequence of the peony PlERF15 gene was compared with that of the Arabidopsis thaliana ERF family.

[0031] Figure 3 To identify tobacco plants transgenic with the PlERF15 gene based on PCR detection. 1: wild-type line; 2-4: PlERF15 transgenic lines.

[0032] Figure 4 Identification of tobacco plants transgenic with the PlERF15 gene based on qRT-PCR detection: where ** indicates P<0.01.

[0033] Figure 5 A phenotypic comparison of wild-type and PLERF15 transgenic tobacco plants under high-temperature stress was conducted: wild-type tobacco leaves wilted, while PLERF15 transgenic tobacco maintained normal growth.

[0034] Figure 6 Comparison of hydrogen peroxide (H2O2) accumulation in leaves of wild-type and PlERF15 transgenic tobacco plants under high temperature stress.

[0035] Figure 7 The superoxide anion (O2) content in leaves of wild-type and PlERF15 transgenic tobacco plants under high temperature stress ·- Comparison of accumulated amounts.

[0036] Figure 8Comparison of relative electrical conductivity (REC) of leaves in wild-type and PlERF15 transgenic tobacco plants under high temperature stress: where ** indicates P < 0.01.

[0037] Figure 9 Comparison of chlorophyll fluorescence parameters Fv / Fm between wild-type and PlERF15 transgenic tobacco plants under high temperature stress: where ** indicates P<0.01. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below through specific embodiments. However, it should be noted that the following embodiments are only used to describe the content of the invention and do not constitute a limitation on the scope of protection of the present invention.

[0039] Unless otherwise specified, the experimental methods in the following examples were performed according to conventional procedures. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] Example 1: Cloning of the full-length cDNA sequence of the peony PlERF15 gene

[0041] Obtaining the full-length sequence of the PlERF15 gene: Peony leaves were used as material, and total RNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa). cDNA was produced by reverse transcription using the PrimeScript RT Reagent Kit (TaKaRa). The reverse transcription system consisted of: 1 μL RNA, 1 μL Oligo(dT)18, 1 μL dNTP Mixture (10 mM each), 2 μL 5× PrimeScript Buffer, 0.25 μL RNase Inhibitor, 0.25 μL PrimeScript RTase, and 4.5 μL RNase-free ddH2O. The reverse transcription program was: 42℃ for 60 min, followed by a 70℃ extension for 15 min. PCR amplification was then performed. The PCR amplification system consisted of: 2 μL cDNA, 2 μL dNTP Mixture (10 mM each), and 2.5 μL 10× Vazyme Lamb Buffer (Mg). 2+The reaction mixture consisted of 1 μL of forward primer (5'-AAATCATAAACAGAAACG-3' (SEQ ID NO.3)), 1 μL of reverse primer (5'-AACATTCTATTTCACCTT-3' (SEQ ID NO.4)), 0.25 μL of Vazyme LAmp DNAPolymerase (5 U / μL), and 16.3 μL of RNase-free ddH2O. The reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 60 s, for a total of 35 cycles; and 72℃ extension for 10 min. The product was detected by 1% agarose gel electrophoresis, yielding a band of approximately 1000 bp. Figure 1 ).

[0042] The full-length cDNA sequence of the peony heat-resistance gene PlERF15 (SEQ ID NO.1)

[0043] TCAGACTGCAATCGCGTGTCGCCCTTCATAAACAGAAACGATTCCATGGATTACTCTTTTTCACACCACCCAAACTTTAATTTCTCGCCGGAATGTTCACAGGAATCTTTCACATGGGATGAGCTCCTTTTCGATCACACTTCTCTTCCATTCAACTTCAATGACTCACAAGAGATGTTACTGTTTGATGTTCTAGCAGAAGGAGCTCGAGAGTCATCGG AACAGCATCTTCCGCCGGAATCAAGGAGGAAGAGGTCACTTCTAATGTCAAACAAGAAGAACCCAAGAAGGAAAAGTCCTACAGAGGCGTACGGAGGCGGCCGTGGGGGAAATTCGCGGCGGAGATAAGGGATTCCACTAGACATGGCATCCGGGTTTGGCTTGGAACGTTTGATAGCGCGGAGGCAGCCGCTCTGGCGTACGACCAAGCTGCATTTTCC ATGAGGGGATCCATGGCCGTTCTTAATTTTCCGGTGGAGAAGGTCCGGGAGTCGCTCCGGGAGATGAAGTATGGGTGTGAGGAAGGATGTTCGCCGGTGGTGGCACTCAAGAAGAGACACTCAATGAGAGTGAAGCCGACGGTTAATAAGAAGAAGAGTAAAAGAAAAGAGGTAGGGTCGGCACAAAATGTGATGATATTAGAGGATTTGGGAGCTGATTATTTAGAGCAACTTCTAAACTTATCAGAAAGTGCTACTCCTTGGTGAATTAATTGTCATGTCCCATCTTCCTTTTCTTTTTTCTTTTTTTTTTTTTTTTTAATGTTTAATATAATCTTGGAGTGAAGATCCTTGGGTAGAGCAGATATGAGGGATATCATGGAAGGGTTTTGACCAATTCCTTGTAAAGGTGAAATAGAATGTTAAGGGCGACACGCGATGCA

[0044] The amino acid sequence (SEQ ID NO.2) deduced from the cDNA sequence of the peony heat-resistant gene PlERF15 is as follows: MDYSFSHHPNFNFSPECSQESFTWDELLFDHTSLPFNFNDSQEMLLFDVLAEGARESSETASSAGIKEEEVTSNVKQEEPKKEKSYRGVRRRPWGKFAAEIRDSTRHGIRVWLGTFDSAEAAALAYDQAAFSMRGSMAVLNFPVEKVRESLREMKYGCEEGCSPVVALKKRHSMRVKPTVNKKKSKRKEVGSAQNVMILEDLGADYLEQLLNLSESATPW

[0045] Example 2: Comparison of the amino acid sequence of peony PlERF15 protein with the Arabidopsis thaliana ERF family.

[0046] The amino acid sequences of the Arabidopsis ERF family were downloaded from the Arabidopsis Information Resource (TAIR) database (https: / / www.arabidopsis.org / ). The amino acid sequences of these family members, along with those of the PlERF15 protein, were represented in FASTA format. A phylogenetic tree was then constructed using MEGA 7.0 software with the Neighbor Joining algorithm and 1000 self-reviews. The most homologous amino acid sequences were observed, showing that it clustered with Arabidopsis AtERF15 in the same class. Figure 2 ).

[0047] Example 3: Expression of the peony PlERF15 gene overexpression vector in tobacco

[0048] Construction of the peony PlERF15 gene overexpression vector: Primers containing BamHI and KpnI restriction sites were designed for amplifying the PlERF15 sequence (forward primer PlERF15-F: 5'-caggtcgactctagaggatccATGGATTACTCTTTTTCACACCACC-3' (SEQ ID NO.5), downstream primer PlERF15-R: 5'-ttcgagctcagatctggtaccCCAAGGAGTAGCACTTTCTGATAAGT-3' (SEQ ID NO.6)). PCR amplification system: 12.5 μL 2×Phanta Flash MasterMix (Vazyme), 1 μL Forward Primer, 1 μL Reverse Primer, 2 μL DNA template, 8.5 μL ddH2O. Reaction program: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 52℃ annealing for 5s, 72℃ extension for 10s, for a total of 35 cycles; 72℃ extension for 1min. After the reaction, the PCR reaction solution was analyzed by agarose gel electrophoresis, and the large fragment containing the restriction enzyme sites of PlERF15 was recovered using the TSP601-DNA gel recovery kit (Tsingke). The binary expression vector pCAMBIA1301 plasmid was double-digested with BamHI and Kpn I. The reaction system was: 2.0μL 10×CutSmart Buffer, 4μL pCAMBIA1301 plasmid, 0.4μL BamHI (20000U / mL), 0.4μL Kpn I (20000U / mL), 13.2μL ddH2O; reaction at 37℃ for 1h. The double enzyme digestion products were analyzed by agarose gel electrophoresis, and the large fragment of plasmid pCAMBIA1301 was recovered and purified using the TSP601-DNA gel recovery kit (Tsingke). The plus One-Step PCR Cloning Kit (Novoprotein) uses homologous recombination to ligate two recovered products. The reaction mixture consists of 1.0 μL of 5× reaction buffer and 0.3 μL of... Plus recombinase, 3.0 μL pCAMBIA1301 large fragment, and 1.0 μL PlERF15 large fragment; ligation was performed in a 50℃ metal bath for 15 min, followed by cooling on ice. 5 μL of the ligation product was then transformed into Trelief. TM5α competent cells (Tsingke) were cultured overnight at 37°C on LB plates (containing 50 mg / L Kan). Positive single clones were picked and expanded, and the plasmid pCAMBIA1301-PlERF15 was extracted. Double enzyme digestion and sequencing were then performed for verification until the pCAMBIA1301-PlERF15 overexpression vector was successfully constructed.

[0049] PlERF15 large fragment sequence:

[0050] ATGGATTACTCTTTTTCACAACCACCCAAACTTTAATTTCTCGCCGGAATGTTCACAG

[0051] GAATCTTTCACATGGGATGAGCTCCTTTTCGATCACACTTCTCTTCCATTCAACTTC

[0052] AATGACTCACAAGAGATGTTACTGTTTGATGTTCTAGCAGAAGGAGCTCGAGAGTC

[0053] ATCGGAAACAGCATCTTCCGCCGGAATCAAGGAGGAAGAGGTCACTTCTAATGTC

[0054] AAACAAGAAGAACCCAAGAAGGAAAAGTCCTACAGAGGCGTACGGAGGCGGCC

[0055] GTGGGGGAAATTCGCGGCGGAGATAAGGGATTCCACTAGACATGGCATCCGGGTT

[0056] TGGCTTGGAACGTTTGATAGCGCGGAGGCAGCCGCTCTGGCGTACGACCAAGCTG

[0057] CATTTTCCATGAGGGGATCCATGGCCGTTCTTAATTTTCCGGTGGAGAAGGTCCGG

[0058] GAGTCGCTCCGGGAGATGAAGTATGGGTGTGAGGAAGGATGTTCGCCGGTGGTGG

[0059] CACTCAAGAAGAGACACTCAATGAGAGTGAAGCCGACGGTTAATAAGAAGAAGA

[0060] GTAAAAGAAAAGAGGTAGGGTCGGCACAAAATGTGATGATATTAGAGGATTTGGG

[0061] AGCTGATTATTTAGAGCAACTTCTAAACTTATCAGAAAGTGCTACTCCTTGGTGA

[0062] Transformation of peony PlERF15 gene overexpression vector into tobacco: 5 μL of pCAMBIA1301-PlERF15 overexpression vector plasmid was transformed into GV3101 (pSoup-p19) competent cells (TOLOBIO). The cells were then cultured on YEB plates (containing 50 mg / L Rif and 50 mg / L Kan) at 28°C for 2 days. Positive clones were picked and cultured overnight at 28°C and 200 rpm in YEB liquid medium (containing 50 mg / L Rif and 50 mg / L Kan). 2 mL of the culture was added to 50 mL of liquid YEB containing the same antibiotics (50 mg / L Rif and 50 mg / L Kan), and cultured under the same conditions until OD. 600=0.3-0.4. Pour the shaken bacteria into a 50mL centrifuge tube, centrifuge at 5000rpm for 10min at room temperature, and discard the supernatant. First, add an appropriate amount of acetylsyleugenone (20mg / mL) to a sterilized small Erlenmeyer flask, then add 5mL of MS0 (MS0 liquid basal medium, without agar and sucrose) to the centrifuge tube to dissolve the bacteria. Mix well with a pipette, pour into the small Erlenmeyer flask containing an appropriate amount of acetylsyleugenone, and then add MS0 to 50mL. Add 50mL of MS0 (MS0 liquid basal medium, without agar and sucrose) to another sterilized small Erlenmeyer flask for later use. Take sterile tobacco seedling leaves, cut them into small pieces (about 1cm × 1cm), and place them in a 50mL Erlenmeyer flask containing MS0. Place 100-150 leaves in the flask. Pour the leaves into a beaker lined with gauze. Add the filtered leaves to a flask containing 50mL MS0 + 100μL acetylsuccine (100μmol / mL). Incubate for 8 minutes, gently shaking constantly during incubation. After incubation, filter out the bacterial solution, remove the leaves, and blot off excess bacterial solution with sterile filter paper. Inoculate the leaves into co-culture medium [MS + 3.0mg / L 6-BA + 0.1mg / L NAA + 30g / L sucrose + 6.66% agar] and incubate in the dark for 3 days. After co-culture, transfer to resistant bud selection and differentiation medium [MS + 3.0mg / L 6-BA + 0.1mg / L NAA + 30g / L sucrose + 6.66% agar + 100mg / L acetylsuccine (100μmol / mL)]. Selection and culture were carried out in [Cb + 25 mg / L Hyg], with subculture every two weeks until shoot differentiation occurred. When the adventitious shoots reached 2 cm or more, they were cut off and transferred to rooting selection medium [1 / 2 MS + 0.3 mg / L IBA + 30 g / L sucrose + 6.66% agar + 50 mg / L Cb + 8 mg / L Hyg] for rooting selection. After 4-6 months of culture, PLERF15 gene-transgenic tobacco could be obtained.

[0063] Example 4: Identification of tobacco plants transfected with the peony PlERF15 gene

[0064] PCR identification: DNA was extracted from leaves of wild-type tobacco and the transgenic tobacco obtained in Example 3 using the NuClean Plant Genomic DNA Kit (CWBIO). Based on this, PCR amplification was performed using the tobacco NtActin (AB158612) gene as an internal control (Forward Primer: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO.7), Reverse Primer: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO.8)). Specific primers for the PlERF15 gene were also designed (Forward Primer: 5'-GAGTCATCGGAAACAGCA-3' (SEQ ID NO.9), Reverse Primer: 5'-ACGCCTCTGTAGGACTTTT-3' (SEQ ID NO.10)). Reaction system: 12.5 μL 2×Rapid TaqMaster Mix (Vazyme), 1 μL Forward Primer, 1 μL Reverse Primer, 2 μL DNA template, 8.5 μL ddH2O. Reaction procedure: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 52℃ annealing for 15 s, 72℃ extension for 5 s, for a total of 35 cycles; 72℃ extension for 5 min. After the reaction, the PCR reaction solution was analyzed by gel electrophoresis. Figure 3 It can be seen that a single, bright NtActin band was detected in both wild-type tobacco and tobacco transgenic with the peony PlERF15 gene. However, regarding the amplified PlERF15 band, a single, bright, and clear band was detected only in tobacco transgenic with the PlERF15 gene, and was not detected in wild-type tobacco.

[0065] qRT-PCR identification: Total RNA was extracted from three lines of transgenic tobacco and wild-type tobacco using the MiniBEST Plant RNA Extraction Kit (TaKaRa). The total RNA was reverse transcribed into cDNA using the HiScript III RT SuperMix for qPCR (+gDNAwiper) (Vazyme) kit. The reaction mixture consisted of 1.0 μL RNA, 4.0 μL 4×gDNAwiper Mix, and 11.0 μL RNase-free dH2O. The reaction conditions were 42℃ for 2 min. After the reaction, 4.0 μL 5×HiScript III qRT SuperMix was added to the reaction mixture from step 1. The reaction conditions were 37℃ for 15 min followed by 85℃ for 5 s. The cDNA obtained from the reverse transcription was then... qRT-PCR was performed using the SYBR qPCR SuperMix Plus (Novoprotein) kit. Based on this, the tobacco NtActin (AB158612) gene was used as an internal control (Forward Primer: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO.7), Reverse Primer: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO.8)). Specific primers for the PlERF15 gene were also designed (Forward Primer: 5'-GAGTCATCGGAAACAGCA-3' (SEQ ID NO.9), Reverse Primer: 5'-ACGCCTCTGTAGGACTTTT-3' (SEQ ID NO.10)) for qRT-PCR detection. Reaction system: 2 μL cDNA, 12.5 μL 2× SYBR qPCR SuperMix Plus, 1 μL Forward Primer, 1 μL Reverse Primer, 8.5 μL ddH2O. Reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles; 72℃ extension for 10 min. After the reaction, 2... -△△Ct The method was used to analyze the relative expression levels of the gene. qRT-PCR identification results showed that PlERF15 had a significantly high expression level in transgenic tobacco. Figure 4 ).

[0066] Example 5: Identification of the heat tolerance of tobacco plants transfected with the peony PlERF15 gene

[0067] Two months after transplanting, three lines of transgenic tobacco plants obtained in Example 4 and wild-type tobacco plants were subjected to high-temperature stress at 42°C and 24 hours of light. After 72 hours, wilting and other symptoms of high-temperature damage were observed in the leaves of wild-type tobacco, while the transgenic tobacco plants containing the peony PlERF15 gene did not exhibit these symptoms and maintained normal growth, indicating that the transgenic tobacco plants containing the PlERF15 gene have strong high-temperature tolerance. Figure 5 ).

[0068] Example 6: Determination of H2O2 accumulation in tobacco plants under high temperature stress

[0069] The accumulation of H2O2 was observed using diaminobenzidine (DAB) staining. A DAB staining solution with a concentration of 0.1 mg / mL and a pH of 5.0 was prepared using 50 mM Tris-acetate buffer. After soaking the leaves in the staining solution in the dark for 24 hours, the leaves were removed and placed in a boiling water bath with 95% (v / v) ethanol. Photographs were taken after 15 minutes. Figure 6 It can be seen that the leaves of wild-type tobacco are darker in color, while the leaves of the transgenic PLERF15 tobacco obtained in Example 4 are significantly lighter in color, indicating that the transgenic PLERF15 tobacco accumulated less H2O2.

[0070] Example 7 O2 in tobacco plants under high temperature stress ·- Accumulation measurement

[0071] O2 was observed using the nitroblue tetrazolium (NBT) staining method. ·- The accumulation amount and specific operation were based on the instructions of the live cell oxidative stress ROS in situ staining kit (Shanghai Haling Company) with slight modifications. The specific steps are as follows: ① Dissolve 50 mg NBT in 100 mL of Tris buffer (pH 7.4) to obtain the NBT staining working solution, and store it at 4℃ in the dark; ② Collect wild-type tobacco leaves and transgenic tobacco leaves identified in Example 4, wash them slightly with pure water, blot off excess water on filter paper, and immerse them in the NBT staining working solution. Stain at room temperature in the dark for 6 hours until the positive areas turn deep blue; ③ Carefully remove the leaves with tweezers, rinse them 3-5 times with pure water, blot off excess water on filter paper, and then immerse them in 95% (v / v) alcohol at 40℃ for 16 hours, changing the 95% (v / v) alcohol several times during this period; ④ Remove the leaves with tweezers, rinse them back and forth in pure water 3-5 times, blot off excess water on filter paper, and then take pictures. Figure 7 It can be seen that the leaves of the PLERF15 transgenic tobacco are significantly lighter in color compared to those of wild-type tobacco, indicating that the PLERF15 transgenic tobacco accumulates less O2. ·- .

[0072] Example 8: Determination of relative electrical conductivity of tobacco plants under high temperature stress

[0073] Weigh 0.1g of wild-type tobacco leaves obtained using a 1cm diameter punch and round leaf slices of transgenic tobacco obtained in Example 4. Place them in a syringe containing an appropriate amount of deionized water, plug the syringe tip, and evacuate until the leaves sink to the bottom. Then pour them together into a glass test tube, add deionized water to a total volume of 20mL. Let stand at room temperature for 4 hours, shake well, and then measure the solution conductivity C1 using a conductivity meter (DDS-307A, Shanghai Leici Instrument Co., Ltd.). Next, seal the test tube, boil in a water bath for 30 minutes, and measure the solution conductivity C2 after cooling to room temperature. Calculate the relative conductivity of the leaves for each treatment using the following formula: Relative conductivity (%) = C1 / C2 × 100%. Figure 8 It can be seen that the relative conductivity of the transgenic PLERF15 tobacco is significantly lower than that of wild-type tobacco, indicating that the transgenic PLERF15 tobacco has a lower relative conductivity.

[0074] Example 9: Determination of chlorophyll fluorescence parameters in tobacco plants under high temperature stress

[0075] Wild-type tobacco leaves and transgenic tobacco leaves identified in Example 4 were placed on a display table. Chlorophyll fluorescence parameters of the labeled leaves, after being left to stand in the dark for 2 hours, were measured and imaged using a chlorophyll fluorescence in vivo imaging system (PlantView 230F, Guangzhou Boluteng Biotechnology Co., Ltd.). Photochemical efficiency (Fv / Fm) was calculated using the instrument's built-in data. Figure 9 It can be seen that, compared with wild-type tobacco, tobacco transgenic with the PlERF15 gene has a significantly higher Fv / Fm.

[0076] In summary, this invention provides a full-length cDNA sequence of the peony PlERF15 gene and its application in plant heat resistance. By transforming the constructed PlERF15 gene overexpression vector into tobacco for expression, reactive oxygen species accumulation is reduced, relative conductivity is lowered, and chlorophyll fluorescence parameter Fv / Fm is improved, thus creating a new tobacco germplasm with strong heat resistance.

Claims

1. Peony PlERF15 protein, characterized in that, The amino acid sequence of the peony PlERF15 protein is shown in SEQ ID NO.

2.

2. Peony encoding the protein of claim 1 PlERF15 Genes, characterized by, The peony PlERF15 The gene sequence is shown in SEQ ID NO.

1.

3. An expression cassette, recombinant vector, recombinant cell, or recombinant bacterial strain, characterized in that, It contains the peony as described in claim 2. PlERF15 Gene.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector is the peony described in claim 2. PlERF15 The gene is obtained by introducing it into a plant expression vector.

5. The method for constructing the recombinant vector according to claim 4, characterized in that, Includes the following steps: Expanding peony PlERF15 Genes are obtained by linking them to an expression vector.

6. The peony PlERF15 protein of claim 1, and the peony of claim 2 PlERF15 The application of the gene, the expression cassette of claim 3, the recombinant vector, the recombinant cell, or the recombinant strain in improving the heat resistance of plants, wherein the plant is tobacco.

7. A method for obtaining plants with heat resistance, characterized in that, Includes the following steps: 1) To make the plant contain the peony PlERF15 protein as described in claim 1; or 2) To cause the plant to express the peony as described in claim 2 PlERF15 Gene; the plant in question is tobacco.

8. The method according to claim 7, characterized in that, It includes steps such as genetic modification, hybridization, backcrossing, or asexual reproduction.

9. A method for identifying a plant with heat-resistant capabilities obtained by the method of claim 7 or 8, characterized in that, Includes the following steps: 1) To identify whether the plant contains the peony PlERF15 protein as described in claim 1; 2) To identify whether the plant contains the peony as described in claim 2. PlERF15 Gene; the plant in question is tobacco.

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