CN114958864A - Paeonia lactiflora PlWRKY47 gene and application thereof in high temperature resistance of plants - Google Patents

Abstract

The invention discloses a peonyPlWRKY47Genes and application thereof in the aspect of high temperature resistance of plants. The invention also discloses the peonyPlWRKY47A protein encoded by a gene. The invention also discloses the amplification of the peonyPlWRKY47The sequences of the primer pairs of the genes are shown as SEQ ID NO.6 and SEQ ID NO. 7. The invention also discloses a watchA cassette, a recombinant vector, a recombinant cell, or a recombinant strain. The invention is realized byPlWRKY47The gene over-expression vector is transformed into tobacco for expression, so that the active oxygen accumulation of plants, particularly the tobacco is reduced, the relative conductivity is reduced, the net photosynthetic rate and the chlorophyll fluorescence parameter Fv/Fm are improved, and a new tobacco germplasm with strong high-temperature resistance is created.

Description

Paeonia lactiflora PlWRKY47 gene and application thereof in high temperature resistance of plants

Technical Field

The invention belongs to the technical field of peony cultivation, and particularly relates to peony PlWRKY47 and application thereof in high temperature resistance of plants.

Background

High temperature is one of the main environmental factors that restrict the growth and development of plants. In recent years, high-temperature hot damage weather caused by global warming is frequent, high-temperature stress increasingly restricts the growth, development and distribution of plants, the yield and quality of a plurality of economic crops are seriously reduced, the development of the planting industry is directly threatened, and great influence is caused on agricultural economic benefit.

Paeonia lactiflora Pall is perennial root herbaceous flower of Paeonia in Paeoniaceae, is listed as ' flower phase ' in the middle of the traditional famous flower in China, is called ' two kings of flowers together with ' flower king ' peony, is favored to be cold and dry due to nature, and is widely cultivated in northern areas such as Shandong, Henan and the like in China. With the international rise of peony cut flowers, peony production is widely carried out in southern areas such as Shanghai, Jianghe and Shanghai. However, in the south, the high temperature and hot summer weather in summer has long duration and high strength, so that the peony main cultivated species is slightly burnt and wilted by stems and leaves, yellowed leaves and scorched edges after being moved from the south in the north; the leaf withering and death in advance and the underground root system dysplasia seriously affect the ornamental effect after flowering and flowering in the next year, and greatly limit the cultivation application of the peony in south (Zhao Daqixia, Hanchenxia, pottery. different peony varieties are identified in heat resistance. Yangzhou university notice (agricultural and life science edition), 2015,36(4): 105-.

Early studies showed that peony can significantly change its physiology, biochemistry and cell structure to resist the damage caused by high temperature stress, so as to establish a new metabolic stability equilibrium state to adapt to the high temperature stress (Zhang Jiaping, Lidanqing, Nie crystal, Xiayiping. the physiological and biochemical response and heat resistance evaluation of peony under high temperature stress Nuclear agro paper, 2016b,30(9): 1848-1856; Wu YQ, Zhao DQ, Han CX, Tao J. Biochemical and molecular responses of Bacillus root to high temperature stress, Canadian Journal of Plant Science,2016,96: 474-484; Zhao DQ, Han outer CX, Zhou CH, Tao J. Shade Li anode high temperature stress-induced degradation of peony root stress, J. biological and biological stress of Yeast stress, J. Zhao ML, J. 919J. biological stress of biological stress, J. Zhao J. Italy, J. 919, J. biological stress of peony root stress, J. Zhao J. No. 919, J. biological stress, J. Zhao J. Foreign cell structures & channels, 2019,24: 247-type 257). In recent years, with the rapid development of molecular biology, heat signal transduction pathways and transcriptional regulation networks of plants responding to high temperature stress have been studied in large quantities. However, peony lacks genomic information and is relatively slow to study at the high temperature resistant molecular level. At present, only the transgenic research that the heat shock protein gene PlHSP70 of the peony can resist high temperature capability is seen (ZHao DQ, Xia X, Su JH, Wei MR, Tao J. overexpression of human peony root HSP70 con. high temperature tolerance. BMC genome, 2019,20: 70). While regulation at the transcriptional level directly affects gene expression in eukaryotes, transcription factors as key molecular switches play a crucial role in plant response to high temperature stress (Javed T, Shabbir R, Ali A, Afzal I, Zaheer U, Gao SJ. transcription factors in plant stress responses: Challenges and potential for research improvement. plants,2020,9: 491). However, the research on the high temperature resistance function of the transcription factor in the peony has not been reported.

WRKY transcription factors are a specific transcription regulator in plants, and are of great interest because of their wide involvement in the regulation of plant biological processes (Wani SH, Anand S, Singh B, Bohra A, Joshi R. WRKY transcription factors and plant stress responses, latest disorders and function promoters. plant Cell Reports,2021,40: 1071-1085). WRKY transcription factors play an important role in plants responding to high temperature stress (Cheng ZY, Luan YT, Meng JS, Sun J, Tao J, ZHao DQ. WRKY transcription factor responses to high-temperature stress. plants,2021,10: 2211). The transgenic verification of the former shows that Arabidopsis thaliana AtWRKY30(El-Esawi MA, Al-Ghamdi AA, Ali HM, Ahmad M. overexpression of AtWRKY30 transcription factor and gravity stress in wheat (Triticum aestivum L.). Genes,2019,10:163), wheat TaWRKY33 (GH, Xu JY, Wang YX, Liu JM, Li PS, Chen M, et Al. Dry-stress WRKY transformation factors, TaWRKY1 and WRWRKY 33 transcription factor and/or maize transcription factor and expression in Plant, BMC 16, Zhang TF 2016, Zhang 2022, Zhang stress, Zhang III, Zhang-stress, Zhang III, and Z III, and Zhang III, Zhang-stress. In the case of the Plant WRKY47, its function in abiotic stress is mainly focused on regulating drought stress (Rainer J, Wang SH, Peleg Z, Blumwald E, Chan RL. the Plant transformation factor OsWRKY47 is a positive regulator of the stress to water transformation strain Plant Biology,2015,88: 401) and aluminum (Li CX, Yan JY, Ren JY, Sun L, Xu C, GLi X, et al. RKY transformation factor copolymers of cell walls modification genes. Journal of integration Biology,2020,62:1176, Fe 2 transformation factor 1242, B J. Pat. No. 4. B. J. of transformation Plant tubes, W. 12. J. Pat. No. 4. B. J. of transformation genes, W. 12. J. Pat. 4. of growth genes, W. 12. J. Pat. No. 4. B. Pat. 4. of growth of Plant growth genes, W. 12. J. Pat. 4. of growth genes, B. Pat. 4. of growth genes, III. B. Pat. 4. of growth genes, tao MZ, Meng Y, Zhu XY, Qian LW, Shah A, et al, the role of WRKY47gene in regulating the release of gene in Arabidopsis thaliana plant Biotechnology Reports,2020,14: 121-.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a peony PlWRKY47 gene cDNA full-length sequence and application thereof in plant high temperature resistance, and provides a new gene family member for creating high temperature resistant plant materials by using WRKY transcription factors.

The invention also aims to solve the technical problem of providing the protein encoded by the peony PlWRKY47 gene.

The invention also aims to solve the technical problem of providing an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain containing the peony PlWRKY47 gene.

The invention also aims to solve the technical problem of providing the peony PlWRKY47 gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain and the application of the recombinant vector in the aspect of changing the high temperature resistance of plants.

The technical problem to be solved by the invention is to provide a method for obtaining plants with high temperature resistance.

The invention finally solves the technical problem of providing a method for identifying whether the plant has high temperature resistance.

The technical scheme is as follows: in order to solve the technical problem, the invention provides a peony PlWRKY47 gene, wherein the full-length sequence of the cDNA of the peony PlWRKY47 gene is shown as SEQ ID No. 1.

The invention also comprises the protein coded by the peony PlWRKY47 gene, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.

Wherein, SEQ ID NO.1 in the sequence table consists of 1947 bases. The peony PlWRKY47 gene can encode PlWRKY47 protein, and the protein has an amino acid sequence shown in SEQ ID NO.2, wherein the SEQ ID NO.2 in the sequence list consists of 507 amino acids.

The invention also comprises a primer pair for amplifying the peony PlWRKY47 gene, wherein the sequences of the primer pair are shown as SEQ ID NO.6 and SEQ ID NO. 7.

The invention also comprises an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain, which contains the Paeonia lactiflora PlWRKY47 gene.

The recombinant vector includes, but is not limited to, binary expression vector pCAMBIA 1301.

The invention also comprises the peony PlWRKY47 gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain and the application of the recombinant vector in the aspect of changing the high temperature resistance of plants. Firstly, the existing plant binary expression vector pCAMBIA1301 can be used for constructing a recombinant expression vector containing a PlWRKY47 gene. Then, the plant expression vector carrying the gene PlWRKY47 of the invention is transferred into agrobacterium EHA105 cells, and then is transformed into plant tissues by a leaf disc method, and the transformed host plant is tobacco. Further, primers designed in the construction of the eukaryotic expression vector are SEQ ID NO.6 and SEQ ID NO. 7.

The invention also provides a construction method of the recombinant vector, which comprises the following steps: amplifying the fragment of the Paeonia lactiflora PlWRKY47 gene, and connecting the fragment with a binary expression vector pCAMBIA1301 to obtain the Paeonia lactiflora pall.

The present disclosure also includes a method of obtaining a plant with high temperature tolerance, comprising the steps of:

1) allowing the plant to comprise the peony PlWRKY47 gene; or

2) So that the plants express the protein coded by the peony PlWRKY47 gene.

The invention also relates to a method for identifying whether the method is a plant with high temperature resistance, which is characterized by comprising the following steps:

1) identifying whether said plant comprises said peony PlWRKY47 gene; or the like, or, alternatively,

2) identifying whether said plant expresses the protein encoded by said peony PlWRKY47 gene.

Wherein, the plant includes but is not limited to tobacco.

Further, transformed tobacco plants were screened by RT-PCR and qRT-PCR validation. Further, RT-PCR and qRT-PCR detection are carried out, tobacco NtActin (AB158612) is taken as an internal reference gene, and primers are designed as follows: an upstream primer NtActin-F: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO. 8); a downstream primer NtActin-R: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO. 9); the primer of the PlWRKY47 gene is designed as follows: upstream primer PlWRKY 47-F: 5'-TACTGAGCCAAGGACTACA-3' (SEQ ID NO. 10); downstream primer PlWRKY 47-R: 5'-CCTGAGATTCTGGTTTTC-3' (SEQ ID NO. 11).

The invention constructs an overexpression vector of the Paeonia lactiflora PlWRKY47 gene, transfers the pCAMBIA1301-PlWRKY47 overexpression vector into tobacco by adopting an agrobacterium-mediated leaf disc method, and puts the plant at 42 ℃ for 72h after culturing for 2 months, so that the tobacco plant with the PlWRKY47 gene can normally grow, while wild tobacco leaves are withered and wilted, which shows that the overexpression Paeonia lactiflora PlWRKY47 gene has the function of changing the high temperature resistance of the plant.

The full-length cDNA sequence (SEQ ID NO.1) of the peony high-temperature resistant gene PlWRKY47 of the invention

The invention discloses an amino acid sequence (SEQ ID NO.2) deduced from the cDNA sequence of peony high-temperature resistant gene PlWRKY47

Has the beneficial effects that: compared with the prior art, the invention has the following advantages: according to the invention, the constructed PlWRKY47 gene overexpression vector is transformed into tobacco for expression, so that the active oxygen accumulation of plants, especially the tobacco is reduced, the relative conductivity is reduced, the net photosynthetic rate and chlorophyll fluorescence parameter Fv/Fm are improved, and a new tobacco germplasm with strong high temperature resistance is created.

Drawings

FIG. 1: detecting RACE result of the whole-length cDNA of the Paeonia lactiflora PlWRKY47 gene; wherein, M: DL2000 marker; 1: 3' -RACE amplification product; 2: 5' -RACE amplification product.

FIG. 2: the peony PlWRKY47 gene amino acid sequence is compared with Arabidopsis thaliana WRKY family.

FIG. 3: identifying a tobacco plant with a PlWRKY47 gene based on RT-PCR detection: wherein, the left picture is the PCR result of the NtActin primer; the right panel shows the PCR result of the PlWRKY47 primer; m: DL2000 marker; 1-2: a transgenic tobacco plant; 3: wild type tobacco.

FIG. 4 is a schematic view of: identifying a tobacco plant with a PlWRKY47 gene based on qRT-PCR detection: wherein different lower case letters indicate significant difference (p < 0.05).

FIG. 5: phenotype comparison of wild type and PlWRKY47 transgenic tobacco plants under high temperature stress: wherein, the wild tobacco leaves are dried and wilted; the tobacco which is transferred with the PlWRKY47 gene keeps a normal growth state.

FIG. 6: leaf hydrogen peroxide (H) of wild type and PlWRKY47 transgenic tobacco plants under high-temperature stress ₂ O ₂ ) And (5) comparing the accumulated amount.

FIG. 7: leaf superoxide anion (O) of wild type and PlWRKY47 transgenic tobacco plants under high-temperature stress ₂ ^·- ) And (5) comparing the accumulated amount.

FIG. 8: leaf relative conductivity (REC) of wild type and PlWRKY47 transgenic tobacco plants under high temperature stress is compared: wherein different lower case letters indicate significant difference (p < 0.05).

FIG. 9: comparison of net photosynthetic rates (Pn) of wild type and PlWRKY47 transgenic tobacco plants under high temperature stress: wherein different lower case letters indicate significant difference (p < 0.05).

FIG. 10: comparing chlorophyll fluorescence parameters Fv/Fm of wild type and PlWRKY47 transgenic tobacco plants under high-temperature stress: wherein different lower case letters indicate significant difference (p < 0.05).

Detailed Description

The technical solutions of the present invention are further described in detail by the following specific examples, but it should be noted that the following examples are only used for describing the content of the present invention and do not limit the scope of the present invention.

The experimental methods not specifically described in the following examples were carried out according to the conventional procedures, and materials, reagents and the like used in the following examples were commercially available unless otherwise specified. Carbenicillin (Cb), hygromycin (Hyg).

Example 1 cloning of full-Length sequence of Paeonia lactiflora PlWRKY47 Gene cDNA

Obtaining a cDNA sequence at the 3' end of the PlWRKY47 gene: the peony leaves were selected as a material, and total RNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa). The first strand of cDNA was produced by reverse transcription using 3' Full RACE Core Set Ver.2.0(TaKaRa) in the reverse transcription system: 1 μ L of RNA, 1 μ L of 3' -RACE Adaptor, 1 μ L of dNTP mix (10mM each), 2 μ L of 5 XM-MLV Buffer, 0.25 μ L of RNase Inhibitor, 0.25 μ L of Reverse Transcriptase M-MLV (RNase H) ^- )、4.5μL RNase Free ddH ₂ O; reverse transcription program: the reaction was carried out at 42 ℃ for 60min and at 70 ℃ for 15 min. On this basis, 3' -RACE was subjected to two separate PCR amplifications. The first round of PCR amplification system is: mu.L of cDNA, 8. mu.L of 1 × cDNA Dilution Buffer II, 2. mu.L of 3' -RACE Outer Primer, 2. mu.L of Gene specific Outer Primer (10. mu.M) (5'-CAACTACTCTGCTCTTCG-3' (SEQ ID NO.3)), and 5. mu.L of 10 × LAPCR Buffer II (Mg ⁺ Plus)、0.5μL LADNA pdymerase、30.5μL RNase Free ddH ₂ And O. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 120s, and circulating for 20 times; extension at 72 ℃ for 10 min. The second round of PCR amplification system is: 1 μ L of first round PCR amplification product, 8 μ L dNTP mix (2.5mM each), 2 μ L3 ' -RACE Inner Primer, 2 μ L Gene Specific Inner Primer (5'-GACAAGTCAAGTCCAATG-3' (SEQ ID NO.4)), 5 μ L10 × LA PCR Buffer II (Mg ⁺ Plus)、0.5μL LA DNA pdymerase(5U/μL)、31.5μL RNase Free ddH ₂ And O. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 120s, and circulating for 30 times; extension for 10min at 72 ℃. The products were detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 1.

Obtaining a cDNA sequence at the 5' end of the PlWRKY47 gene: using SMARTer ^TM RACE cDNA Amplification Kit User Manual (Clontech) reverse transcription produces the first strand of cDNA, the reverse transcription reaction is divided into three steps, first, system one is performed: 1 μ L of RNA, 1 μ L of 5' -RACE CDS Ptimer A, 9 μ L of deinized H ₂ And O. The reaction procedure is as follows: after 3min at 72 ℃ the reaction was carried out for 2min at 42 ℃. Then carrying out a system II: mu.L of the first reaction Mixture, 4. mu.L of 5 XFrist-stand Buffer, 0.5. mu.L of Dithiothreitol (100mM), 1. mu.L of dNTP mix (20mM each), 0.5. mu.L of RNase Inhibitor, 2. mu.L of SMAR Tcribe Reverse Transcriptase Transcriptase, and 1. mu.L of L SMARTER IIA Oligonudeatide. The reaction procedure is as follows: after reaction at 42 ℃ for 90min, the reaction was carried out at 70 ℃ for 10 min. And finally, carrying out a system III: 20. mu.L of the reaction mixture of system two and 50. mu.L of Tricine-EDTA Buffer. The reaction procedure is as follows: standing at 25 deg.C for 15min, and diluting cDNA. On the basis, 5' -RACE carries out PCR amplification, and the reaction system is as follows: 2.5. mu.L of 5'cDNA, 25. mu.L of 2 XSeqAmp Buffer, 1. mu.L of SeqAmp DNA Polymerase (1.25U/50. mu.L), 5. mu.L of 10 XUPM, 1. mu.L of 5' Gene Specific Primer (5'-TGAATTTCTGGGCAGGTAACATTGGACTT-3' (SEQ ID NO.5)), 15.5. mu.L of RNase Free ddH ₂ And O. The reaction conditions are as follows: reacting at 94 ℃ for 30s and at 72 ℃ for 3min, and circulating for 5 times; reacting at 94 ℃ for 30s, at 70 ℃ for 30s and at 72 ℃ for 3min, and circulating for 5 times; reacting at 94 ℃ for 30s, annealing at 68 ℃ for 30s, extending at 72 ℃ for 3min, and circulating for 25 times. The products were checked by electrophoresis on a 1% agarose gel, the results of which are shown in FIG. 1.

Example 2 comparison of amino acid sequence of Paeonia lactiflora PlWRKY47 Gene with Arabidopsis thaliana WRKY family

The Arabidopsis thaliana WRKY family amino acid sequences are downloaded from The Arabidopsis Information Resource (TAIR) (https:// www.arabidopsis.org /) database, The Arabidopsis thaliana WRKY family amino acid sequences and The peony PlWRKY47 gene amino acid sequences are represented in a FASTA format and stored as TXT files, and then an evolutionary tree is constructed by MEGA 7.0 software, The algorithm is Neightbor-Joining, The self-evaluation is performed for 1000 times, The most homologous amino acid sequences can be observed, and The results show that The Arabidopsis thaliana WRKY family amino acid sequences are clustered with Arabidopsis thaliana AtWRKY47 (FIG. 2).

Example 3 expression of Paeonia lactiflora PlWRKY47 Gene overexpression vector in tobacco

Construction of an overexpression vector of Paeonia lactiflora PlWRKY47 gene: designed to contain the cleavage sites Bsa I andEcoRV primers for amplification of the sequence PlWRKY47 (upstream primer PlWRKY 47-F: 5'-cagtggtctcacaacATGGACTTATCACATACCCA-3' (SEQ ID NO.6), downstream primer PlWRKY 47-R: 5'-cagtggtctcatacaATCAGTAGAGAACTGGTGCA-3' (SEQ ID NO. 7)). PCR amplification System: 12.5 μ L2 XPPhanta Flash Master Mix (Vazyme), 1 μ L Forward Primer, 1 μ L Reverse Primer, 2 μ L DNA template (using Nuclean Plant Genomic DNA Kit (CWBIO) to extract Paeonia lactiflora DNA as template), 8.5 μ L ddH ₂ And O. Reaction procedure: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 5s, and extension at 72 ℃ for 10s for 35 cycles; extension at 72 ℃ for 1 min. After the reaction, the PCR reaction solution was subjected to agarose gel electrophoresis analysis, and the large fragment of PlWRKY47 containing the enzyme cleavage site was recovered using TSP601-DNA gel recovery kit (Tsingke). Taking a binary expression vector pCAMBIA1301 plasmid, carrying out double enzyme digestion by Bsa I (Biorun) and EcoRV (NEB), wherein the reaction system is as follows: 2.0. mu.L of 10 × CutSmart Buffer, 7. mu.L of pCAMBIA1301 plasmid, 0.4. mu.L of Bsa I (20000U/mL), 0.4. mu.L of EcoRV (20000U/mL), 10.2. mu.L of ddH ₂ O; the reaction is carried out for 0.5h at 37 ℃. The double digestion products were analyzed by agarose gel electrophoresis, and the large fragment of the purified plasmid pCAMBIA1301 was recovered using TSP601-DNA gel recovery kit (Tsingke). By using

The plus One step PCR Cloning Kit (Novoprotein) Kit adopts a homologous recombination method to connect two recovered products, and the reaction system is as follows: 4.0. mu.L of 5 Xreaction buffer, 1.0. mu.L

plus recombinase, 9. mu.L pCAMBIA1301 large fragment, 6. mu.L PlWRKY47 large fragment, 7.0. mu.L ddH ₂ O; connecting in 50 deg.C metal bath for 15min, cooling on ice, and converting Trelief into 5 μ L of connection product ^TM 5 alpha competent cells (Tsingke), then culturing overnight at 37 ℃ on an LB plate (containing Kan 50mg/L), picking positive single clone for amplification culture, extracting plasmids pCAMBIA1301-PlWRKY47, and then carrying out double enzyme digestion and sequencing verification until the construction of the pCAMBIA1301-PlWRKY47 over-expression vector is successful.

Paeonia lactiflora PlWRKY47 gene excessTransforming tobacco with the expression vector: mu.L of pCAMBIA1301-PlWRKY47 overexpression vector plasmid was used to transform GV3101(pSoup-p19) competent cells (TOLOBIO), and then cultured on YEB plates (containing Rif 50mg/L and Kan 50mg/L) at 28 ℃ for 2d, and positive single clones were picked up in YEB liquid medium (containing Rif 50mg/L and Kan 50mg/L) and cultured at 28 ℃ and 200rpm overnight. 2mL of the shaken bacterial solution was added to 50mL of YEB (containing Rif 50mg/L and Kan 50mg/L) containing the same antibiotics, and cultured under the same conditions until OD was reached ₆₀₀ 0.3-0.4. The shaken bacteria are poured into a 50mL centrifuge tube, centrifuged at 5000rpm for 10min at room temperature, and the supernatant is discarded for later use. Adding appropriate amount of acetosyringone (20mg/mL) into sterilized small triangular flask, and adding 5_ mL MS into centrifuge tube ₀ (MS liquid minimal medium, no agar and sucrose) dissolving thallus, beating with gun, pouring into small triangular flask containing appropriate amount of acetosyringone, and adding MS ₀ To 50 mL. Add 50mL MS to another sterilized Erlenmeyer flask ₀ And (5) standby. Taking aseptic seedling leaves of tobacco, cutting into small pieces (about 1cm × 1cm), adding 50mL MS ₀ Cutting 100 pieces of 150 leaves into a small triangular flask, placing into the flask, pouring the leaves into a beaker covered with gauze, adding 50mL MS into the filtered leaves ₀ + 100. mu.L acetosyringone (100. mu. mol/mL) in three vials, infected for 8min with constant gentle shaking; after infection, bacterial liquid is filtered, leaves are taken out, excess bacterial liquid on the surfaces of the leaves is sucked dry by sterile filter paper, and the leaves are inoculated in a co-culture medium (MS +3.0 mg/L6-BA +0.1mg/L NAA +30g/L sucrose + 6.66% agar)]Culturing in dark for 3 d; after the co-culture is finished, transferring into a resistant bud screening differentiation culture medium [ MS +3.0 mg/L6-BA +0.1mg/L NAA +30g/L sucrose + 6.66% agar +100mg/L Cb +25mg/L Hyg]Carrying out selective culture for one subculture for two weeks until the buds are differentiated; when the meristem adventitious bud reaches more than 2cm, the adventitious bud is cut off and transferred into a rooting screening medium [1/2MS +0.3mg/L IBA +30g/L sucrose + 6.66% agar +50mg/L Cb +8mg/L Hyg]And (5) carrying out rooting screening. After 4-6 months of culture, the tobacco with the PlWRKY47 gene can be obtained.

Example 4 identification of tobacco plants transformed with the Paeonia lactiflora PlWRKY47 Gene

And (3) PCR identification: extracting tobacco by Nuclean Plant Genomic DNAkit (CWBIO) kitLeaf DNA. On the basis, the tobacco NtActin (AB158612) gene is used as an internal reference (Forward Primer: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO.8) and Reverse Primer: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO.9)), and specific primers (Forward Primer: 5'-TACTGAGCCAAGGACTACA-3' (SEQ ID NO.10) and Reverse Primer: 5'-CCTGAGATTCTGGTTTTC-3' (SEQ ID NO.11)) of the PlWRKY47 gene are designed for PCR amplification. Reaction system: 12.5 μ L of 2 × Rapid Taq Master Mix (Vazyme), 1 μ L Forward Primer, 1 μ L Reverse Primer, 2 μ L DNA template, 8.5 μ L ddH ₂ And O. Reaction procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 52 ℃ for 15s, and extension at 72 ℃ for 5s for 35 cycles; extension at 72 ℃ for 5 min. And after the reaction is finished, carrying out gel electrophoresis detection on the PCR reaction solution. As can be seen from FIG. 3, a single and bright NtActin band was detected in both wild type tobacco and transgenic tobacco with PlWRKY47 gene, while a single, bright and clear band was detected only in transgenic tobacco with PlWRKY47 gene and not in wild type tobacco with respect to the amplified PlWRKY47 band.

qRT-PCR identification: total RNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa) and reverse transcribed into cDNA using the HiScript III RT Supermix for qPCR (+ gDNA wiper) (Vazyme) Kit, in the following reaction scheme: 1.0. mu.L of RNA, 4.0. mu.L of 4 XgDNA wiper Mix, 11.0. mu.L of RNase Free dH ₂ O; the reaction conditions are as follows: the reaction was carried out at 42 ℃ for 2 min. After the reaction is finished, adding 4.0 mu L of 5 XHiScript III qRT SuperMix into the reaction solution in the first step; the reaction conditions are as follows: the reaction was carried out at 37 ℃ for 15min and at 85 ℃ for 5 s. Using cDNA obtained by reverse transcription

The SYBR qPCR Supermix Plus (Novoprotein) kit was used for qRT-PCR detection. On the basis, the tobacco NtActin (AB158612) gene is used as an internal reference (Forward Primer: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO.8) and Reverse Primer: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO.9)), and specific primers (Forward Primer: 5'-TACTGAGCCAAGGACTACA-3' (SEQ ID NO.10) and Reverse Primer: 5'-CCTGAGATTCTGGTTTTC-3' (SEQ ID NO.11)) of the PlWRKY47 gene are designed to carry out qRT-PCR detection.Reaction system: 2. mu.L of cDNA, 12.5. mu.L

SYBR qPCR SuperMix Plus、1μL Forward Primer、1μL Reverse Primer、8.5μL ddH ₂ And (O). Reaction procedures are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 5s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; extension at 72 ℃ for 10 min. After the reaction is finished, use 2 ^-△△Ct The method performs analysis of relative expression levels of genes. The qRT-PCR identification result shows that the PlWRKY47 has a remarkably high expression level in the transgenic tobacco (figure 4).

Example 5 identification of high temperature resistance of tobacco plants with Paeonia lactiflora PlWRKY47 gene

After the transgenic tobacco and the wild type tobacco obtained in the embodiment 4 are transplanted for 2 months, the tobacco plants are respectively placed under the conditions of 42 ℃ and 24 hours of illumination for high-temperature stress, after 72 hours, the high-temperature damage symptoms such as withering and wilting of leaves of the wild type tobacco can be observed, and the high-temperature damage symptoms of the transgenic peony PlWRKY47 gene tobacco do not appear, and the transgenic peony PlWRKY47 gene tobacco still keeps a normal growth state, which indicates that the transgenic PlWRKY47 gene tobacco has stronger high-temperature resistance, and the result is shown in a figure 5.

Example 6H of tobacco plants under high temperature stress ₂ O ₂ Measurement of accumulation amount

H observation by Diaminobenzidine (DAB) staining method ₂ O ₂ The accumulated amount of (3). DAB staining solution was prepared at a concentration of 0.1mg/mL and pH 5.0 using 50mM Tris-acetate buffer. After fully soaking the leaves in the dark for 24 hours with a staining solution, the leaves were taken out and put into 95% (v/v) alcohol for boiling water bath, and pictures were taken after 15 min. As can be seen from FIG. 6, the leaf color of the wild tobacco is darker, while the leaf color of the tobacco with the gene PlWRKY47 of the peony is obviously lighter, which indicates that the tobacco with the gene PlWRKY47 accumulates less H ₂ O ₂ . The tobacco leaves in this example are the transgenic tobacco and wild-type tobacco obtained in example 4, and the leaves after high temperature stress after 2 months of growth were grown by the method of example 5.

Example 7O of tobacco plants under high temperature stress ₂ ^·- Measurement of accumulation amount

Observation of O by fluorescent Probe method ₂ ^- The accumulated amount is specifically operated according to the specifications of a living cell oxidative stress ROS in-situ staining kit (Shanghai Harlin Co.) and is slightly modified, and the specific steps are as follows: dropping 100 mu L of cleaning solution on a glass slide, pinching 2 stainless steel double-sided razor blades to quickly cut fresh leaves on filter paper, and avoiding main vein; dipping the cut sample by a fine-head brush pen, placing the sample in glass slide cleaning liquid, and adjusting the position; thirdly, after all the leaf samples are placed, completely sucking the cleaning solution on the glass slide as much as possible, then adding 10 mu L of a fluorescence stain, namely, ethidium Dihydrobromide (DHE), and incubating for 20min at 37 ℃; iv was observed under a fluorescence microscope (Axio Imager D2, ZEISS, germany) and photographed. As can be seen from FIG. 7, the fluorescence in the wild tobacco leaf is stronger, while the leaf color of the tobacco leaf with the gene of PlWRKY47 of peony is obviously weaker, which indicates that the tobacco with the gene of PlWRKY47 accumulates less O ₂ ^· -. The tobacco leaves in this example are the transgenic tobacco and wild-type tobacco obtained in example 4, and the leaves after high temperature stress after 2 months of growth were grown by the method of example 5.

Example 8 relative conductivity determination of tobacco plants under high temperature stress

0.1g of a blade wafer obtained by a puncher with the diameter of 1cm is weighed and put into an injector containing a proper amount of deionized water, and the front end of the injector is blocked and vacuumized until the blade is submerged under water. Then poured into a glass test tube together and deionized water was added to make a total volume of 20 mL. Standing at room temperature for 4h, shaking, and measuring the conductivity C1 of the solution with a conductivity meter (DDS-307A, Shanghai Lei magnetic apparatus Co., Ltd.). The tube was then sealed, placed in a boiling water bath for 30min, and after cooling to room temperature at the same time the conductivity of the solution was determined to be C2. Each treatment calculated the relative conductivity of the blades according to the following equation: relative conductivity (%) ═ C1/C2 × 100%. As can be seen from FIG. 8, the relative conductivity of the tobacco leaves of the PlWRKY47 transgenic peony is significantly lower compared with that of the wild type tobacco, indicating that the PlWRKY47 transgenic tobacco has less relative conductivity. The tobacco leaves in this example are the transgenic tobacco and wild-type tobacco obtained in example 4, and the leaves after high temperature stress after 2 months of growth were grown by the method of example 5.

Example 9 determination of photosynthetic parameters of tobacco plants under high temperature stress

The photosynthetic parameters were measured using a portable photosynthetic apparatus (LI-6400, Li-Cor, USA) and the net photosynthetic rate was automatically recorded by the system. As can be seen from FIG. 9, the net photosynthetic rate of the PlWRKY47 transgenic tobacco is significantly higher compared with the wild type tobacco, indicating that the PlWRKY47 transgenic tobacco has higher net photosynthetic rate. The tobacco leaves in this example are the transgenic tobacco and wild-type tobacco obtained in example 4, and the leaves after high temperature stress after 2 months of growth were grown by the method of example 5.

Example 10 chlorophyll fluorescence parameter determination of tobacco plants under high temperature stress

The leaves were clamped with a leaf clamp, and chlorophyll fluorescence parameters of the leaves labeled after standing in the dark for 2 hours were measured with a chlorophyll fluorescence meter (PAM-2500, Walz, Germany). The photochemical efficiency (Fv/Fm) is calculated by adopting an instrument with data processing software PAM Win. As can be seen from FIG. 10, the transgenic tobacco of PlWRKY47 has significantly higher Fv/Fm compared with wild type tobacco. The tobacco leaves in this example are the transgenic tobacco and wild-type tobacco obtained in example 4, and the leaves after high temperature stress after 2 months of growth were grown by the method of example 5.

In conclusion, the invention provides a full-length sequence of the cDNA of the PlWRKY47 gene of peony and application thereof in the aspect of high temperature resistance of plants, and the constructed PlWRKY47 gene overexpression vector is transformed into tobacco for expression, so that the accumulation of active oxygen is reduced, the relative conductivity is reduced, the net photosynthetic rate and chlorophyll fluorescence parameter Fv/Fm are improved, and a new tobacco germplasm with strong high temperature resistance is created.

Sequence listing

<110> Yangzhou university

<120> Paeonia lactiflora PlWRKY47 gene and application thereof in high temperature resistance of plants

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1947

<212> DNA

<213> Paeonia lactiflora Pall > thermostable gene PlWRKY47(Paeonia lactiflora Pall.)

<400> 1

taaccttgct tcttcttaca aattcaagac catggcaaac tactcgtcaa aaacatacat 60

ataaatatat ccatcccctc atccgatcgt aaactcccag aaaatggact tatcacatac 120

ccaagagccc tttggggtgg ttgggagttc cactaattac aacagagaac atgccattga 180

agagttggac ttcttctcta gtgatcatca caaggacgga gtttcaagat cgaatactga 240

gccaaggact acacaattac tgagtaaaac aaaggaacca ttcacaaaat ctgggggact 300

gaatcttctt accttgggtt cagctggttc acagccctgg aaaaatgaag agaaacccat 360

aacccaagtg agactattac aggtcaggct agagcagctt aaagaagaaa accagaatct 420

caggagcatg ttggatcaga ttacaaacaa ctactctgct cttcgaaggc aagtactatt 480

ggcgatgcaa caaggagcgt gtgagagtga tcgtcaacag aagaaagagg agatatataa 540

tgacaagtca agtccaatgt tacctgccca gaaattcatg gattcaccat ctggtgcaat 600

agatatcaac gagccttcac aatcaactga taatacacca gaacgattaa aggcatctct 660

aacaaataat atggaagtaa tgtcaaggaa aaggaatcct gatcttgata caattcaaat 720

aagcaggaaa aggctgtgtg tagacaacaa tggaggccct gatcaaacat cgaagtgttg 780

gggtggtcaa gtggaaagtt caaacatggc acaagcaaca aattcaaaag agcatgtacc 840

tgaggtatcc ggtaggaagg caagggtgtc aataagagca cggtctgaag tgtctatgat 900

aagcgatggt tgtcaatgga gaaaatacgg tcagaagatg gcaaagggta acccttgtcc 960

acgtgcttac tatcgttgca ctatggccgc tggatgctcc gttcgtaagc aggtgcaaag 1020

atgtgcagag gacaagacca ttctcatcac aacatatgaa ggaaatcata accaccctct 1080

tccagcagca gccacagcca tggctaacac aacatcagca gcagtatcca tgctcctctc 1140

cggctcaatc actagcaatc atgccctaac aaactccggc ttcttctcac attatgcttc 1200

caccatggcc acactatcaa cttcttcacc gtctccaaca attacactcg acttcactct 1260

ccctccaaat cccatgcaaa accaacaatc cttactgccc tcaccattct acccttcaca 1320

ttttaatgga atcccacaac aaatagaaca tcccttacac agtgtcccat ctaagcttcc 1380

aattattcca ctagagcaaa tagcacaaaa gcatccttca atggtcgata cagtcacatc 1440

agctattacc actgacccta atttcacagt ggcgctagct gcagcgattt cgttaataat 1500

aggaaaaacg cagagcaata atgattacag ccatagcagc aatggtgctc ctaactctgc 1560

ttttagaatg catacacagt cagcatcacc aaattcggaa gtcatgcacc agttctctac 1620

tgattagcag caatgcactg aaacatggtg agagagcatc tcttatttaa agaaagttgt 1680

atggctacga ggaagggagg tgatatattt tgatatattc atatatatcg attggacatc 1740

agagatcttt tattttgcac aaaatggtag gtactacatt gtcgttcaaa tttctgtgtt 1800

ttttatttgt gcggacatat cgtttcgtct cttttaagtc atgtacaggg aaacagctta 1860

taaggatatg tagactaatg aacgtcataa taagatctta tactgagttg atatttattt 1920

gcggaaagaa tttacaaaaa aaaaaaa 1947

<210> 2

<211> 507

<212> PRT

<213> Paeonia lactiflora Pall > thermostable gene PlWRKY47(Paeonia lactiflora Pall.)

<400> 2

Met Asp Leu Ser His Thr Gln Glu Pro Phe Gly Val Val Gly Ser Ser

1 5 10 15

Thr Asn Tyr Asn Arg Glu His Ala Ile Glu Glu Leu Asp Phe Phe Ser

20 25 30

Ser Asp His His Lys Asp Gly Val Ser Arg Ser Asn Thr Glu Pro Arg

35 40 45

Thr Thr Gln Leu Leu Ser Lys Thr Lys Glu Pro Phe Thr Lys Ser Gly

50 55 60

Gly Leu Asn Leu Leu Thr Leu Gly Ser Ala Gly Ser Gln Pro Trp Lys

65 70 75 80

Asn Glu Glu Lys Pro Ile Thr Gln Val Arg Leu Leu Gln Val Arg Leu

85 90 95

Glu Gln Leu Lys Glu Glu Asn Gln Asn Leu Arg Ser Met Leu Asp Gln

100 105 110

Ile Thr Asn Asn Tyr Ser Ala Leu Arg Arg Gln Val Leu Leu Ala Met

115 120 125

Gln Gln Gly Ala Cys Glu Ser Asp Arg Gln Gln Lys Lys Glu Glu Ile

130 135 140

Tyr Asn Asp Lys Ser Ser Pro Met Leu Pro Ala Gln Lys Phe Met Asp

145 150 155 160

Ser Pro Ser Gly Ala Ile Asp Ile Asn Glu Pro Ser Gln Ser Thr Asp

165 170 175

Asn Thr Pro Glu Arg Leu Lys Ala Ser Leu Thr Asn Asn Met Glu Val

180 185 190

Met Ser Arg Lys Arg Asn Pro Asp Leu Asp Thr Ile Gln Ile Ser Arg

195 200 205

Lys Arg Leu Cys Val Asp Asn Asn Gly Gly Pro Asp Gln Thr Ser Lys

210 215 220

Cys Trp Gly Gly Gln Val Glu Ser Ser Asn Met Ala Gln Ala Thr Asn

225 230 235 240

Ser Lys Glu His Val Pro Glu Val Ser Gly Arg Lys Ala Arg Val Ser

245 250 255

Ile Arg Ala Arg Ser Glu Val Ser Met Ile Ser Asp Gly Cys Gln Trp

260 265 270

Arg Lys Tyr Gly Gln Lys Met Ala Lys Gly Asn Pro Cys Pro Arg Ala

275 280 285

Tyr Tyr Arg Cys Thr Met Ala Ala Gly Cys Ser Val Arg Lys Gln Val

290 295 300

Gln Arg Cys Ala Glu Asp Lys Thr Ile Leu Ile Thr Thr Tyr Glu Gly

305 310 315 320

Asn His Asn His Pro Leu Pro Ala Ala Ala Thr Ala Met Ala Asn Thr

325 330 335

Thr Ser Ala Ala Val Ser Met Leu Leu Ser Gly Ser Ile Thr Ser Asn

340 345 350

His Ala Leu Thr Asn Ser Gly Phe Phe Ser His Tyr Ala Ser Thr Met

355 360 365

Ala Thr Leu Ser Thr Ser Ser Pro Ser Pro Thr Ile Thr Leu Asp Phe

370 375 380

Thr Leu Pro Pro Asn Pro Met Gln Asn Gln Gln Ser Leu Leu Pro Ser

385 390 395 400

Pro Phe Tyr Pro Ser His Phe Asn Gly Ile Pro Gln Gln Ile Glu His

405 410 415

Pro Leu His Ser Val Pro Ser Lys Leu Pro Ile Ile Pro Leu Glu Gln

420 425 430

Ile Ala Gln Lys His Pro Ser Met Val Asp Thr Val Thr Ser Ala Ile

435 440 445

Thr Thr Asp Pro Asn Phe Thr Val Ala Leu Ala Ala Ala Ile Ser Leu

450 455 460

Ile Ile Gly Lys Thr Gln Ser Asn Asn Asp Tyr Ser His Ser Ser Asn

465 470 475 480

Gly Ala Pro Asn Ser Ala Phe Arg Met His Thr Gln Ser Ala Ser Pro

485 490 495

Asn Ser Glu Val Met His Gln Phe Ser Thr Asp

500 505

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

caactactct gctcttcg 18

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacaagtcaa gtccaatg 18

<210> 5

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgaatttctg ggcaggtaac attggactt 29

<210> 6

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cagtggtctc acaacatgga cttatcacat accca 35

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cagtggtctc atacaatcag tagagaactg gtgca 35

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcctcatgca attcttcg 18

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acctgcccat ctggtaac 18

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tactgagcca aggactaca 19

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cctgagattc tggttttc 18

Claims (10)

1. PeonyPlWRKY47Gene characterized in that said peonyPlWRKY47The full-length sequence of the gene cDNA is shown as SEQ ID NO. 1.

2. The peony of claim 1PlWRKY47The gene coded protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. Amplifying the peony of claim 1PlWRKY47The primer pair of the gene is characterized in that the sequence of the primer pair is shown as SEQ ID NO.6 and SEQ ID NO. 7.

4. An expression cassette, recombinant vector, recombinant cell or recombinant strain comprising the peony of claim 1PlWRKY47A gene.

5. The recombinant vector according to claim 4, wherein the recombinant vector comprises binary expression vector pCAMBIA 1301.

6. The peony of claim 1PlWRKY47Use of a gene, an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain according to claim 4, or a recombinant vector according to claim 5 for modifying the ability of a plant to withstand high temperatures.

7. The method for constructing a recombinant vector according to claim 5, comprising the steps of: amplifying the peony of claim 1PlWRKY47The fragment of the gene is connected with a binary expression vector pCAMBIA1301 to obtain the gene.

8. A method for obtaining a plant with high temperature resistance, which is characterized by comprising the following steps:

1) allowing a plant to contain the peony of claim 1PlWRKY47A gene; or

2) Allowing a plant to express the peony of claim 2PlWRKY47A protein encoded by the gene.

9. A method for identifying a plant having the ability to tolerate high temperatures according to the method of claim 8, comprising the steps of:

1) identifying whether the plant comprises peony of claim 1PlWRKY47A gene; or the like, or, alternatively,

2) identifying whether the plant expresses the peony of claim 2PlWRKY47A protein encoded by the gene.

10. The method of claim 8 or 9, wherein the plant comprises tobacco.

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