Dianthus caryophyllus heat shock transcription factor as well as coding gene DcHsfA2 and application thereof

Abstract

The invention discloses a carnation heat shock transcription factor as well as a coding gene DcHsfA2 and application thereof. The dianthus caryophyllus heat shock transcription factor provided by the invention is a protein with an amino acid sequence as shown in SEQ ID NO: 2. The invention also provides an encoding gene DcHsfA2 of the carnation heat shock transcription factor. The invention also provides a method for preparing the transgenic plant, which comprises the following steps: introducing the coding gene into a starting plant to obtain the transgenic plant, compared with the starting plant, the heat resistance of the transgenic plant is improved. According to the invention, a molecular foundation is laid for improving the heat resistance of dianthus caryophyllus through a genetic engineering means, and a theoretical reference is provided for deeply researching the biological function and mechanism of the heat shock transcription factor in regulating and controlling the adversity stress resistance of plants.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a carnation heat shock transcription factor and a coding gene DcHsfA2 thereof and applications thereof. Background Art

[0002] Dianthus caryophyllus L. is a perennial herbaceous plant of the genus Dianthus in the family Caryophyllaceae, also known as carnation and musk carnation. Dianthus caryophyllus is widely loved by the public for its varied flower colors and profound flower language, and has high ornamental and commercial value. Dianthus caryophyllus is one of the most widely used species in the international cut flower market, and can also be used as a material for arranging flower beds. Dianthus caryophyllus grows best in an environment with good air circulation, coolness, and sufficient light. High temperature will affect its growth and development, thereby affecting its ornamental value. Heat shock transcription factors (Hsfs) play a very important role when plants are subjected to abiotic stress, especially heat stress. Therefore, exploring the function of Hsfs in various abiotic stresses of carnation is extremely important for the production of carnation.

[0003] Carnations are loved by people for their slender stems, bright colors and compact petals. At present, carnations have great commercial value in the domestic market. The optimum temperature for carnation growth and development is 16-20℃, with night temperature around 10℃. High temperatures in summer can lead to problems such as accelerated growth, thin stems and small flowers; on the other hand, they can lead to more diseases and insect pests, which seriously affect the growth and development of carnations. At present, there have been relevant studies on the heat tolerance of carnation, mainly focusing on the analysis of the expression profile of carnation under heat stress (Wan XL, Zhou Q, Wang YY, et al. Identifification of heat-responsive genes in carnation (Dianthus caryophyllus L.) by RNA-seq [J]. Frontiers in Plant Science, 2015, 6 (519).) and the identification of the Hsf gene family in carnation (Li W, Wan XL, Yu JY, et al. Genome-wide identification, classification, and expression analysis of the Hsf gene family in carnation (Dianthus caryophyllus) [J]. International journal of molecular sciences, 2019, 20 (20): 5233.).The related functions of HsfA2 have been verified in plants such as Arabidopsis (Li CG, Chen QJ, Gao XQ, et al. AtHsfA2 modulates expression of stress responsive genes and enhances tolerance to heat and oxidative stress in Arabidopsis [J]. Science in China Ser. C Life Sciences, 2005, 4 (6): 540-550.) and tall fescue (Wang XY, Huang WL, Liu J, et al. Molecular regulation and physiological functions of a novel FaHsfA2c cloned from tall fescue conferring plant tolerance to heat stress [J]. Plant Biotechnology Journal, 2017, 15: 237-248.), and HsfA2 plays an important role in regulating their heat tolerance. However, the research on the function of heat shock transcription factor HsfA2 in carnation is limited.

[0004] Hsfs are directly or indirectly involved in the regulation of abiotic stress in plants. Although researchers have previously verified the function of heat shock transcription factor HsfA2 in Arabidopsis, tomato and lily, no one has studied the heat shock transcription factor of carnation. Summary of the invention

[0005] In view of this, the present invention proposes a carnation heat shock transcription factor and its encoding gene DcHsfA2 and applications.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides a protein, the amino acid sequence of the protein is shown in SEQ ID NO:2.

[0008] The present invention also provides a gene encoding the protein, and the nucleotide sequence of the gene encoding the protein is as shown in SEQ ID NO:1.

[0009] The gene was named DcHsfA2, and the protein encoded by it was named DcHsfA2. The specific information is as follows: the DcHsfA2 gene sequence is shown in SEQ ID NO: 1, sequence 1 contains 1080 nucleotides, which encodes the protein shown in SEQ ID NO: 2, and sequence 2 consists of 359 amino acids.

[0010] The expression cassette, recombinant expression vector or recombinant bacteria containing the encoding gene also fall within the protection scope of the present invention.

[0011] The invention also provides a method for preparing transgenic plants.

[0012] The method for preparing transgenic plants provided by the present invention comprises the following steps: introducing the coding gene shown in SEQ ID NO: 1 into a starting plant to obtain a transgenic plant; compared with the starting plant, the heat resistance of the transgenic plant is improved.

[0013] The coding gene is introduced via a recombinant expression vector, which is obtained by inserting the coding gene into the multiple cloning site of the expression vector pCAMBIA1300.

[0014] The improved heat resistance of the transgenic plants is manifested as follows: after heat stress, compared with the starting plants, the accumulation of malondialdehyde in the transgenic plants is reduced, the accumulation of relative conductivity is reduced, the superoxide dismutase content is increased, the peroxidase content is increased, the active oxygen content is reduced, the survival rate is increased, and / or the relative expression of heat-related endogenous genes is increased.

[0015] The plant is Arabidopsis thaliana.

[0016] The application of the protein in improving the heat resistance of plants also falls within the protection scope of the present invention.

[0017] The application of the encoding gene in improving the heat resistance of plants also falls within the protection scope of the present invention.

[0018] The plant is Arabidopsis thaliana.

[0019] Beneficial effects:

[0020] (1) The present invention obtains the gene sequence of the DcHsfA2 open reading frame, the ORF region is 1080bp long, and encodes a total of 359 amino acids. The isoelectric point of the amino acid sequence encoded by DcHsfA2 is 5.05, the molecular weight is 40.5538KDa, and the average value of the total average hydrophilicity (Gravy) is -0.548. Subcellular localization shows that DcHsfA2 is localized in the cell nucleus. The relative expression of the DcHsfA2 gene is significantly upregulated under 42°C stress.

[0021] (2) Heterologous transformation of Arabidopsis with DcHsfA2 can improve the basic heat tolerance of transgenic Arabidopsis. Overexpression of DcHsfA2 can reduce the content of reactive oxygen species in transgenic Arabidopsis under heat stress, increase the activity of antioxidant enzymes, and reduce electrolyte permeability and the degree of membrane lipid peroxidation. DcHsfA2 can increase the relative expression levels of heat shock protein genes AtHSP70 and AtHSP18.2 in transgenic plants, indicating that DcHsfA2 can improve the heat tolerance of plants by regulating the transcription of some heat shock proteins.

[0022] The present invention is the first to conduct a preliminary study on the function of DcHsfA2 in carnation. Based on the previous transcriptome data, the HS-related gene DcHsfA2 was screened out, a plant overexpression vector was constructed and transformed, and the function of DcHsfA2 was studied in depth. The present invention lays a molecular foundation for improving the heat resistance of carnation by genetic engineering means, and provides a theoretical reference for in-depth research on the biological functions and mechanisms of heat shock transcription factors in regulating plant resistance to adverse stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] For purposes of illustration and not limitation, the present invention will now be described in terms of preferred embodiments thereof, with particular reference to the accompanying drawings, in which:

[0024] Figure 1 The results of RNA electrophoresis test; Maker is the standard DNA, and lane 1 is the extracted RNA sample;

[0025] Figure 2 The electrophoresis detection results of DcHsfA2 ORF region; Maker is the standard DNA, and lane 1 is the amplified DcHsfA2 ORF region band;

[0026] Figure 3 Evolutionary analysis and conservative motif analysis of DcHsfA2; Figure 3 A in the middle is the result of the evolutionary tree analysis of DcHsfA2; Figure 3 Middle B is the result of DcHsfA2 conserved motif analysis;

[0027] Figure 4 is the result of DcHsfA2 amino acid sequence alignment;

[0028] Figure 5 This is the result of DcHsfA2 subcellular localization;

[0029] Figure 6 The expression results of DcHsfA2 under heat stress were analyzed by qRT-PCR;

[0030] Figure 71-7 represent the PCR test results of the positive colonies of the overexpression vector, and 8 represents the plasmid test results;

[0031] Figure 8 The results of RT-PCR detection of the relative expression of DcHsfA2 in homozygous transgenic Arabidopsis lines;

[0032] Fig. 9 The results of heat stress analysis of sterile transgenic Arabidopsis seedlings are shown in Figure 2. Fig. 9 A in the middle shows the phenotype of sterile transgenic Arabidopsis seedlings before and after heat stress; Fig. 9 Middle B is the survival rate of sterile transgenic Arabidopsis seedlings before and after heat stress;

[0033] Fig.10 The results of heat stress phenotype analysis of transgenic Arabidopsis potted seedlings are shown in Figure 2. Fig.10 A in the middle shows the phenotype of transgenic Arabidopsis potted seedlings before and after heat stress; Fig.10 Middle B is the survival rate of transgenic Arabidopsis potted seedlings after heat stress;

[0034] Fig.11 are related physiological indicators of transgenic Arabidopsis thaliana under heat stress; Fig.11 A in the middle is the MDA content; Fig.11 Where B is the relative conductivity; Fig.11 C in the middle is SOD activity; Fig.11 D in the middle is POD activity;

[0035] Fig.12 For the DcHsfA2 overexpressing strain O2 ·- and H2O2 accumulation;

[0036] Fig.13 The results of endogenous heat shock protein expression analysis in transgenic plants overexpressing DcHsfA2 are shown in Figure 2. Fig.13 A in the middle is the relative expression level of AtHsp18.2; Fig.13 Middle B is the relative expression level of AtHsp70; Fig.13 Center C is the relative expression level of AtHsp90. DETAILED DESCRIPTION

[0037] 1 Test materials

[0038] 1.1 Plant materials

[0039] Dianthus caryophyllus ‘Imagine’ tissue culture seedlings (Feng Yi, Xue Li, Liu Qinghua, et al. Cloning of the heat shock transcription factor gene DcHsfB1 of Dianthus caryophyllus and its expression response to different abiotic stresses [J]. Acta Physiologica Sinica, 2019, 55(7): 975-982.) under a light intensity of 120 μmol·m -2 ·s -1The tissue culture was carried out under the conditions of a photoperiod of 16 / 8h (light / dark) and a temperature of 24°C.

[0040] Columbia ecotype Arabidopsis thaniana (Sun Y, HuD, Xue P, et al. Identification of the DcHsp20 gene family in carnation (Dianthuscaryophyllus) and functional characterization of DcHsp17.8 in heattolerance. Planta, 2022, 256(1):1-17.).

[0041] 1.2 Strains and vectors used

[0042] Escherichia coli competent cells E. coli DH5α (D9057A) and pMD18-T vector (6011) were purchased from TaKaRa Biotech (Dalian) Engineering Co., Ltd. Yeast AH109 competent cells were purchased from Qingdao Bokang Biotechnology Co., Ltd. Agrobacterium competent GV3101 (AYBIO-G6039) was purchased from Angyu Biotechnology Co., Ltd. The plant overexpression vector pCAMBIA1300 (Jiang XQ, Zhang CQ, LüPT, et al. RhNAC3, astress-associated NAC transcription factor, has a role in dehydration tolerance through regulating osmotic stress-related genes in rose petals. Plant Biotechnol Journal, 2014, 12(1): 38-48.) was provided by our laboratory.

[0043] 1.3 Enzymes and chemical reagents used

[0044] Chemical reagents used: RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. RT reagent Kit With gDNA Eraser (Perfect Real Time) and PCR Mix were purchased from TaKaRa Biotechnology (Dalian) Engineering Co., Ltd. Real-time quantitative analysis was performed using Premix Ex TaqTMII (purchased from TAKARA). Nucleic acid and protein stain, agarose, MS culture medium, gentamicine (Gentamicine), acetosyringone (Acetosyringone, AS), morpholineethanesulfonic acid (MES hydrate, MES), magnesium chloride (MgCl2), LB broth, etc. were all purchased from Qingdao En Biological Reagent Co., Ltd. The remaining chemicals were all domestic analytical grade.

[0045] Enzymes used: DNA Marker, ampicillin (Amp), kanamycin (Kana) were purchased from Sigma; DNA restriction endonucleases (XbaI, PstI), T4 DNA ligase, TaqDNA polymerase were purchased from TaKaRa Biotechnology (Dalian) Engineering Co., Ltd.; Taq PCR Master Mix was purchased from TaKaRa Biotechnology (Dalian) Engineering Co., Ltd.

[0046] 2 Test methods

[0047] 2.1 Cloning of the ORF region of the DcHsfA2 gene from Carnation

[0048] 2.1.1 Extraction of total RNA from Carnation

[0049] RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) was used to extract total RNA from sterile seedlings of Carnation according to the steps in the kit instructions. DNA in plant materials was removed using DNaseI. The extracted RNA solution was stored at -80°C.

[0050] RNA purity test: OD260 / OD280 ratio is an indicator to measure the degree of protein contamination. Take 2 μl RNA solution and test the OD260 / OD280 ratio of the extracted RNA under a nucleic acid protein detector.

[0051] RNA integrity detection: The integrity was detected by agarose gel electrophoresis (electrophoresis conditions: gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150 V, 15 min).

[0052] 2.1.2 Reverse transcription to obtain the first strand of cDNA

[0053] use RT reagent KitWith gDNAEraser (PerfectReal Time) kit was used to reverse transcribe cDNA to obtain the first strand of cDNA. The reverse transcription product was diluted 10 times and stored at -20°C for future use.

[0054] 2.1.3 PCR amplification and product detection

[0055] The specific primers DcHsfA2 (XbaI, PstI) with restriction sites used in the experiment were designed by Primerpremier5.0 software, and the primers were synthesized and sequenced by Qingdao Qingke Zixi Biotechnology Co., Ltd. (primer list see Table 1). PCR amplification system see Table 2.

[0056] Table 1 Primer sequences

[0057]

[0058]

[0059] Table 2 PCR reaction system

[0060]

[0061] The PCR reaction program was: 94°C pre-denaturation for 4 min→94°C denaturation for 30 sec→61°C annealing for 30 sec→72°C extension for 1 min→35 cycles→72°C extension for 10 min→12°C∞.

[0062] After the PCR reaction, the PCR product was detected by 1% agarose gel electrophoresis. The spotting method was dry spotting, and the dye was EB stain (ethidium bromide). 3.5μl DNA Marker (DL2000, the corresponding sizes of the bands from top to bottom were 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp) and 3μl Londing Buffer were added to the first lane, and 4μl PCR product and 1μl Londing Buffer were added to the detection well, and then electrophoresis was performed and gel imaging was observed.

[0063] 2.2 Bioinformatics analysis of DcHsfA2

[0064] The amino acid sequences with high homology to DcHsfA2 of Carnation were downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the amino acid sequences were aligned using Gene Doc. The physicochemical properties were analyzed using the Prot Param online tool (http: / / web.expasy.org / protpararn / ). The amino acid sequences of DcHsfA2 of Carnation and the HSF family of Arabidopsis thaliana (https: / / www.arabidopsis.org / ) were aligned using clustal X software, and the phylogenetic tree was constructed using the Neighbor-joining method using MEGA5.0 software. The conservation of the DcHsfA2 motif was analyzed online using MEME (http: / / meme-suite.org / tools / meme).

[0065] 2.3D Subcellular Localization of HsfA2

[0066] Homozygous transgenic Arabidopsis lines grown in substrate soil for about two weeks were selected, the root tips were placed on a glass slide, and an appropriate amount of clean water was dripped on it with a pipette, and then a coverslip was covered. The green fluorescent protein signal was observed under 488nm excitation light using laser confocal microscopy (LEICATCS SP5) to analyze the subcellular localization of DcHsfA2.

[0067] 2.4 Analysis of the expression pattern of DcHsfA2 under heat stress

[0068] The sterile carnation seedlings with uniform growth after subculture were subjected to 42℃ high temperature stress treatment, and samples were taken at 1, 2, 4, 8, and 12 hours. The sterile carnation seedlings cultured under normal culture conditions were used as the control group (Control check, ck), and the experimental group and treatment group materials were quickly frozen with liquid nitrogen, marked, and stored at -80℃ for later use.

[0069] RNA was extracted from the treated samples using an RNA extraction kit, and the first strand of cDNA was synthesized using a reverse transcription kit (refer to 2.1.1 and 2.1.2 for specific methods). The synthesized cDNA was used as a template, DcGAPDH was used as an internal reference gene, and DcHsfA2-2 was used as a primer for the target fragment (Table 1). –ΔΔCt The relative expression of DcHsfA2 was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). All data are the average of 3 independent replicates. SAS 8.0 statistical software was used for variance analysis, and P<0.05 (*) and P<0.01 (**) indicated significant differences.

[0070] 2.5 Construction of plant overexpression vector

[0071] 2.5.1 Recovery of target gene fragment and ligation with pMD18-T vector

[0072] For the target bands with the correct size and brightness, use the SanPrep column DNA gel recovery kit and refer to the instructions for gel recovery. Take 1 μl of the recovered product for electrophoresis detection and connect it with pMD18-T according to the reaction system in Table 3. After connecting at 16°C overnight, take out the connected product and store it in a refrigerator at 4°C.

[0073] Table 3 pMD18-T Vector connection system

[0074]

[0075]

[0076] 2.5.2 Transformation of E. coli and identification of recombinant clones

[0077] The ligation product was heat-shocked and transformed into E. coli DH5α, inverted at 37°C, and cultured overnight for 12-14 hours. Colony PCR detection was performed, the system was shown in Table 4, and the PCR detection primers were M13 F and M13 R (primer sequences were shown in Table 1).

[0078] Table 4 RT-PCR reaction system

[0079]

[0080] 2.5.3 Sequence alignment and plasmid extraction

[0081] The positive clones were selected and sent to Qingdao Qingke Zixi Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed and compared with the known reference sequences in the genome using DNAMAN software to obtain successful clone samples.

[0082] Take 100 μl of the successfully cloned bacterial solution and put it into 15-20 ml LB liquid culture medium containing Amp for large-scale shaking. Take 100 μl of pCAMBIA1300-super and put it into a conical flask containing 15-20 ml LB liquid culture medium containing Kana for large-scale shaking.

[0083] The plasmids of the correctly sequenced bacterial solution and pCAMBIA1300-super were extracted using the SanPrep column plasmid DNA small-scale extraction kit according to the instructions. The extracted plasmids were tested by electrophoresis, and the obvious and correct bands were recovered by gel recovery using the SanPrep column DNA gel recovery kit.

[0084] 2.5.4 Ligation of target plasmid and pCAMBIA1300

[0085] The target gene and expression vector pCAMBIA1300 were digested by double enzyme digestion at 37°C for 5 h. The enzyme digestion system is shown in Table 5.

[0086] Table 5 Enzyme digestion system

[0087]

[0088]

[0089] The digested product was recovered by gel extraction. The gel extraction product and pCAMBIA1300-super were ligated with T4 DNA Ligase at 16°C overnight. The ligation system is shown in Table 6.

[0090] Table 6 Connection system

[0091]

[0092] The ligation product was transformed into competent E. coli, and a single colony was selected for bacterial solution PCR. The PCR reaction system is shown in Table 7. The bacterial solution with the correct band size was selected for sequencing. The bacterial solution with successful sequencing was propagated by shaking and then the plasmid was extracted.

[0093] Table 7 Bacterial liquid PCR system

[0094]

[0095] 2.5.5 Agrobacterium transformation and positive bacteria detection

[0096] In a sterile environment, take 50μl of GV1301 Agrobacterium and add 1μl of plasmid DNA, stir the bottom of the tube to mix, place on ice for 5min, freeze in liquid nitrogen for 5min, bath in 37℃ water for 5min, and bath in ice for 5min. Then add 350μl of LB liquid culture medium and culture at 28℃ for 2h. Centrifuge at 6000rpm for 1min, take 100μl of supernatant and mix by blowing, take 50μl of transformation liquid and evenly apply it on the LB plate containing Kana, and culture at 28℃ for 2-3d.

[0097] Pick 15 independent colonies of appropriate size, transfer to plates and perform PCR detection according to the above steps. The detection system is shown in Table 7. The PCR reaction program is: 94℃ pre-denaturation for 4min→94℃ denaturation for 30sec→61℃ annealing for 30sec→72℃ extension for 1min→35 cycles→72℃ extension→5min→12℃∞. Select strains with brighter bands and appropriate size and shake them in large quantities. Mix the bacterial solution with glycerol in a 1:1 ratio and store at -80℃.

[0098] 2.6 Functional verification of DcHsfA2 in Arabidopsis

[0099] 2.6.1 Heterologous transformation of Arabidopsis thaliana and screening of homozygous lines

[0100] Arabidopsis culture: Vermiculite and perlite are mixed in the matrix soil and sterilized at 121℃ for 30min. Soak the sterilized matrix soil with water and divide it into small pots for later use. Sow wild-type (WT) Arabidopsis in the sterilized matrix soil with a toothpick and cover it with film (spray an appropriate amount of water on the inside of the plastic wrap to keep it moist). Use a toothpick to make holes in the film for ventilation. When the seeds grow into seedlings, remove the film and water them every two days.

[0101] Heterologous transformation of Arabidopsis thaliana: The inflorescence dip method was used to transform Arabidopsis thaliana (Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal, 1998, 16(6): 735-743).

[0102] Disinfection of Arabidopsis seeds: Take the harvested T0 generation Arabidopsis seeds and place them in a 2.0 ml centrifuge tube containing 8% sodium hypochlorite for sterilization for 4 minutes, and shake them upside down during this period. After pouring out the sodium hypochlorite, add 95% ethanol to the centrifuge tube, sterilize for 1 minute and repeat 3 times. After pouring out the ethanol, add 1 ml of double distilled water to wash, and repeat 3 times.

[0103] Screening of single-copy strains: Transgenic Arabidopsis was sown in MS solid medium containing 30 μg / ml Hyg, and Arabidopsis seedlings that could germinate and grow normally were transplanted into matrix soil for culture. Arabidopsis strains screened to the T2 generation were sown in MS solid medium, 30 seeds were sown in each culture dish, and each strain was repeated in 4 dishes, a total of 120 seeds were cultured and WT was used as a control. After the seeds germinated, a chi-square test was performed, and those that met the 3:1 ratio were considered to be T2 generation strains with single copy insertions. Each group of experiments was repeated 2-3 times.

[0104] Screening of homozygous lines: After sterilizing the T3 seeds, sow them in MS solid medium containing 30 μg / ml Hyg, and count the number of positive seedlings in each line. Lines with 100% positive results are considered homozygous lines. Each group of experiments is repeated 2-3 times.

[0105] 2.6.2 Detection of relative expression levels of transgenic Arabidopsis

[0106] Total RNA of transgenic Arabidopsis was extracted and reverse transcribed into cDNA (the same method as above). The expression level of DcHsfA2 gene in Arabidopsis was detected by semi-quantitative RT-PCR method, and AtActin was used as an internal reference gene for leveling. The primer list is shown in Table 1.

[0107] 2.6.3DcHsfA2 heat resistance functional verification

[0108] Study on the heat tolerance of sterile seedlings of DcHsfA2 transgenic Arabidopsis: Columbia wild-type (WT) Arabidopsis and pCAMBIA1300 empty-type Arabidopsis (VC) were used as the control group (ck) and T3 generation homozygous transgenic Arabidopsis seeds were sterilized, sown in MS medium, inverted in a 4°C refrigerator for 3 days, and then placed in a light incubator for culture. The culture conditions were: light intensity of 120 μmol·m -2 ·s -1 , photoperiod 16 / 8h (light / dark), temperature 22℃. After 5 days, they were placed at 45℃ for 2h, and the survival rate was counted after 7 days of recovery. Each strain was repeated 3-6 dishes, and 20-30 seeds were sown in each dish for each strain. Each group of experiments was repeated 3 times.

[0109] Study on heat tolerance of DcHsfA2 transgenic Arabidopsis potted seedlings: Transgenic Arabidopsis and ck group Arabidopsis that had grown normally for about 10 days in MS medium were transplanted into substrate soil. After 2 weeks, they were subjected to 45℃ high temperature treatment for 2 hours. After 7 days of recovery, they were photographed and the phenotypes were observed. Each strain was replicated 10 times, and each group of experiments was repeated 3 times.

[0110] Determination of physiological indicators of transgenic Arabidopsis under heat stress: Seedlings (transgenic lines and ck group lines) grown in nutrient soil for about 3 weeks were transferred to 42°C for high temperature treatment for 6 hours and then the relevant physiological indicators were measured. Malondialdehyde (MDA) was measured according to Heath and Packer (Heath RL, Packer L. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 1968, 125 (1): 189-198.). Relative conductivity (Electrolyte leakage) was measured according to Sairam (Sairam RK, Srivastava GC. Changes inantioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress [J]. Plant Sci, 2002, 162 (6): 897-904.) method. The superoxide dismutase (SOD) assay was performed according to Giannopolitis (Giannopolitis CN, Ries SK. Superoxide dismutases: I. Occurrence in higher plants [J]. Plant Physiol, 1977, 59 (2): 309-314.) and the peroxidase (POD) assay was performed according to Maehly and Chance (Maehly AC, Chance B. The assay of catalases and peroxidases. Methods Biochem. Anal. 1954, 1: 357-424.).ROS chemical tissue staining: ROS chemical tissue staining was performed according to Fukao's method (Fukao T, Yeung E, Bailey-Serres J. The submergence tolerance regulator SUB1A mediates crosstalk between submergence and drought tolerance in rice [J]. Plant Cell, 2011, 23: 412-427.). ·- Nitroblue tetrazolium (NBT) staining was used for staining, and 3,3'-diaminobenzidine tetrahydrochloride (DAB) staining was used for H2O2 staining. The plants were photographed under a Stemi DV4 (Carl Zeiss, Gottingen, Germany) stereo microscope.

[0111] Analysis of endogenous gene expression in DcHsfA2 transgenic Arabidopsis under heat stress: 3-week-old transgenic Arabidopsis seedlings and ck group Arabidopsis seedlings were subjected to heat stress (42°C, 6h) at the same time, and total RNA was extracted from the plants. Real-time fluorescence quantitative PCR (qRT-PCR) was used to determine the relative expression of Arabidopsis-related endogenous genes under abiotic stress using AtActin as an internal reference gene. The primer sequences of Arabidopsis-related endogenous genes are shown in Table 1.

[0112] 3 Results and analysis

[0113] 3.1 Cloning and sequence analysis of the ORF region of the DcHsfA2 gene

[0114] 3.1.1 Quality detection of total RNA from Carnation

[0115] The results of electrophoresis of RNA extraction products are shown in Figure 1 The 18S and 28S bands of RNA detected by electrophoresis were clear and without tails, and the brightness of 28S was about 2 times that of 18S, indicating that the extracted RNA was relatively complete. The OD260 / OD280 was 2.1 detected by UV spectrophotometer, indicating that the purity was good and could be used for subsequent experimental operations.

[0116] 3.1.2 Cloning and analysis of the ORF region of the DcHsfA2 gene

[0117] According to the DcHsfA2 fragment in the database genome, forward and reverse primers DcHsfA2-F (XbaI) and DcHsfA2-R (PstI) containing restriction sites were designed. PCR amplification was performed using the first strand of cDNA as a template. Electrophoresis gel detection showed that the PCR product band was a bright band of 1080 bp, which was consistent with the expected result. The results are shown in Figure 2 .

[0118] 3.1.3DcHsfA2 bioinformatics analysis

[0119] The invention separates a heat-related gene from Carnation 'Imagine'. The target fragment is connected to the pMD18-T vector and sequenced. The ORF region is 1080 bp (shown in SEQ ID NO: 1), encoding 359 amino acids (shown in SEQ ID NO: 2). The gene is named DcHsfA2, and the protein encoded by the gene is named DcHsfA2. The protein belongs to the Hsfs family member. The physical and chemical property analysis shows that the isoelectric point (Theoretical, pI) is 5.05, the molecular weight (Molecular weight) is 40.5538KDa, the average value of the total average hydrophilicity (Gravy) is -0.548, and the N-terminus of the sequence is methionine (Met).

[0120] Phylogenetic analysis showed that DcHsfA2 belongs to the A2 HSFs family and has the closest genetic distance to Arabidopsis AtHsfA2, indicating that DcHsfA2 has the highest homology with AtHsfA2 ( Figure 3 A). DcHsfA2 was analyzed for conserved motifs with the other 21 HSF amino acid sequences of Arabidopsis, among which the DBD domain contained Motif2, Motif4, and Motif 1, the OD domain contained Motif5 and Motif3, and the NES contained Motif 8. Neither DcHsfA2 nor AtHsfA2 contained Motif 6 and Motif7, indicating that DcHsfA2 and AtHsfA2 had the highest homology, and the results were consistent with the phylogenetic tree analysis ( Figure 3 (middle B).

[0121] The amino acid sequences with high homology to DcHsfA2 were downloaded from NCBI, namely AtHsfA2 (NM-001124916), SIHsfA2a (XP_025888472), OsHsfA2a (NP-001044160), and OsHsfA2b (NP-001059028). Gene Doc was used to perform amino acid multiple sequence alignment ( Figure 4). Analysis shows that DcHsfA2 contains five typical domains of the HsfA family, namely the relatively conservative DBD domain, HR-A / B with two adjacent hydrophobic seven-fold repeat sequences and rich in leucine, and NLS, AHA and NES. Among them, the 42nd to 237th amino acids represent the DBD domain, the 148th to 180th and 201st to 212th amino acids represent two hydrophobic seven-peptide repeat regions: HR-A and HR-B, respectively, the 228th to 242nd amino acids represent the NLS domain, the ETLLSATLDN and GNWGEDVED sequences are the AHA1 motif and the AHA2 motif, respectively, and the 348th to 355th amino acids represent the NES.

[0122] 3.2 Analysis of basic characteristics of DcHsfA2

[0123] 3.2.1 Subcellular localization of DcHsfA2

[0124] The root tips of homozygous Arabidopsis thaliana of the T3 generation grown in substrate soil for about two weeks were selected and the subcellular localization of DcHsfA2 was observed using laser confocal microscopy. Figure 5 As shown in the picture, it can be seen that GFP is located in the cell nucleus of the root tip of Arabidopsis thaliana, so DcHsfA2 is a nuclear localized protein.

[0125] 3.2.2 Expression pattern of DcHsfA2 under heat stress

[0126] After 1-4 h of heat stress, the relative expression of DcHsfA2 was upregulated by about 200 to 300 times compared with the ck group, and the relative expression of the gene decreased after 8 h of stress ( Figure 6 ). This suggests that DcHsfA2 plays an important role in plant response to heat stress.

[0127] 3.3 Overexpression vector construction

[0128] The cloned ORF region of DcHsfA2 was double-digested with XbaI and PstI enzymes and then ligated with the vector pCAMBIA1300-super, heat-shocked, and colony PCR was performed. The results were as follows: Figure 7 Most of the colony detection bands were bright and consistent with expectations, and the plasmid control was accurate, indicating that the plant overexpression vector was successfully constructed.

[0129] 3.4 Functional verification of DcHsfA2 overexpression in Arabidopsis

[0130] 3.4.1 Screening of homozygous transgenic Arabidopsis lines

[0131] The homozygous transgenic Arabidopsis lines were screened and the relative expression of the DcHsfA2 gene was analyzed. Figure 8As shown. After amplification, the transgenic lines OE3, OE4, and OE5 obtained specific bands, while the wild-type Arabidopsis (WT) and the empty vector plant (VC) did not show corresponding bands. Therefore, OE3, OE4, and OE5 were selected for the next experiment.

[0132] 3.4.2DcHsfA2 improves the heat tolerance of transgenic lines

[0133] 3.4.2.1DcHsfA2 improves the heat tolerance of sterile transgenic Arabidopsis seedlings

[0134] The growth of ck group Arabidopsis and overexpressed Arabidopsis after about 5 days of growth was analyzed. Under normal conditions, the growth was good ( Fig. 9 After 7 days of high temperature stress and recovery, although the survival rates of ck group Arabidopsis and overexpression Arabidopsis were reduced, the survival rate of overexpression Arabidopsis was higher than that of ck group ( Fig. 9 (B), indicating that DcHsfA2 improves the heat tolerance of transgenic Arabidopsis sterile seedlings.

[0135] 3.4.2.2DcHsfA2 increases the heat tolerance of transgenic Arabidopsis seedlings

[0136] Analysis of heat tolerance of transgenic Arabidopsis thaliana potted seedlings ( Fig.10 ), the growth conditions of the ck group and the transgenic lines under normal conditions were good and consistent. After 2h of heat stress at 45℃, the growth condition of the ck group was poorer than that of the transgenic Arabidopsis ( Fig.10 The survival rates of OE3, OE4, and OE5 were all above 80%, while those of WT and VC strains were all below 60% ( Fig.10 In B), the survival rate of the transgenic lines was significantly higher than that of the control group. From the above experimental results, it can be seen that overexpression of DcHsfA2 in Arabidopsis increases the heat resistance of Arabidopsis potted seedlings.

[0137] 3.4.2.3 Physiological index determination of transgenic Arabidopsis thaliana under heat stress

[0138] To further explore the physiological mechanism of DcHsfA2 in improving the heat tolerance of transgenic Arabidopsis, the relevant physiological indicators of Arabidopsis were measured before and after heat stress. Fig.11 The malondialdehyde (MDA) content of the plants was analyzed. After heat stress, the accumulation of MDA in DcHsfA2 overexpressing plants (OE3, OE4, OE5) was significantly reduced compared with that in WT plants ( Fig.11A), indicating that the cell membrane of transgenic Arabidopsis thaliana showed lower damage degree after heat stress than that of ck group Arabidopsis thaliana, and had higher heat resistance. The relative conductivity of the plants was measured, and the results showed that the relative conductivity of Arabidopsis thaliana increased after heat stress compared with normal conditions, but the accumulation of relative conductivity of the three transgenic lines was significantly lower than that of the control group ( Fig.11 The superoxide dismutase (SOD) and peroxidase (POD) levels of the plants were measured. The results showed that under normal conditions, there was no significant difference in the content of POD and SOD between the OE3, OE4 and OE5 lines and the control line. After heat stress, the content of SOD and POD in the three transgenic lines increased significantly ( Fig.11 Middle C, Fig.11 In conclusion, under heat stress, transgenic Arabidopsis has stronger heat tolerance than wild-type Arabidopsis, so DcHsfA2 increases the heat tolerance of transgenic Arabidopsis.

[0139] 3.4.2.4 ROS chemical tissue staining of transgenic Arabidopsis

[0140] Reactive oxygen is an important component of the plant response to abiotic stress signals. The leaves of transgenic Arabidopsis and control Arabidopsis were stained with NBT and DAB 6 hours after heat stress to observe the superoxide anion (O2 ·- ) and H2O2 accumulation in wild-type, empty vector and overexpression Arabidopsis lines, such as Fig.12 As shown. Under normal conditions, almost no dye deposits were found in the leaves of the three transgenic lines and the control line. After heat stress, all plants showed dyeing, but the content of deposits in the three transgenic lines was significantly less than that in the control line. The above results show that the transgenic lines overexpressing DcHsfA2 can scavenge reactive oxygen species better than the WT and VC group lines.

[0141] 3.4.2.5 Expression analysis of heat-related endogenous genes in transgenic Arabidopsis

[0142] Hsfs, as the terminal component of signal transduction, can induce the expression of Hsp. The expression levels of heat-related endogenous genes AtHsp18.2, AtHsp70 and AtHsp90 in transgenic Arabidopsis were analyzed by qRT-PCR. Fig.13As shown. After heat stress, the relative expression levels of AtHsp18.2 and AtHsp70 were significantly upregulated, and the relative expression levels of AtHsp18.2 and AtHsp70 in the three transgenic lines before and after heat stress were significantly higher than those in WT. The relative expression level of AtHsp90 under normal conditions was almost the same in all lines, but after heat stress, the relative expression level of AtHsp90 in the three transgenic lines increased significantly and was significantly higher than that in WT. This indicates that the overexpression of DcHsfA2 may regulate some heat shock protein genes to work together.

[0143] Abbreviation table

[0144]

[0145]

[0146] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions may occur depending on design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A protein, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO:

2.

2. The gene encoding the protein according to claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

1.

3. An expression cassette, recombinant expression vector or recombinant bacteria containing the coding gene according to claim 2.

4. A method for preparing a transgenic plant, comprising the following steps: introducing the coding gene described in claim 2 into a starting plant to obtain a transgenic plant; compared with the starting plant, the heat resistance of the transgenic plant is improved.

5. The method for preparing a transgenic plant according to claim 4, characterized in that: The coding gene is introduced via a recombinant expression vector, which is obtained by inserting the coding gene into the multiple cloning site of the expression vector pCAMBIA1300.

6. The method for preparing a transgenic plant according to claim 4, characterized in that: The improved heat resistance of the transgenic plants is manifested as follows: after heat stress, compared with the starting plants, the accumulation of malondialdehyde in the transgenic plants is reduced, the accumulation of relative conductivity is reduced, the superoxide dismutase content is increased, the peroxidase content is increased, the active oxygen content is reduced, the survival rate is increased, and / or the relative expression of heat-related endogenous genes is increased.

7. The method for preparing a transgenic plant according to any one of claims 4 to 6, characterized in that: The plant is Arabidopsis thaliana.

8. Use of the protein according to claim 1 in improving the heat resistance of plants.

9. Use of the coding gene according to claim 2 in improving the heat resistance of plants.

10. The use according to claim 8 or 9, characterized in that: The plant is Arabidopsis thaliana.

Images