Application of antheraea assamensis hsf20 gene in improving high temperature stress tolerance

Abstract

This invention belongs to the field of molecular biology technology, specifically relating to anthuriums. AaHSF20 Application of genes in improving heat stress tolerance. This invention provides, for the first time, the application of genes in improving heat stress tolerance in anthuriums. AaHSF20 A gene, the nucleotide sequence of which is shown in SEQ ID NO.1. Furthermore, this invention also provides [a related invention]. AaHSF20 Gene-related biomaterials and the AaHSF20 protein. Results showed that... (The text abruptly ends here, likely due to anthurium orientalis). AaHSF20 Genes, biomaterials, and the AaHSF20 protein can be used in plants to improve their tolerance to high-temperature stress, cultivate heat-resistant plant germplasm, and construct heat-intolerant plant models. Therefore, the technical solution of this invention can be used to reveal the molecular mechanism of heat tolerance in Anthurium, develop high-temperature stress mitigation technologies, and provide key gene resources and theoretical support for the molecular improvement of heat tolerance in Anthurium and precision breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to anthuriums. AaHSF20 Application of genes in improving tolerance to high temperature stress. Background Technology

[0002] Anthurium ( Anthurium andraeanum Anthurium is a perennial evergreen tropical ornamental flower belonging to the genus Anthurium in the family Araceae. It is also known as flamingo flower or flamingo flower. It originated in the tropical rainforests of Central and South America. With its advantages such as long flowering period, rich flower colors, and good storage and transportation resistance, it has become the world's second largest tropical commercial flower. It is widely used in high-end floral art and interior decoration, and the potted plant and cut flower market is huge.

[0003] Anthuriums thrive in warm, humid, and semi-shaded environments with diffused light, with an optimal growth temperature of 20-28℃. They are intolerant of strong sunlight and drought. Against the backdrop of global warming and frequent extreme summer heat, anthurium production in greenhouse cultivation in my country has been severely impacted. Temperatures above 35℃ can cause anthuriums to lose flower color, experience stunted growth, wither and wrinkle leaves and bracts, and even collapse, sometimes resulting in the death of the entire plant. This not only reduces the yield and ornamental quality of anthuriums but also significantly increases planting and production costs.

[0004] Current research has revealed that plants can resist high-temperature stress and enhance their heat tolerance through a series of physiological and biochemical reactions, cell signal transduction, and gene regulation mechanisms. Transcription factors are core components regulating plant high-temperature stress responses. Heat shock transcription factors (HSFs) are key heat-response transcription factors in plants, specifically binding to the heat shock elements of target gene promoters, activating the expression of heat shock proteins and downstream stress-resistance genes, maintaining cellular protein homeostasis, and enhancing plant heat tolerance. Typical plant HSF families contain multiple conserved domains such as DBD, OD, and NLS, and can be divided into three classes based on structural characteristics: HSFA, HSFB, and HSFC. The HSFA subfamily possesses transcriptional activation functions and is a key member regulating plant responses to abiotic stresses. Currently, the HSF gene family has been systematically identified in Arabidopsis thaliana, rice, maize, and various ornamental plants such as peony and chrysanthemum. Numerous studies have confirmed that HSFA genes play a central role in regulating plant tolerance to high temperatures and oxidative stress. However, the high-temperature response regulatory mechanisms of members of the Anthurium HSF gene family have not yet been systematically reported. Summary of the Invention

[0005] The purpose of this invention is to provide anthuriums AaHSF20 The application of genes in improving tolerance to high temperature stress provides scientific guidance for developing technologies to alleviate high temperature stress in Anthurium and creating heat-resistant Anthurium germplasm.

[0006] This invention provides red anthurium AaHSF20 Genes, the red anthurium AaHSF20The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] This invention provides the aforementioned anthurium AaHSF20 The gene encodes the AaHSF20 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] This invention provides anthuriums containing the above-described technical solution. AaHSF20 Gene-derived biological materials, wherein the biological materials comprise at least one of the following (1)-(3): (1) Expression vector super1300-AaHSF20-cGFP; (2) Silencing vector TRV2-AaHSF20; (3) A silencing system containing the silencing carrier TRV2-AaHSF20 described in (2).

[0009] Preferably, the silencing system is prepared by mixing Agrobacterium tumefaciens containing TRV1 and Agrobacterium tumefaciens containing the silencing carrier TRV2-AaHSF20 (2) at a volume ratio of 1:1.

[0010] This invention provides the anthurium described in the above technical solution. AaHSF20 Application of the gene or the expression vector super1300-AaHSF20-cGFP in Arabidopsis thaliana; The application is: overexpression of Anthurium. AaHSF20 After gene modification, improve the high temperature stress tolerance of Arabidopsis thaliana and / or cultivate high temperature tolerant Arabidopsis thaliana germplasm.

[0011] This invention provides the anthurium described in the above technical solution. AaHSF20 Application of the gene or the silencing vector TRV2-AaHSF20 or the silencing system in Anthurium; The application is: Silent Red Palm AaHSF20 After gene sequencing, a heat-intolerant Anthurium plant model was constructed.

[0012] This invention provides a method for constructing a heat-intolerant Anthurium plant model, comprising the following steps: Plants were infected using the silencing system described in the above technical solution and then cultured to construct a heat-intolerant plant model.

[0013] This invention provides a method for improving the tolerance of plants to high temperature stress, comprising the following steps: The red palm described in the above technical solution AaHSF20 Genes were introduced into target plants to improve their tolerance to high-temperature stress. The plant in question is Arabidopsis thaliana.

[0014] Preferably, the plant's tolerance to high temperature stress is improved by increasing the activity of antioxidant enzymes in the target plant; The antioxidant enzyme activity includes one or more of the following: superoxide dismutase activity, peroxidase activity, and catalase activity.

[0015] Beneficial effects: This invention provides for the first time information related to the heat resistance of anthuriums. AaHSF20 A gene, the nucleotide sequence of which is shown in SEQ ID NO.1. Based on this, the present invention provides... AaHSF20 The gene-encoded AaHSF20 protein, and AaHSF20 Gene-related biomaterials can be used in plants. By enhancing the properties of plants... AaHSF20 Increasing gene expression or the content of AaHSF20 protein can not only improve plant tolerance to high-temperature stress but also cultivate heat-resistant plant germplasm. This can be achieved by increasing the expression level of the gene containing Anthurium. AaHSF20 By infecting plants with a gene silencing system and then culturing them, heat-intolerant plant models can be obtained. Therefore, the technical solution of this invention can not only cultivate heat-tolerant germplasm, but also fully reveal the molecular mechanism of heat tolerance in Anthurium andraeanum and develop technologies to alleviate high-temperature stress through plant models. This provides key gene resources and theoretical support for the molecular improvement of heat tolerance in Anthurium and precision breeding. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The present invention provides the treatment of different high-temperature treatment times in the bracts of Anthurium. AaHSF20 Changes in gene expression levels; Figure 2 The VIGS silence provided by this invention AaHSF20 The influence of genes on the circular phenotype of Anthurium bracts; Figure 3 The VIGS silence provided by this invention AaHSF20 The silencing efficiency and its effects on the physiological and biochemical indicators of anthurium bracts (where a is the silencing efficiency verified by qRT-PCR; b is SOD activity; c is POD activity; d is CAT activity; e is MDA content; f is anthocyanin content). Figure 4 The control group (WT) and the control group provided by the present invention AaHSF20Phenotypic comparison of Arabidopsis thaliana plants overexpressing (OE) under normal temperature (RT) and high temperature (HT) stress conditions; Figure 5 The high temperature stress effect on wild-type (WT) and AaHSF20 Overexpression (OE) in Arabidopsis seedlings AaHSF20 Effects of relative expression levels and antioxidant enzyme activity (where a is the result of RT-qPCR detection 1 h after treatment). AaHSF20 Relative expression level; b is superoxide dismutase (SOD) measured after 3 h of treatment; c is peroxidase (POD) measured after 3 h of treatment; d is catalase (CAT) activity measured after 3 h of treatment. Detailed Implementation

[0018] In this invention, unless otherwise specified, the equipment, reagents and methods used are conventionally selected.

[0019] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1 1. Materials and Methods 1.1 Plant materials and growth conditions The 'Pink Crown' varietal of Anthurium was used as the experimental material. The 'Pink Crown' varietal was planted in soil and cultured in a 25℃ incubator (12h light / 12h dark).

[0021] 1.2 Gene Cloning RNA was extracted from FHG bracts (i.e., bracts of the Anthurium 'Pink Crown' variety) after 6 hours of high-temperature treatment using the CTAB+LiCl precipitation method. Total RNA was then reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (catalog number R212, Nanjing Novizan Biotechnology Co., Ltd.). Primers were designed using the genomic reference sequence of the Anthurium 'Alabama' variety. The genomic reference sequence for the Anthurium 'Alabama' variety was obtained from the National Gene Bank Life Big Data Platform (https: / / www.cngb.org / ) and the Anthurium Genome Database (code: CNP0006988).

[0022] Using the designed primers (SEQ ID NO.3 and SEQ ID NO.4), the complete coding sequence of AaHSF20 was cloned, specifically including: AaHSF20-CDS-F(SEQ ID NO.3):5’-ATGGAAGGCAGTGTAATGGTTGC-3’; AaHSF20-CDS-R(SEQ ID NO.4):5’-TCAAGGCTCCCCGCCC-3’; AaHSF20 The amino acid sequence of AaHSF20 protein is as SEQ ID As shown in NO.2, specifically: MAAAGEEMEGSVMVAKEEVEAREAVPLPPQSPRPMPGLNEAGPPPFLTKTFEMVEDPATDAVVSWSSGRNSFVVWDAHAFATTLLRKHFKHGNFSSFIRQLNTYGFRKVDPDRWEFANEAFLGGQKHLLKHIRRRRNPPQHAQQQQRAGEAACLELGQFGSLETDVERLRRDRSVLLH EIVKLRHQQEGSRAQLAAMEERLQGTERKQRQMMAFLARALENPSFFCQLVHRSGEQRKQLEGGPSRKRRLTANPSSEDLSGVEEGVVTVGAEMETLLTALDGEVGTSSWDQGGEAETLLNSGDHPNLGAVTDVLWEEILREDLLGGSSAEDGEVSEVDVEELAARPYEWGEDVEDLVEQMVFLGGEP*.

[0023] 1.3 VIGS momentary silence of the round bracts of Anthurium Using restriction endonucleases EcoR I and Bam The tobacco mosaic virus TRV2 plasmid was digested with HI double enzymes and then cloned. AaHSF20 Using the cDNA obtained after reverse transcription of the required gene CDS sequence as a template, primers were designed based on the genomic reference sequence of the 'Alabama' cultivar, and the desired target fragment was cloned. The primer sequences are as follows: AaHSF20-TRV-F (SEQ ID NO.5): 5'-CTGTGAGTAAGGTTACCGAATTCGCAGCAGCAGGGAAATAGGAAG-3'; AaHSF20-TRV-R (SEQ ID NO. 6): 5'-GTGAGCTCGGTACCGGATCCCACCTCCTCCTTCGCAACCA-3'.

[0024] The TRV2-AaHSF20 construct was obtained by ligation using the homologous recombinase ClonExpressII One Step Cloning Kit (catalog number C112, Nanjing Novizan Biotechnology Co., Ltd.) and used for VIGS experiments. The helper vector TRV1, empty vector TRV2, and recombinant vector TRV2-AaHSF were transformed into *Agrobacterium tumefaciens* GV3101 competent cells, respectively. Positive single colonies were picked and inoculated into LB broth containing the corresponding antibiotics and cultured with shaking at 28°C. The bacterial pellet was collected by centrifugation and resuspended in infection buffer (1M MgCl2, 1M MES, 0.05M acetylsyleugenone) to OD200. 600 =2.0. Agrobacterium resuspensions containing equal volume ratios (1:1) TRV1 and TRV2, and TRV1 and TRV2-AaHSF20 were mixed separately and co-cultured at 22°C in the dark for 3 hours before being used to infect the circular bracts of Anthurium.

[0025] Take bracts from Anthurium during its peak flowering period, cut circular pieces using a 1cm diameter punch, mix thoroughly, and randomly divide into two portions. Immerse each circular piece completely in Agrobacterium infection solution that has been treated in the dark for 3 hours, and place it in a vacuum pump. Increase the system pressure to the maximum, maintain for 6 minutes, then slowly release the pressure (for approximately 10 minutes). Repeat this cycle twice. After infection, rinse the circular pieces three times with deionized water and pre-culture at 16℃ in the dark for 2 days. Then transfer them to a photoperiod of 25℃ (12h light) / 22℃ (12h dark) for 1 day. Finally, heat-treat the circular pieces at 42℃ for 10 hours to assess their heat resistance. Samples were taken at 42℃ for 30 minutes for RT-qPCR to detect VIGS efficiency, samples were taken at 42℃ for 3 hours for SOD, POD, CAT, and MDA activities, and samples were taken at 42℃ for 6 hours for anthocyanin content determination.

[0026] 1.4 Arabidopsis heterologous overexpression AaHSF20 Using restriction endonucleases Kpn I and Xba The super1300-cGFP plasmid was double-digested with enzyme I and then mixed with the cloned... AaHSF20 The gene CDS fragment was ligated using the homologous recombinase ClonExpressII One Step Cloning Kit (catalog number C112, Nanjing Novizan Biotechnology Co., Ltd.) to obtain the super1300-AaHSF20-cGFP construct, which was used for heterologous overexpression experiments in Arabidopsis thaliana.

[0027] This experiment employed the Agrobacterium-mediated floral dip method for genetic transformation of Arabidopsis thaliana. Agrobacterium strain GV3101 containing the target gene was cultured in LB broth containing the appropriate antibiotic at 28°C with shaking. Subsequently, the bacterial cells were collected by centrifugation at 4200 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in the infection buffer (MS, sucrose, and 0.05% Silwet L-77). The bacterial concentration was adjusted to OD0.05. 600 =0.8.

[0028] Arabidopsis plants in good growth condition and about 5 weeks into bolting were selected and thoroughly watered the day before transformation. On the day of transformation, mature flowers and pods that had opened were removed. The inflorescence of the plant was inverted and immersed in the Agrobacterium-containing solution for 2 minutes. The plant was then removed, covered with plastic wrap to maintain humidity, and cultured in the dark for 24 hours. Afterward, the plastic wrap was removed, and the plant was cultured under normal long-day conditions (16 hours light / 8 hours dark). Once the plants matured, T0 generation seeds were collected and dried for one week. Positive transgenic plants were then screened using 25 mg / L hygromycin. Seeds from the selected T1 generation plants were harvested individually and further screened on 1 / 2 MS medium containing 40 mg / L hygromycin to obtain T2 generation plants. The same method was used to screen up to the T3 generation to obtain stable homozygous transgenic plants.

[0029] The received T3 generation transgenic positive seeds were sterilized and sown on 1 / 2 MS medium. After vernalization at 4°C for 3 days, 4-week-old Arabidopsis seedlings were subjected to high-temperature treatment at 40°C (16h light) / 32°C (8h darkness) for 2 days. Phenotypic changes were then observed. Furthermore, samples were taken at 1h and 3h after high-temperature treatment. The former was used for RT-qPCR to detect the expression levels of relevant genes, and the latter for measuring the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT).

[0030] 1.5 RT-qPCR Detection Method Real-time quantitative PCR (RT-qPCR) amplification using SYBR Green I dye was performed using the ChamQ Blue Universal SYBR qPCR Master Mix kit (catalog number Q312, Nanjing Novizan Biotechnology Co., Ltd.). 2 -ΔΔCT The method calculates the relative expression level of the target gene.

[0031] 1.6 Assay of Superoxide Dismutase (SOD) Activity SOD activity was determined using the nitroblue tetrazolium (NBT) photoreduction method. 0.1 g of anthurium bract tissue was accurately weighed, and 1.8 mL of pre-cooled 0.05 mol / L phosphate buffer (pH 7.0) was added. The mixture was ground on ice and extracted for 2 hours. After centrifugation at 10000 g for 15 min at 4 °C, the supernatant was collected as the enzyme solution.

[0032] The assay was performed in 96-well plates, with a total reaction volume of 200 μL per well, specifically containing 150 μL phosphate buffer (pH 7.8), 30 μL 130 mmol / L Met, 30 μL 750 μmol / L NBT, 30 μL 100 μmol / L EDTA-Na2, 30 μL 20 μmol / L riboflavin, 50 μL enzyme solution, and 50 μL distilled water. Phosphate buffer was used as a control instead of enzyme solution. After reacting under 4000 lx light for 20 min, the absorbance was immediately measured at 560 nm. One unit of SOD activity (U) is expressed as the inhibition of 50% of NBT photoreduction. SOD enzyme activity was calculated using the following formula: SOD (U / g) = [(A ck A E [1 / 2 × A] / (1 / 2 × A) ck ×m×V t ) Among them, A ck A represents the absorbance of the control tube. E V is the absorbance of the sample tube, V is the total volume of the sample solution (mL), and m is the fresh weight of the sample (g). t The amount of enzyme solution used during the assay is in mL.

[0033] 1.7 Peroxidase (POD) Activity Assay POD activity was determined using the guaiacol method. The enzyme extraction method was the same as that used in the SOD activity determination section 1.5 above.

[0034] The reaction system consisted of 100 μL of 2% H₂O₂, 100 μL of 0.05 mol / L guaiacol solution, and 20 μL of enzyme solution. Phosphate buffer (pH 7.0) was used as a control. After adding the enzyme solution, the reaction was allowed to proceed at room temperature for 1 min. The reaction was then terminated by rapidly adding 50 μL of 1 mol / L H₂SO₄, and the absorbance was immediately measured at 470 nm. Enzyme activity units (U) were defined as the amount of enzyme produced per minute. A 470 A change of 0.01 represents one unit. The specific calculation formula is as follows: POD(U / g) = ( A 470 ×V t ) / (m×V s(×0.01×t) in, A 470 V represents the change in absorbance during the reaction time. t To determine the total volume of the system (mL), m is the fresh weight of the sample (g), and V s t represents the amount of enzyme solution used during the assay (mL), and t represents the reaction time (min).

[0035] 1.8 Determination of malondialdehyde (MDA) content This study used the thiobarbituric acid (TBA) method to determine the malondialdehyde (MDA) content in samples. Approximately 0.1 g of anthurium bract tissue was accurately weighed, and 1.6 ml of 5% TCA was added. The mixture was thoroughly ground and homogenized. The homogenate was centrifuged at 4000 rpm for 10 min at 4℃. 500 μL of the supernatant was collected, and an equal volume of 0.67% TBA was added. After mixing, the mixture was boiled in a water bath for 30 min, cooled, and centrifuged again at 4000 rpm for 10 min. The absorbance of the supernatant was measured at 532 nm, 600 nm, and 450 nm. The MDA content was calculated using the following formula: MDA (μmol / g) = [6.452 × (A) 532 -A 600 -0.559×A 450 ]×V÷W Where V is the volume of the extract (ml) and W is the volume of the leaf (g).

[0036] 1.9 Catalase (CAT) Activity Assay The activity of catalase (CAT) in samples was determined by ultraviolet absorption spectrometry using a catalase (CAT) activity assay kit (micro-method, catalog number BC0205) from Beijing Solarbio Science & Technology Co., Ltd. The principle is that H2O2 has a characteristic absorption peak at 240 nm, and CAT can catalyze the decomposition of H2O2. Enzyme activity is calculated by measuring the decrease in absorbance per unit time at 240 nm in the reaction system.

[0037] Sample preparation: Take approximately 0.1 g of tissue and add 1 mL of extraction buffer (included in the Catalase (CAT) activity assay kit from Beijing Solarbio Science & Technology Co., Ltd.) for homogenization in an ice bath. Centrifuge at 8000 g at 4℃ for 10 min, collect the supernatant, and place on ice for testing.

[0038] Assay Procedure: A 96-well UV plate was used for the assay. Before assay, the CAT detection working solution was incubated in a 25°C water bath for 10 min. 10 μL of sample supernatant and 190 μL of preheated working solution were added sequentially to the wells of the plate, mixed immediately, and the time was started. Using a microplate reader, the absorbance A1 was measured at 240 nm at 5 s and the absorbance A2 was measured at 1 min 5 s.

[0039] Enzyme activity unit is defined as: one activity unit per minute that catalyzes the degradation of 1 μmol H2O2 in a reaction system per gram of tissue. CAT activity is calculated based on tissue sample mass. The calculation formula is: CAT (U / g) = 764.5 × A÷W×F in, A=A1 A2; W is the sample mass (g); F is the sample pretreatment dilution factor.

[0040] 2.0 Anthocyanin Content Determination The anthocyanin detection kit (microplate method) from Shanghai Yuanye Biotechnology Co., Ltd. was used. The detection principle is that anthocyanins are dissolved in organic solvents, and anthocyanins are crudely extracted using organic solvents. Anthocyanins are red in acidic solutions, and the color intensity is directly proportional to the anthocyanin content. They have a maximum absorption wavelength of 530 nm, and their absorbance is measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0041] The concentration of anthocyanins at an OD value of 0.1 is defined as one unit (U). The calculation formula is as follows: Anthocyanin content (U / g) = (A 530nm A 空白 ) ÷ (0.1 × W) Where W is the fresh weight of the sample (g).

[0042] 2. Experimental Results 2.1 AaHSF20 Gene expression patterns in response to high temperatures For Anthurium plants in good growth condition, treatments were performed at 30 min, 2 h, 6 h, 12 h, 24 h, and 48 h before treatment (room temperature 25℃ control, light intensity 12000 Lx / 0 Lx, humidity 75%, recorded as 0 h) and under high temperature treatment (high temperature treatment conditions: photoperiod 12 h / 12 ​​h; day / night temperature 38℃ / 30℃; light intensity 12000 Lx / 0 Lx, humidity 75%). (The 30 min, 2 h, 6 h, and 12 h treatments were conducted under 38℃ light conditions). The results were detected using RT-qPCR. AaHSF20 Gene expression levels under high temperature conditions are shown in the following figures. Figure 1 (This figure is based on transcriptome sequencing data, and the expression level is normalized to FPKM value).

[0043] like Figure 1 As shown, relative to 0h, AaHSF20 The gene expression was significantly upregulated at 30 min, 2 h, 6 h, 12 h, 24 h, and 48 h after high temperature, with the highest expression level at 6 h, indicating that... AaHSF20Gene expression levels are induced by high temperatures.

[0044] 2.2 AaHSF20 Positive regulation of the heat resistance of Anthurium discs Transiently silencing gene in the bracts of Anthurium using the tobacco boll virus (TRV)-mediated viral-induced gene silencing (VIGS) system. AaHSF20 The expression, the result is as follows Figure 2 As shown (in) Figure 2 In the middle, will carry AaHSF20 The recombinant VIGS vector (TRV2-AaHSF20) containing the gene fragment was injected into discs of Anthurium bracts using the Agrobacterium tumefaciens infiltration method. An empty vector (TRV) was used as a negative control. Phenotyping was performed at 42℃ for 10 hours. (Disc diameter = 1 cm).

[0045] Combination Figure 2 It can be seen that at room temperature (25℃), there was no significant phenotypic difference between the control group and the silent group. However, after heat stress treatment, compared with the control group, transient silencing... AaHSF20 This accelerated the fading of the anthurium bracts.

[0046] The silencing efficiency was further detected by qRT-PCR, and the results are shown in [see figure]. Figure 3 (exist Figure 3 In this study, data are presented as mean ± standard deviation (n=3). An asterisk (*) above a bar indicates a significant difference compared to the TRV control group (*). p <0.05,** p <0.01, *** p <0.001, **** p <0.0001; two-tailed t-test).

[0047] Combination Figure 3 a found that in the control group AaHSF20 The expression level was significantly higher in the expression group than in the silence group, and the measured SOD ( Figure 3 b), POD ( Figure 3 c), MDA ( Figure 3 d), CAT ( Figure 3 The levels of peroxidases such as e-phosphorus oxidase were also lower in the silent group compared to the control group. Additionally, anthocyanin levels were also measured, and the silent group showed a decrease in anthocyanin content (…). Figure 3 The f) was significantly lower than that of the control group. These results indicate that AaHSF20 Transient silencing within the bracts of Anthurium reduces its resistance to heat stress. In conclusion, AaHSF20 It has played a positive role in regulating the heat resistance of Anthurium.

[0048] 2.3 AaHSF20 Positive regulation of heat resistance in Arabidopsis thaliana This study successfully constructed the super1300-AaHSF20-cGFP plant expression vector for heterologous overexpression in Arabidopsis thaliana. Stable homozygous T3 transgenic Arabidopsis lines were obtained through Agrobacterium-mediated inflorescence infection transformation and successive hygromycin resistance screening. Under high-temperature stress treatment at 42℃ for 12 h, compared with the wild-type control (WT), Arabidopsis plants overexpressing the AaHSF20 gene (OE) exhibited less wilting, indicating that overexpression of this gene can enhance the heat tolerance of Arabidopsis thaliana. Figure 4 ,exist Figure 4 The control group (RT) was set at 22℃ (16h light / 8h darkness), while the high-temperature treatment (HT) was set at 40℃ (16h light / 32℃ (8h darkness) for 2 days. The figure shows a representative photograph of the plants after 2 days of treatment (scale bar = 1cm).

[0049] High temperature stress on wild-type (WT) and AaHSF20 Overexpression (OE) in Arabidopsis seedlings AaHSF20 The effects of relative expression levels and antioxidant enzyme activity are shown in the figure. Figure 5 (exist Figure 5 In this study, data are expressed as mean ± standard deviation (SD) (n = 3 independent biological replicates). Significant differences between WT and OE under the same conditions are indicated by an asterisk (*). p <0.05,** p <0.01, two-tailed t-test).

[0050] Combined with molecular-level detection results, it shows that ( Figure 5 (a) After high-temperature treatment for 1 hour, the overexpressing plants (OE) AaHSF20 Gene expression levels were significantly upregulated; after 3 hours of high-temperature treatment, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in overexpressing plants (OE) were significantly increased. Figure 5 b, Figure 5 C and Figure 5 The values ​​of d) were significantly higher than those of wild-type control (WT). These results indicate that... AaHSF20 Genes may participate in the plant's response to high-temperature stress by regulating the activity of antioxidant enzymes, thereby positively regulating the heat tolerance of Arabidopsis thaliana.

[0051] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Overexpressing red palm AaHSF20 The gene may contain anthuriums AaHSF20 The application of gene overexpression vectors in Arabidopsis thaliana is characterized by, The application is: overexpression of Anthurium. AaHSF20 After gene modification, improve the high temperature stress tolerance of Arabidopsis thaliana and / or cultivate high temperature tolerant Arabidopsis thaliana germplasm; The red palm AaHSF20 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The red palm AaHSF20 The gene encodes the AaHSF20 protein, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. Silent Red Palm AaHSF20 The gene may contain anthuriums AaHSF20 The application of gene silencing vectors or silencing systems containing said silencing vectors in Anthurium, characterized in that, The application is: Silent Red Palm AaHSF20 After gene generation, a heat-intolerant Anthurium plant model was constructed; The red palm AaHSF20 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. A method for constructing a heat-intolerant Anthurium plant model, characterized in that, Includes the following steps: After infecting Anthurium plants with the silencing system described in claim 3, the Anthurium plants are cultured to silence them. AaHSF20 After gene sequencing, a heat-intolerant Anthurium plant model was constructed.

5. A method for improving the tolerance of plants to high temperature stress, characterized in that, Includes the following steps: Overexpressing red palm AaERF105 Genes were then used to improve the high-temperature stress tolerance of target plants. The plant in question is Arabidopsis thaliana. The red palm AaHSF20 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that, By increasing the activity of antioxidant enzymes in target plants, the plant's tolerance to high temperature stress can be improved. The antioxidant enzyme activity includes one or more of the following: superoxide dismutase activity, peroxidase activity, and catalase activity.

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