A gene encoding a plant heat shock transcription factor, PvHsf16, and its applications.

Abstract

This invention discloses a gene, PvHsf16, encoding a plant heat shock transcription factor, and its applications. This gene originates from differentially expressed genes upregulated in members of the Hsf gene family of switchgrass under cadmium (Cd) stress. By constructing a yeast expression vector for PvHsf16, PvHsf16 cells exhibited significantly greater cadmium tolerance, indicating that this gene enhances yeast cadmium resistance. PvHsf16 is located in the cell nucleus. Overexpression of the PvHsf16 gene in Arabidopsis thaliana, compared to the wild type under CdCl2 treatment, showed enhanced cadmium tolerance in the PvHsf6 overexpressing lines, with significantly increased chlorophyll content, root length, and biomass. The PvHsf16 gene provides a genetic resource for cultivating switchgrass germplasm suitable for both energy grass and cadmium remediation, and can be applied to the remediation of cadmium-polluted areas, playing an important role in researching phytoremediation technologies and improving soil heavy metal pollution.

Description

Technical Field

[0001] This invention relates to a gene PvHsf16 encoding a plant heat shock transcription factor and its applications, belonging to the field of plant genetic engineering technology. Background Technology

[0002] Plant heat shock transcription factors (Hssfs) play a crucial role in plant resistance to abiotic damage and various important biological processes. Although Hssfs vary in sequence size, eukaryotic Hsfs share a typical structure, consisting of a conserved DNA binding domain (DBD), oligomerization domain (OD or HR-A / B), nuclear localization signal (NLS) sequence, nuclear export signal (NES) sequence, and a C-terminal transcription activation domain (CTD). Based on the number of inserted amino acids in the HR-A / B region, plant Hsfs are classified into three classes: A, B, and C, each further subdivided into several subclasses. Class A and C members have 21 and 7 amino acids inserted between HR-A and HR-B, respectively, while class B Hsf members have a compact structure between HR-A and HR-B with no inserted amino acid sequences. Hsfs can recognize conserved heat shock element (HSE) binding motifs in the promoters of their response genes, including motifs encoding heat shock proteins (HSPs). Plant Hsfs have been confirmed as regulators of plant tolerance to biotic and abiotic stresses.

[0003] Cadmium (Cd) is a non-essential heavy metal element that is highly toxic to humans, animals, and plants. Soil Cd pollution is non-degradable and has long-term adverse effects on soil fertility and crop growth. Cd can also enter organisms through the food chain, posing significant human health risks. Cd stress significantly reduces seed germination and rooting rates. Accumulation of Cd in plant tissues inhibits water and nutrient absorption, leading to physiological and metabolic disorders, including reduced photosynthetic intensity, enzyme inactivation, and the development of symptoms such as dwarfism, decline, and growth retardation. These effects ultimately result in decreased crop quality and yield, and even death. The remediation of soil Cd pollution is urgent and cannot be delayed.

[0004] Switchgrass (Panicum virgatum L.) is a perennial, tall, C4 herbaceous plant originating in North America. Compared to traditional crops, switchgrass is highly adaptable, insect-resistant, requires less fertilizer, and has high yields, with a maximum yield reaching 74.1 t·hm². 2 Switchgrass not only boasts a large biomass but also exhibits extremely high levels of lignin and cellulose in its cell wall dry matter, making it a model plant for producing bioenergy, ethanol, and methane. As early as 1992, it was selected by the U.S. Department of Energy (USDOE) as a model bioenergy crop. Cadmium (Cd), a major heavy metal pollutant, can be absorbed by crops from the soil environment and transported to edible parts such as leaves and grains. Cd in these grains can cause serious and long-term diseases in humans. Planting cover vegetation on Cd-contaminated land is an effective remediation strategy. Perennial bioenergy grasses, generally possessing high biomass and robust root systems, are suitable candidate plants for replanting in Cd-contaminated areas, capable of restoring ground cover vegetation and even gradually restoring the ecosystem. Switchgrass is well-suited for planting on marginal lands; its strong root system effectively mitigates soil erosion, and repeated planting and harvesting of switchgrass (biomass) can absorb and reduce Cd levels in the soil. The ash (residue) after combustion or cell fermentation is collected (utilized) without posing a risk of environmental pollution. Studies have shown that switchgrass has a certain tolerance to Cd, but Cd also has a significant inhibitory effect on its growth. Whether Cd stress-responsive genes can be extracted from switchgrass is of great significance for implementing molecular genetic improvement of switchgrass, creating innovative switchgrass germplasm materials that serve both as an energy grass and for Cd remediation, and for remediating Cd- and other heavy metal-contaminated land and restoring ecological functions. Summary of the Invention

[0005] Objective of the Invention: To address the shortcomings of existing technologies, the first objective of this invention is to provide a gene, PvHsf16, encoding a plant heat shock transcription factor. The second objective of this invention is to improve the application of the gene PvHsf16 in enhancing plant cadmium tolerance.

[0006] Technical solution: In order to achieve the above-mentioned objective, the present invention provides a gene PvHsf16 encoding a plant heat shock transcription factor, the nucleotide sequence of which is shown in SEQ NO.1.

[0007] The expression protein of the gene PvHsf16, which encodes a plant heat shock transcription factor, described in this invention is subcellularly located in the cell nucleus, and its amino acid sequence is shown in SEQ NO.2.

[0008] The present invention also includes an expression vector containing the gene PvHsf16, which encodes the plant heat shock transcription factor described in the present invention.

[0009] The present invention also includes host cells containing the gene PvHsf16 encoding the plant heat shock transcription factor described in the present invention or the expression vector of the gene PvHsf16 encoding the plant heat shock transcription factor described in the present invention.

[0010] The present invention also includes a host bacterium containing the gene PvHsf168, which encodes the plant heat shock transcription factor described in the present invention.

[0011] The present invention relates to the application of the gene PvHsf16, which encodes a plant heat shock transcription factor, in improving cadmium tolerance in plants.

[0012] The present invention relates to the application of the gene PvHsf16, which encodes a plant heat shock transcription factor, in promoting plant growth under cadmium stress or in the preparation of products that promote plant growth under cadmium stress.

[0013] The application of the gene PvHsf16, which encodes a plant heat shock transcription factor, in the construction of transgenic materials resistant to cadmium stress, as described in this invention.

[0014] The application of the gene PvHsf16, which encodes a plant heat shock transcription factor, in cadmium stress-tolerant plant breeding.

[0015] A method to alter plant tolerance to cadmium stress by overexpressing or suppressing the expression of the gene PvHsf16 in plants.

[0016] An agent for improving plant tolerance to cadmium stress, comprising an expression promoter or expression activator of the gene PvHsf16 encoding a plant heat shock transcription factor.

[0017] This invention utilizes qRT-PCR analysis of the expression of 51 Hsf gene family members of switchgrass under different abiotic stress treatments (see...). Figure 1 Nine Cd-induced upregulated Hsf genes, including PvHsf3, PvHsf4, PvHsf16, PvHsf19, PvHsf23, PvHsf24, PvHsf35, PvHsf64, and PvHsf45, were screened out under CdCl2 treatment. These genes were then used in yeast heterologous transformation experiments to assess cadmium tolerance. Yeast cells overexpressing PvHsf3 and PvHsf16 exhibited cadmium-tolerant phenotypes, with PvHsf16 cells showing significantly greater cadmium tolerance compared to PvHsf3 cells. PvHsf16 is located in the cell nucleus. Compared to the wild-type Arabidopsis treated with CdCl2, the PvHsf6 overexpression lines showed enhanced cadmium tolerance, with significantly increased chlorophyll content, root length, and biomass.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0019] (1) This invention provides a plant heat shock transcription factor encoding gene PvHsf16 that can improve cadmium tolerance. There are currently no related reports on this gene. This gene is derived from differentially expressed genes in the Hsf gene family members of switchgrass under cadmium stress. By constructing a yeast expression vector of PvHsf16, the gene described in this invention can be verified to improve yeast cadmium resistance by using yeast spot plate experiments.

[0020] (2) In this invention, the PvHsf16 gene was overexpressed in Arabidopsis thaliana and it was found that the gene could significantly improve the transgenic cadmium tolerance of Arabidopsis thaliana. After cadmium stress treatment, the chlorophyll content, biomass and root length of the transgenic plants were significantly higher than those of WT (P<0.05).

[0021] (3) The PvHsf16 gene described in this invention provides gene resources for cultivating switchgrass germplasm that can be used for both energy grass and cadmium remediation. It can be applied to the treatment of cadmium-polluted areas and plays an important role in the research of plant bioremediation technology and the improvement of soil heavy metal pollution. Attached Figure Description

[0022] Figure 1 Heatmap of Hsf family gene expression in switchgrass under different concentrations of cadmium (CdCl2) stress;

[0023] Figure 2 Phenotypic diagram for validating the Hsfs gene of switchgrass in yeast heterologous transformation under cadmium (CdCl2) stress;

[0024] Figure 3 Subcellular localization and functional characteristics of the PvHsf16 gene;

[0025] Figure 4 The image shows a positive result from PCR detection of the PvHsf16 gene in Arabidopsis thaliana (detection of the hygromycin resistance gene HPT, 598bp).

[0026] Figure 5 Basta selection and qRT-PCR identification of three Arabidopsis thaliana lines overexpressing the PvHsf16 gene;

[0027] Figure 6 Phenotypic comparison of seeds from three Arabidopsis thaliana lines overexpressing the PvHsf16 gene (T3 generation) and wild-type Arabidopsis thaliana seeds after 10 days of culture on 1 / 2 MS solid medium containing different concentrations of cadmium (CdCl2).

[0028] Figure 7The figures show a comparison of chlorophyll content, root length, and biomass of wild-type Arabidopsis thaliana plants and three Arabidopsis thaliana plants overexpressing the Hsf16 gene under different concentrations of cadmium (CdCl2) stress. (a) represents chlorophyll content, (b) represents average root length, and (c) represents average biomass per plant. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0030] Experimental materials and reagents:

[0031] (1) Strains and vectors:

[0032] Yeast strain YCF1 and entry vector pEND-linker were both purchased from Invitrogen.

[0033] Escherichia coli DH5α was purchased from Kangwei Century Biotechnology Co., Ltd.

[0034] (2) Enzymes and kits:

[0035] The restriction endonuclease PvuⅠ was purchased from NEB.

[0036] The PCR product recovery kit, EZNA Gel Extraction Kit-Spin, was purchased from OMEGA.

[0037] Ampicillin (Amp), kanamycin (Kan), rifampin (Rif), hygromycin B (HygB), etc., were purchased from Sigma-Aldrich.

[0038] The plasmid extraction kit, HiPure Plasmid Micro Kit, was purchased from Magen.

[0039] YPDA solid culture medium was purchased from Coolaber, catalog number PM2021;

[0040] YPDA culture medium was purchased from Coolaber, catalog number PM2011;

[0041] The SD / -Ura with Agar yeast culture medium with two deficiencies was purchased from Coolaber (product number PM2272).

[0042] LB liquid medium BL1058A was purchased from Biosharp.

[0043] All other chemical reagents and consumables were purchased from Nanjing Shoude Equipment Co., Ltd.

[0044] (3) Reaction primers:

[0045] vHsf16-F: AGCT GAATTC ATGGAGGCGGGCGGCGGG;

[0046] PvHsf16-R: AGCT AAGCTT GGTTTTCTCCGCCGAGGTGA;

[0047] M13-F: TGTAAAACGACGGCCAGT;

[0048] M13-R: CAGGAAACAGCTATGACC;

[0049] The above was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0050] Example 1: Cloning of the PvHsf16 gene

[0051] (1) PCR amplification to obtain the target gene fragment

[0052] The genome sequence information of *P. virgatum* was downloaded from the JGI website (https: / / phytozome-next.jgi.doe.gov / info / Pvirgatum_v4_1), and 51 *PvHsf* members were identified, numbered PvHsf1-51. Primers were designed and synthesized based on the coding sequence of the PvHsf16 gene: PvHsf16-F: AGCT GAATTC ATGGAGGCGGGCGGCGGG, PvHsf16-R: AGCTAAGCTT GGTTTTCTCCGCCGAGGTGA. Using gDNA as a template, the target gene fragment was amplified by PCR using Q5 high-fidelity DNA polymerase. The PCR reaction system for Q5 high-fidelity DNA polymerase amplification is shown in Table 1 below. The PCR reaction program was: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 63℃ annealing for 10 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 5 min; 10℃, 10 min.

[0053] Table 1 High-fidelity DNA polymerase PCR amplification reaction system for target gene

[0054]

[0055]

[0056] (2) Recovery of PCR products

[0057] PCR products were recovered using the EZNA Gel Extraction Kit-Spin, following the kit's instructions:

[0058] ① Cut off the gel block containing the target fragment, place it in a 1.5mL centrifuge tube, and add an equal volume of Binding Buffer.

[0059] ② Adjust the metal bath to 60℃, place the centrifuge tubes on the metal bath for 7-10 minutes, and invert the centrifuge tubes every 2-3 minutes to fully dissolve the gel.

[0060] ③ Place the adsorption column into a 2 mL collection tube.

[0061] ④ Once the gel block has completely dissolved, transfer all the liquid to the adsorption column at 10,000g for 1 minute. Discard the waste liquid and return the adsorption column to the collection tube. If there is too much liquid, it can be transferred in stages until all the liquid has been transferred to the adsorption column.

[0062] ⑤ Add 300uL Binding Buffer to the centrifuge column, centrifuge at 13000g for 1min, discard the supernatant, and put the adsorption column back into the collection tube.

[0063] ⑥ Add an appropriate volume of anhydrous ethanol to the SPW Wash Buffer, add 700 μL of SPW Wash Buffer to the adsorption column, centrifuge at 13000 g for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.

[0064] ⑦ Repeat step ⑥.

[0065] ⑧ Put the collection tube containing the waste liquid back into the centrifuge, centrifuge at 13000g for 2 minutes, place the adsorption column into a new centrifuge tube, and leave it in the air for 2-3 minutes.

[0066] ⑨ Add 30 µL of solution buffer (preheated to 65°C), let stand at room temperature for 3 minutes, and centrifuge at 13000g for 1 minute.

[0067] ⑩ Measure the concentration of recovered DNA using an ELISA reader and store it in a -20°C refrigerator.

[0068] The full-length PvHsf16 was obtained by PCR amplification.

[0069] (3) Ligation of the target gene fragment with the entry vector pEND-Linker

[0070] The double-digested entry vector pEND-linker was ligated with the PvHsf16 target gene fragment, and the reaction system is shown in Table 2 below:

[0071] Table 2. Ligation reaction system between target gene fragment and pEND-Linker entry vector

[0072]

[0073] Mix the above liquids thoroughly and react at 16°C for 2-3 hours.

[0074] (4) Transformation of the ligation product into competent E. coli DH5α cells:

[0075] ① Take an ice box and remove the E. coli DH5α competent cells from the -80℃ ultra-low temperature freezer and place them on ice to thaw.

[0076] ② Add 10 μL of the ligation product of the target gene fragment and the entry vector pEND-Linker to the competent cells when they are just thawed, and place on ice for 30 min.

[0077] ③ Open the metal bath, adjust the temperature to 42℃, heat shock at 42℃ for 1 min, and place on ice for 2-3 min. Add competent cells to 1 mL of LB liquid medium (antibiotic-free), and activate at 37℃ and 180 rpm for 30 min.

[0078] ④ Centrifuge at 7000 rpm for 1 min at room temperature, discard the supernatant in a clean bench, leaving a small amount of resuspended precipitate, spread the resuspended bacterial solution on the resistant medium (LB + 50 mg / L kanamycin), and incubate overnight at 37°C.

[0079] ⑤ Select 3-5 single clones and verify them by PCR.

[0080] Positive clones were selected from transformed E. coli competent cells for PCR detection. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min 30 s, 30 cycles; 72℃ extension for 7 min; 10℃, 10 min.

[0081] The PCR detection reaction system is shown in Table 3 below.

[0082] Table 3. PCR detection reaction system for positive clones

[0083]

[0084] PCR products were analyzed by gel electrophoresis. Plasmids were extracted from bacterial cultures of the same size as the target band using the HiPure Plasmid Micro Kit and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of PvHsf16 is 1347 bp, as shown in SEQ NO.1, and the amino acid sequence of the PvHsf16 expressed protein is shown in SEQ NO.2.

[0085] SEQ NO.1: PvHsf16 gene CDS coding sequence

[0086]

[0087] MEAGGGGASSLPPFLSKTYEMVDNPATDAVVAWTPPGTSFVVANQAEFCRDLLPKYFKHNNFSSFVRQLNTYGFRKIDPEQWEFANEDFIRGQRHRLKNIHRRKPIFSHSSH NQVSAPLADHERREYEEEIEKLKCENADLISELEKNAEKKLDMERRMQELENKLISLEDRQKNLIAYVRDIVRAPGFRSSFVQQTDHHGKKRRLLIPASLHQDANTEGNQIVH GGLTNPPVYRESFDKMESSLNSLENFFREASEAFDISYDDGVPGPSSAIVITELHSSGESDPVVPSPPSRMHTSSAGAGDSLSRMHTSSAGAGDSLSSHDISGSTSCAESPP LPQMQSCTDSRAKVSEIDVNLEPAITETGPSRDQPAEDHPSLAPANDGFWEQFLTEQPGSNAHQGVQSERRDGDSKDDRTRTGDQENLWRGKKNVEQMTEKLGHLTSAEKT*.

[0088] Example 2: Yeast heterologous expression and cadmium tolerance analysis

[0089] An overexpression vector for the PvHsf16 gene was constructed using LR recombination technology.

[0090] (1) Construction of yeast expression vector

[0091] The specific method for extracting plasmids from positive clones with correct sequencing results is as follows:

[0092] ① Collect bacterial cells: Collect 1-5 mL of Escherichia coli DH5α liquid, centrifuge at 10000g at room temperature for 1 min, discard the waste liquid, and collect the bacterial cells.

[0093] ② Add 250 μL Buffer P1 (add an appropriate volume of RNase A according to the requirements of the Magen HiPure Plasmid Micro Kit, i.e., add 0.167 mg RNase A per 250 ml Buffer P1), vortex for 1 min, and fully lyse the bacterial cells.

[0094] ③ Add 250 μL of Buffer P2 and slowly invert the container 8-10 times.

[0095] ④ Add 250 μL Buffer NP3, mix immediately, invert 8-10 times, centrifuge at 13000g for 10 min at room temperature.

[0096] ⑤ Insert the adsorption column into the collection tube, transfer the supernatant into the collection tube (if not all of it can be transferred at once, it can be transferred in several times), centrifuge at 13000g for 1 min at room temperature, remove the waste liquid, and put the adsorption column back into the collection tube.

[0097] ⑥ Add 500 μL Buffer PW1, centrifuge at 13000 g at room temperature for 1 min, remove the waste liquid, and put the adsorption column back into the collection tube.

[0098] ⑦ Add 500 μL Buffer PW2, centrifuge at 13000 g for 1 min at room temperature, remove the waste liquid, and put the adsorption column back into the collection tube.

[0099] ⑧ Repeat step ⑦.

[0100] ⑨ Place the adsorption column back into the empty collection tube, centrifuge at 13000g for 2 minutes at room temperature, then place the adsorption column into a new centrifuge tube and air dry for 2-3 minutes.

[0101] ⑩ Add 60 μL of Elution Buffer to the middle of the adsorption membrane, incubate for 2-3 min, centrifuge at 13000 g for 1 min at room temperature.

[0102] ⑩ Use an ELISA reader to detect the concentration and store at -20°C.

[0103] The system was linearized using the restriction endonuclease PvuⅠ, as shown in Table 4 below.

[0104] Table 4. Linearization treatment system for restriction endonuclease PvuⅠ

[0105]

[0106] Reaction conditions: 37℃, enzyme digestion for 1 h. Gel electrophoresis was performed for detection, and the target fragment was recovered from the gel. The target fragment was then recombined with the expression vector pGAD426 using LR recombination. PCR primers M13-F: TGTAAAACGACGGCCAGT; M13-R: CAGGAAACAGCTATGACC were used for PCR amplification. The reaction system is shown in Table 5 below.

[0107] Table 5 LR Recombination Reaction System

[0108]

[0109] Reaction conditions: 25℃, 1h. Transformed into competent E. coli DH5α cells, positive clones were selected, and the correct expression vector was determined by vector size and PCR detection. The plasmid pGAD426-PvHsf16 was then extracted from the positive clone.

[0110] (2) Preparation of YCF1 (cadmium-sensitive yeast strain) competent cells

[0111] ① Take yeast YCF1 cells from the laboratory -80℃ freezer and place them in YPDA solid medium, and incubate them in a 30℃ incubator for 1-2 days.

[0112] ② Pick single clones and place them in pre-prepared YPDA culture medium, then incubate overnight on a shaker at 28°C and 210 rpm.

[0113] ③ After concentrating the bacterial cells, turn the container to a large shaker and place it in a shaker at 28℃ and 210 rpm. When the OD... 600 When the concentration is 0.8-1.0, remove the bacterial cells for later use.

[0114] ④ Transfer the above bacterial cells, centrifuge at 4℃, 500g, for 4 minutes, and discard the supernatant.

[0115] ⑤ Add 10ml of EZ1 solution to resuspend the bacterial cells, centrifuge at 4℃, 500g, for 4min, and discard the supernatant.

[0116] ⑥ Resuspend the bacterial cells in 1 ml of EZ2 solution, aliquot the competent cells into 10 μl portions, wrap each portion in a paper towel, and store at -80°C.

[0117] (3) Cadmium-sensitive yeast YCF1 conversion steps

[0118] ① Take the prepared YCF1 competent cells. After 2-3 minutes, when the competent cells are initially thawed, add (0.2-1 μg) of pGAD426-PvHsf16 plasmid to the competent cells in a sterile environment and mix gently.

[0119] ② Add 100 μl of EZ3 solution to the above liquid competent cells and mix immediately.

[0120] ③ Place the well-mixed liquid in a 30℃ incubator and incubate for 60-90 minutes. Mix the liquid every 20 minutes during the incubation process, and mix it 2-3 times in total.

[0121] ④ After the culture is completed, spread the liquid evenly on the SD / -Ura with Agar medium and incubate at 30℃ for 2-3 days.

[0122] (4) Cadmium resistance test of yeast strains

[0123] ① Use a toothpick to pick up a single YCF1 clone and inoculate it into 1 mL of liquid-deficient medium (SD / -His-Ura) for growth. Incubate at 28°C with shaking at 210 rpm for 2-3 days.

[0124] ② Once the bacterial culture reaches the plateau phase, dilute it 10-fold with sterile water in a gradient manner.

[0125] ③ Take 5 μL and spot the concentrations sequentially at 100 μmol·L⁻¹ -1 Yeasts were cultured at 30°C for 5-6 days in a monodeficient medium (SD / -Ura) containing CdCl2 and their phenotypes were observed and photographs were taken.

[0126] At 100 μmol·L -1 On CdCl2 SD / -Ura selective medium plates, compared with the control pGAD426-GUS strain, the growth of the PvHsf03 overexpressing yeast strain was phenotypic, and the growth of the PvHsf16 overexpressing yeast strain was significantly enhanced (see...). Figure 2 It exhibits a certain degree of Cd resistance phenotype. The subcellular localization and functional characteristics of the PvHsf16 gene are shown in the diagram. Figure 3 .

[0127] Example 3: Arabidopsis heterologous transformation to PvHsf16 and cadmium tolerance identification

[0128] (1) The PvHsf16 gene was inserted into the plant expression vector PHB using gene synthesis (completed by Nanjing Qingke Biotechnology Co., Ltd.). Agrobacterium transformation: The recombinant plasmid was transformed into Agrobacterium GV3101 by freeze-thaw method. After overnight culture, single clones on the plate were picked and colony PCR was performed for identification. Positive clones were added to 500 μL of B containing 50 mg / L kan and 50 mg / L rif and cultured overnight for subsequent experiments, or stored with glycerol added at a 1:1 ratio.

[0129] (2) Arabidopsis transformation was carried out by inflorescence infection method. The infected Arabidopsis was placed flat in a light-proof plastic basin, sprayed with a small amount of water, and covered with plastic wrap to prevent moisture evaporation. Newspaper was placed on the plastic wrap to maintain a dark environment. After 16-24 hours of cultivation, the plastic wrap was removed, and the Arabidopsis was taken out and placed in the Arabidopsis room for further cultivation. After the Arabidopsis matured, the seeds were harvested, dried, and stored at 4℃ for later use.

[0130] (3) After drying, the t0 generation seeds were sterilized with 75% ethanol on a clean bench and evenly sprinkled on a solution containing 20 mg / L hygromycin. -1The culture dishes were placed on 1 / 2 MS solid medium and cultured in the dark at 4°C for 2 days. The culture dishes were then transferred to an Arabidopsis thaliana chamber for about one week. Subsequently, the Arabidopsis thaliana plants that showed normal growth and exhibited hyg resistance were transferred to nutrient soil and cultured in the Arabidopsis thaliana chamber. Sixteen positive plants were obtained by PCR detection (PCR positive results are shown in [link to PCR results]). Figure 4 Phenotypic screening was performed using 1 / 2 MS solid medium containing 20 μM Basta (Basta selection results are shown in [link to Basta selection results]). Figure 5 (a) and identified by qRT-PCR (see qRT-PCR identification results). Figure 5 (b) Select OE-5, OE-6 and OE-8 ( Figure 5 In b), 5, 6, and 8) are transgenic Arabidopsis thaliana overexpressing PvHsf16. They were cultured generation after generation until the t3 generation homozygous seeds were harvested and stored at -4℃ for subsequent cadmium tolerance analysis.

[0131] (4) Seeds of OE-5, OE-6, and OE-8 lines (generation t3) and wild-type Arabidopsis thaliana seeds (WT) were sown using sterile toothpicks on 1 / 2 MS solid medium containing 90 μM and 180 μM CdCl2, respectively. Seeds sown on cadmium-free 1 / 2 MS solid medium served as a control. Observations were conducted after 10 days. Figure 6 As shown, wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana OE-5, OE-6, and OE-8 grown on control 1 / 2 MS solid medium all showed normal phenotypes with no significant differences. Both wild-type and transgenic Arabidopsis thaliana plants grown on 1 / 2 MS solid medium containing 90 μM and 180 μM CdCl2 were inhibited, exhibiting stunted growth and shortened root systems. Compared to the WT type, the seedlings of the three transgenic Arabidopsis thaliana lines OE-5, OE-6, and OE-8 were larger, had longer root systems, and showed superior growth compared to the wild type.

[0132] (5) The growth and physiological data of Arabidopsis thaliana seedlings under different concentrations of cadmium stress were statistically analyzed. The results are shown in […]. Figure 7 In the cadmium-free control, there were no significant differences in chlorophyll content, average root length, and average biomass per plant in Arabidopsis thaliana. After treatment with 90 μM and 180 μM dCl2, Arabidopsis growth was inhibited, and the above indicators gradually decreased. However, the indicators of transgenic Arabidopsis OE-5, OE-6, and OE-8 were significantly higher than those of wild-type WT (P<0.05), indicating that transgenic Arabidopsis thaliana had higher tolerance to cadmium stress than wild-type. Therefore, overexpression of PvHsf16 can significantly improve the cadmium resistance of transgenic Arabidopsis thaliana.

Claims

1. A gene encoding a plant heat shock transcription factor PvHsf16 application in improving the cadmium tolerance of Arabidopsis thaliana, said gene PvHsf16 The nucleotide sequence is shown as SEQ NO.

1.

2. A gene encoding a plant heat shock transcription factor PvHsf16 The application of the gene in promoting Arabidopsis thaliana growth under cadmium stress or in the preparation of products that promote Arabidopsis thaliana growth under cadmium stress. PvHsf16 The nucleotide sequence is shown in SEQ NO.

1.

3. A gene encoding a plant heat shock transcription factor PvHsf16 Application in constructing transgenic Arabidopsis thaliana tolerant to cadmium stress, the gene PvHsf16 The nucleotide sequence is shown in SEQ NO.

1.

4. A gene encoding a plant heat shock transcription factor PvHsf16 Application of the gene in cadmium stress-tolerant breeding of Arabidopsis thaliana PvHsf16 The nucleotide sequence is shown in SEQ NO.

1.

5. A method for improving the tolerance of Arabidopsis thaliana to cadmium stress, characterized in that, By overexpressing the gene in Arabidopsis PvHsf16 The gene improves the tolerance of Arabidopsis thaliana to cadmium stress. PvHsf16 The nucleotide sequence is shown in SEQ NO.1.

Images