Gene PvHsf16 for coding plant heat shock transcription factor and application thereof

Abstract

The invention discloses a gene PvHsf16 for coding a plant heat shock transcription factor and application of the gene PvHsf16. The gene is derived from an up-regulated differential expression gene in a member of a switchgrass Hsf gene family subjected to cadmium (Cd) stress treatment. By constructing a PvHsf16 yeast expression vector, PvHsf16 cells show more obvious cadmium resistance, and the gene can improve the cadmium resistance of yeast. PvHsf16 is positioned in a cell nucleus, the PvHsf16 gene is overexpressed in arabidopsis thaliana, and compared with a wild type treated by CdCl2, an arabidopsis thaliana PvHsf6 overexpressed strain has enhanced cadmium tolerance, and the chlorophyll content, the root length and the biomass of the strain are remarkably improved. The PvHsf16 gene provides a gene resource for cultivating the germplasm of the energy grass-cadmium remediation dual-purpose switchgrass, can be applied to the treatment of cadmium polluted areas, and plays an important role in the aspects of researching a plant bioremediation technology, improving the heavy metal pollution condition of soil and the like.

Description

Technical Field

[0001] The invention relates to a gene PvHsf16 encoding a plant heat shock transcription factor and application thereof, belonging to the technical field of plant gene engineering. Background Art

[0002] Heat shock transcription factors (Hsfs) play an important role in plant resistance to stress damage and various important biological processes. Although there are certain differences in the sequence size of Hssf, eukaryotic Hsfs have a typical structure, which is divided into a conserved DNA binding domain (DBD), an oligomerization domain (OD or HR-A / B), a nuclear localization signal (NLS) sequence, a nuclear export signal (NES) sequence and a C-terminal transcription activation domain (CTD). According to the number of inserted amino acids in the HR-A / B region, plant Hsfs are divided into three categories: A, B, and C, and each category is divided into several subcategories. The A and C members have 21 and 7 amino acids inserted between HR-A and HR-B, respectively, while the B Hsf members have a compact structure between HR-A and HR-B and no inserted amino acid sequence. Hsfs can recognize conserved heat shock element (HSEs) binding motifs in the promoters of their response genes, including motifs encoding heat shock proteins (HSPs). Plant Hsfs have been confirmed to be regulatory factors for plant tolerance to biotic and abiotic stresses.

[0003] Cadmium (Cd) is one of the non-essential heavy metal elements for humans, animals and plants and is highly toxic. Soil Cd pollution is non-degradable and will have long-term adverse effects on environmental 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. The accumulation of Cd in plant tissues inhibits the absorption of water and nutrients, 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 lead to reduced crop quality, yield, and even death. The control of soil Cd pollution is imminent and cannot be delayed.

[0004] Switchgrass (Panicum virgatum L.) is a perennial tall C4 herb originating from North America. Compared with traditional crops, switchgrass has strong adaptability, insect resistance, low fertilizer requirements, and high yields, with the highest yield reaching 74.1 t·hm 2 . Switchgrass not only has a large biomass, but also has a high content of lignin and cellulose in the cell wall dry matter. It is a model plant for producing bioheat, ethanol and methane. As early as 1992, switchgrass was selected by the U.S. Department of Energy (USDOE) as a model bioenergy crop. As a major heavy metal pollutant, cadmium (Cd) can be absorbed by crops from the soil environment and transported to edible parts such as leaves and grains. At the same time, Cd in these grains can cause serious and long-term diseases in humans. Planting cover vegetation on Cd-contaminated land is an effective governance strategy. Perennial bioenergy grasses generally have high biomass and strong and developed root systems. They are suitable candidates for replanting in Cd-contaminated areas. They can be used to restore ground cover vegetation and even gradually restore the ecosystem. Switchgrass is very suitable for planting on marginal land. Its strong root system can effectively improve soil erosion. Repeated planting and harvesting of switchgrass (biomass) can absorb and reduce the Cd content in the soil. The ash (residue) after combustion or cell fermentation is collected (utilized) without the 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 it is possible to discover Cd stress response genes from switchgrass is of great significance for implementing molecular genetic improvement of switchgrass, creating energy grass-Cd restoration switchgrass germplasm materials, and treating and repairing land contaminated by Cd and other heavy metals and restoring ecological functions. Summary of the invention

[0005] Purpose of the invention: In view of the deficiencies in the prior art, the first purpose of the present invention is to provide a gene PvHsf16 encoding a plant heat shock transcription factor. The second purpose of the present invention is to provide an application of a gene PvHsf16 encoding a plant heat shock transcription factor in improving plant cadmium tolerance.

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

[0007] The expression protein of the gene PvHsf16 encoding the plant heat shock transcription factor of the present invention is subcellularly localized 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 encoding the plant heat shock transcription factor of the present invention.

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

[0010] The present invention also includes a host bacterium containing the gene PvHsf168 encoding the plant heat shock transcription factor of the present invention.

[0011] The invention discloses an application of the gene PvHsf16 encoding a plant heat shock transcription factor in improving the cadmium tolerance of plants.

[0012] The invention discloses an application of the gene PvHsf16 encoding a plant heat shock transcription factor in promoting plant growth under cadmium stress or in preparing a product for promoting plant growth under cadmium stress.

[0013] The invention discloses an application of the gene PvHsf16 encoding a plant heat shock transcription factor in constructing a transgenic material resistant to cadmium stress.

[0014] The invention discloses an application of the gene PvHsf16 encoding a plant heat shock transcription factor in plant breeding for cadmium stress tolerance.

[0015] A method for changing plant cadmium stress tolerance, which changes the plant cadmium stress tolerance by overexpressing or inhibiting the expression of the gene PvHsf16 in the plant.

[0016] A preparation 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] The present invention uses qRT-PCR analysis to analyze the expression of 51 Hsf gene family members of switchgrass under different abiotic stress treatments (see Figure 1 ), and screened out 9 Cd-induced up-regulated Hsf genes under cadmium (CdCl2) treatment, including PvHsf3, PvHsf4, PvHsf16, PvHsf19, PvHsf23, PvHsf24, PvHsf35, PvHsf64 and PvHsf45, which were used for cadmium tolerance evaluation through yeast heterologous transformation experiments. Among them, yeast cells overexpressing PvHsf3 and PvHsf16 showed a cadmium-resistant phenotype, and PvHsf16 cells showed more significant cadmium tolerance compared with PvHsf3. PvHsf16 is localized in the nucleus. Compared with the wild type under CdCl2 treatment, the Arabidopsis PvHsf6 overexpression line has enhanced cadmium tolerance, and its chlorophyll content, root length and biomass are significantly increased.

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

[0019] (1) The present invention provides a plant heat shock transcription factor encoding gene PvHsf16 that has the ability to improve cadmium tolerance. There is no relevant report on this gene so far. The gene is derived from upregulated differentially expressed genes in members of the Hsf gene family of switchgrass under cadmium stress. A yeast expression vector of PvHsf16 is constructed, and a yeast spot plate experiment is used to verify that the gene of the present invention can improve yeast cadmium resistance.

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

[0021] (3) The PvHsf16 gene described in the present invention provides a genetic resource for cultivating switchgrass germplasm for both energy grass and cadmium restoration, which can be applied to the treatment of cadmium-contaminated areas and plays an important role in the research of plant bioremediation technology and the improvement of soil heavy metal pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the qRT-PCR expression calorimeter of switchgrass Hsf family genes under different concentrations of cadmium (CdCl2) stress;

[0023] Figure 2 This is the phenotype diagram for functional verification of yeast heterologous transformation of switchgrass Hsfs gene under cadmium (CdCl2) stress;

[0024] Figure 3 This is a diagram of the subcellular localization and functional characteristics of the PvHsf16 gene;

[0025] Figure 4 This is the positive image of the PCR test results of Arabidopsis transformation PvHsf16 gene (detecting hygromycin resistance gene HPT, 598bp);

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

[0027] Figure 6 Phenotypic comparison of T3 seeds of three Arabidopsis strains overexpressing the PvHsf16 gene and wild-type Arabidopsis seeds after being cultured on 1 / 2MS solid medium containing different concentrations of cadmium (CdCl2) for ten days;

[0028] Figure 7Comparison of chlorophyll content, root length and biomass of wild-type Arabidopsis plants and three Arabidopsis plants overexpressing the switchgrass Hsf16 gene under different concentrations of cadmium (CdCl2) stress, where (a) is the chlorophyll content, (b) is the average root length, and (c) is the average biomass per plant. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is 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 purchased from Invitrogen;

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

[0034] (2) Enzymes and kits:

[0035] 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), rifampicin (Rif), hygromycin B (HygB), etc. were purchased from Sigma;

[0038] Plasmid extraction kit HiPure Plasmid Micro Kit was purchased from Magen (Meiji Biotechnology);

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

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

[0041] Yeast medium SD / -Ura with Agar was purchased from Coolaber (Coolaber), catalog number PM2272;

[0042] LB liquid medium BL1058A was purchased from Biosharp

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

[0044] (3) Reaction primer:

[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 Biotechnology (Shanghai) Co., Ltd.

[0050] Example 1 Cloning of PvHsf16 gene

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

[0052] The genome sequence information of switchgrass 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. According to the coding sequence of the PvHsf16 gene, synthetic primers were designed: PvHsf16-F: AGCT GAATTC ATGGAGGCGGGCGGCGGG, PvHsf16-R: AGCTAAGCTT GGTTTTCTCCGCCGAGGTGA, and the target gene fragment was amplified by PCR amplification using Q5 high-fidelity DNA polymerase using gDNA as a template. The PCR reaction system of Q5 high-fidelity DNA polymerase amplification is shown in Table 1. The PCR reaction program was: 98℃ pre-denaturation for 3min; 98℃ denaturation for 30s, 63℃ annealing for 10s, 72℃ extension for 1min, 30 cycles; 72℃ extension for 5min; 10℃, 10min.

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

[0054]

[0055]

[0056] (2) PCR product recovery

[0057] The PCR product was recovered using the EZNA Gel Extraction Kit-Spin kit according to the kit procedure, specifically:

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

[0059] ② Adjust the metal bath to 60℃, place the centrifuge tube on the metal bath for 7-10 minutes, and turn the centrifuge tube upside down every 2-3 minutes to fully dissolve the rubber block.

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

[0061] ④ When the gel block is completely dissolved, transfer all the liquid to the adsorption column, 10000g, 1min, pour out the waste liquid, and put the adsorption column back into the collection tube. If there is too much liquid, transfer it in batches until all the liquid is 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 SPW Wash Buffer, add 700ul SPW Wash Buffer to the adsorption column, centrifuge at 13000g for 1min, pour out the waste liquid, and put the adsorption column back into the collection tube.

[0064] ⑦Repeat step ⑥.

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

[0066] ⑨Add 30uLElution Buffer (preheated at 65℃), let stand at room temperature for 3min, and centrifuge at 13000g for 1min.

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

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

[0069] (3) Connect the target gene fragment to the entry vector pEND-Linker

[0070] The double-digested entry vector pEND-linker was connected to the PvHsf16 target gene fragment. The reaction system is as shown in Table 2:

[0071] Table 2 Target gene fragment and entry vector pEND-Linker ligation reaction system

[0072]

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

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

[0075] ① Take out the ice box, take out the E. coli DH5α competent cells from the -80℃ ultra-low temperature freezer and place them on ice to melt.

[0076] ② When the competent medium has just been thawed, add 10uL of the connection product of the target gene fragment and the entry vector pEND-Linker and place it on ice for 30 minutes.

[0077] ③Open the metal bath, adjust the temperature to 42℃, heat shock at 42℃ for 1min, place on ice for 2-3min. Add 1mL LB liquid medium (without antibiotics) to the competent cells, activate at 37℃, 180rpm for 30min.

[0078] ④ Centrifuge at 7000rpm at room temperature for 1min, pour off the supernatant in the clean bench, keep a small amount of resuspended precipitate, spread the resuspended bacterial solution on the resistance culture medium (LB + 50mg / L kanamycin), and culture in a 37℃ incubator overnight.

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

[0080] The positive clones were picked from the transformed E. coli competent cells for PCR detection. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min 30 s, 30 cycles; extension at 72°C for 7 min; and 10°C for 10 min.

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

[0082] Table 3 Positive clone PCR detection reaction system

[0083]

[0084] The PCR products were run on gels, and the bacterial solution with the same size as the target band was selected to extract the plasmid using the plasmid extraction kit 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 resistance analysis

[0089] The overexpression vector of PvHsf16 gene was constructed using LR recombination technology.

[0090] (1) Construction of yeast expression vector

[0091] Extract the positive clone plasmid with correct sequencing results. The specific method is as follows:

[0092] ① Collect bacteria. Collect 1-5 mL of E. coli DH5α liquid, centrifuge at 10,000 g at room temperature for 1 min, discard the waste liquid, and collect the bacteria.

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

[0094] ③Add 250μL Buffer P2 and slowly invert upside down 8-10 times.

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

[0096] ⑤Insert the adsorption column into the collection tube, transfer the supernatant to the collection tube (if the transfer is not completed in one time, it can be transferred in batches), centrifuge at 13000g at room temperature for 1 min, remove the waste liquid, and put the adsorption column back into the collection tube.

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

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

[0099] ⑧Repeat step ⑦.

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

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

[0102] ⑩ Use an ELISA reader to detect the concentration and store in a -20℃ refrigerator.

[0103] Linearization was performed with restriction endonuclease PvuⅠ, and the system is shown in Table 4.

[0104] Table 4 Restriction endonuclease PvuⅠ linearization treatment system

[0105]

[0106] Reaction conditions: 37°C, enzyme digestion for 1h. Gel electrophoresis detection, and gel excision to recover the target fragment. The target fragment and the expression vector pGAD426 were subjected to LR recombination reaction, and PCR amplification was performed using PCR primers M13-F: TGTAAAACGACGGCCAGT; M13-R: CAGGAAACAGCTATGACC. The reaction system is shown in Table 5.

[0107] Table 5LR recombination reaction system

[0108]

[0109] Reaction conditions: 25°C, 1h. Transform competent E. coli DH5α, pick positive clones, and determine the correct expression vector by vector size and PCR detection to extract the positive clone plasmid pGAD426-PvHsf16.

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

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

[0112] ②Pick a single clone and place it in the YPDA culture medium prepared in advance, and place it on a shaker at 28℃ and 210rpm overnight.

[0113] ③ After shaking, transfer the concentrated cells to a shaker and place them in a shaker at 28°C and 210 rpm. 600 =0.8-1.0, remove the bacteria for later use.

[0114] ④ Transfer the above bacteria, centrifuge at 4℃, 500g, 4min, and discard the supernatant.

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

[0116] ⑥Add 1ml EZ2 solution to resuspend the cells, and dispense competent cells into 10μl samples each. Wrap with paper towels and store in a -80℃ refrigerator.

[0117] (3) Transformation steps of cadmium-sensitive yeast YCF1

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

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

[0120] ③ Place the evenly mixed liquid in a 30℃ incubator and culture it for 60-90 minutes. Mix the liquid once every 20 minutes during the culture process, and mix it 2-3 times during the whole process.

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

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

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

[0124] ② When the bacterial solution grows to the plateau stage, dilute it 10 times with sterile water in a gradient manner.

[0125] ③ Take 5 μL and place it at 100 μmol·L -1 The cells were cultured in a CdCl2-deficient medium (SD / -Ura) at 30°C for 5-6 days, and the yeast phenotype was 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 phenotypically enhanced, and the growth of the PvHsf16 overexpressing yeast strain was significantly enhanced (see Figure 2 ), showing a certain Cd-tolerant phenotype. The subcellular localization and functional characteristics of the PvHsf16 gene are shown in Figure 3 .

[0127] Example 3 Arabidopsis heterologous transformation of PvHsf16 and identification of cadmium resistance

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

[0129] (2) Arabidopsis transformation was performed using the 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 water evaporation. Newspaper was placed on the plastic wrap to maintain a dark environment. After culturing for 16-24 hours, the plastic wrap was removed, and the Arabidopsis was taken out and continued to be cultured in the Arabidopsis room. When the Arabidopsis matured, the seeds were collected, dried, and stored at 4°C for later use.

[0130] (3) The dried t0 generation seeds were sterilized with 75% ethanol on a clean bench and evenly sprinkled on a plate containing 20 mg·l hygromycin (hyg). -11 / 2MS solid medium; incubate in dark at 4°C for 2 days, then transfer the culture dish to the Arabidopsis room for culture for about a week; then transfer the hyg-resistant Arabidopsis plants that can grow normally to nutrient soil and culture them in the Arabidopsis room. 16 positive plants were obtained by PCR test (PCR positive test results are shown in Figure 4 ), and phenotypic screening was performed on 1 / 2MS solid medium containing 20 μM Basta (Basta selection results are shown in Figure 5 a) and identified by qRT-PCR (qRT-PCR identification results see Figure 5 b), select OE-5, OE-6 and OE-8 ( Figure 5 5, 6 and 8) in (b) are transgenic Arabidopsis thaliana overexpressing PvHsf16, which were cultured generation by generation until the t3 generation homozygous seeds were harvested and stored at -4°C for the subsequent cadmium resistance function analysis.

[0131] (4) The t3 seeds of OE-5, OE-6, and OE-8 lines and wild-type Arabidopsis seeds (WT) were sown on 1 / 2MS solid medium containing 90 μM and 180 μM CdCl2 using sterilized toothpicks. At the same time, seeds were sown on 1 / 2MS solid medium without cadmium as a control. The results were observed after 10 days. Figure 6 As shown in the figure, the wild-type Arabidopsis and transgenic Arabidopsis OE-5, OE-6 and OE-8 grown on the control 1 / 2MS solid medium were phenotypically normal, with no significant differences. The wild-type and transgenic Arabidopsis plants grown on the 1 / 2MS solid medium containing 90μM and 180μM CdCl2 were inhibited, showing short plant shape and shortened root system. Compared with the WT type, the seedlings of the three transgenic Arabidopsis lines OE-5, OE-6 and OE-8 were larger, with longer root systems, and grew better than the wild type.

[0132] (5) The growth and physiological data of Arabidopsis seedlings under different concentrations of cadmium stress were collected. Figure 7 In the control group without cadmium stress, there was no significant difference in chlorophyll content, average root length and average biomass per plant of Arabidopsis plants; after 90μM and 180μM CdCl2 stress treatment, Arabidopsis growth was inhibited, and the above indicators gradually decreased, but 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 has a higher tolerance to cadmium stress than wild type. Therefore, overexpression of PvHsf16 can significantly improve the cadmium resistance of transgenic Arabidopsis.

Claims

1. A gene PvHsf16 encoding a plant heat shock transcription factor, the nucleotide sequence of which is shown in SEQ NO.

1.

2. The expressed protein of the gene PvHsf16 encoding a plant heat shock transcription factor as claimed in claim 1, wherein the amino acid sequence thereof is shown in SEQ NO.

2.

3. An expression vector containing the gene PvHsf16 encoding the plant heat shock transcription factor according to claim 1.

4. A host cell or host bacteria containing the gene PvHsf16 encoding a plant heat shock transcription factor according to claim 1 or the expression vector of the gene PvHsf16 encoding a plant heat shock transcription factor according to claim 3.

5. Use of the gene PvHsf16 encoding a plant heat shock transcription factor according to claim 1 in improving plant cadmium tolerance.

6. Use of the gene PvHsf16 encoding a plant heat shock transcription factor according to claim 1 in promoting plant growth under cadmium stress or in preparing a product for promoting plant growth under cadmium stress.

7. Use of the gene PvHsf16 encoding a plant heat shock transcription factor according to claim 1 in constructing a transgenic material resistant to cadmium stress.

8. Use of the gene PvHsf16 encoding a plant heat shock transcription factor according to claim 1 in breeding plants for tolerance to cadmium stress.

9. A method for changing plant cadmium stress tolerance, characterized in that: Changing plant cadmium stress tolerance by overexpressing or repressing the expression of the gene PvHsf16 in plants.

10. A preparation for improving plant cadmium stress tolerance, characterized in that The invention relates to an expression promoter or an expression activator of the gene PvHsf16 encoding a plant heat shock transcription factor.

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