Pitaya low-temperature response protein and application thereof

Abstract

The invention relates to a pitaya low-temperature response protein and application thereof, the pitaya low-temperature response protein HubZIP6 is a member of a bZIP gene family, the length is 408 bp, and 135 amino acids are encoded. Through an agrobacterium tumefaciens-mediated arabidopsis thaliana and tomato genetic transformation method, it is found that after HubZIP6 is over-expressed in arabidopsis thaliana and tomato plants, the low-temperature damage degree of a transgenic line is remarkably lower than that of a wild type, and the transgenic line has a high yield by reducing ion permeability and accumulation of malonaldehyde, hydrogen peroxide and superoxide anions. The activity of antioxidant enzymes is increased, and the expression of related cold response genes is promoted, so that the cold resistance of transgenic plants is improved, and the HubZIP6 plays a positive regulation role in the process that the plants respond to low-temperature stress.

Description

Technical Field

[0001] This application relates to the field of plant molecular breeding technology, and in particular to a low-temperature responsive protein in dragon fruit and its application. Background Technology

[0002] Dragon fruit, also known as pitaya, is a fruit belonging to the genus *Pyracantha* in the family Cactaceae. Seleniereus Dragon fruit is a perennial climbing plant native to Central America and is one of the famous tropical fruits. It is a perennial tropical herbaceous fruit that has become popular in my country in recent years. Due to its ease of cultivation, unique appearance, good flavor, and rich content of betaine and other nutrients, it is favored by growers and consumers alike.

[0003] As a tropical fruit tree, dragon fruit has poor cold resistance. Low temperatures often lead to a decline in the yield and quality of dragon fruit, or even crop failure, which is a major limiting factor for the growth, development and industrial development of dragon fruit.

[0004] bZIPs are a class of plant-specific transcription factors, one of the largest transcription factor families discovered to date, widely involved in various biological processes such as plant growth and development, leaf senescence, signal transduction, and stress response. Currently, 75 and 89 bZIPs have been identified in the model plants Arabidopsis thaliana and rice, respectively. bZIP Genes. bZIP transcription factors are involved in various cold response regulatory pathways in plants, but to date, no research has been reported on the role of dragon fruit bZIP transcription factors in low-temperature stress response. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a dragon fruit low-temperature responsive protein and its application.

[0006] The first objective of this invention is to provide a dragon fruit low-temperature responsive protein.

[0007] The second objective of this invention is to provide a dragon fruit low-temperature response gene.

[0008] A third objective of this invention is to provide a biomaterial.

[0009] A fourth objective of this invention is to provide the application of the dragon fruit low-temperature response protein, the dragon fruit low-temperature response gene, or the biomaterial in improving plant cold resistance.

[0010] A fifth objective of this invention is to provide the application of the dragon fruit low-temperature response protein, the dragon fruit low-temperature response gene, or the biomaterial in the creation of highly cold-resistant plant varieties.

[0011] The sixth objective of this invention is to provide a method for improving the cold resistance of plants or creating highly cold-resistant plant varieties.

[0012] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims protection for the following products: A dragon fruit low-temperature responsive protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] SEQ ID NO.2: MMQVSSGESDPRYANMDDKKRKRMISNRESARRSRMKKQQHMDQLLNQVTTLKSEIAQHSQTIDAVSQRYVAVQSENNVLRAKLMELTDRLTSLNSVLKMVEEVGGFSMDIPDVLLEPWHLPCALQPVSNIFQC*.

[0014] A dragon fruit low-temperature response gene, wherein the dragon fruit low-temperature response protein is described.

[0015] Preferably, its nucleotide sequence is as shown in SEQ ID NO.1.

[0016] SEQ ID NO.1: ATGATGCAGGTGAGTTCAGGGTCAGAATCGGACCCTAGATACGCCAACATGGACGACAAGAAGCGCAAGCGGATGATTTCGAACCGCGAGTCGGCTAGGCGATCGAGGATGAAGAAGCAGCAGCATATGGACCAGCTGCTGAACCAGGTGACCACGCTGAAATCAGAGATTGCTCAACATTCTCAGACAATCGATGCCGTCTCG CAGCGCTACGTTGCAGTTCAATCGGAGAACAATGTGCTTCGAGCCAAGCTGATGGAGCTCACTGATAGGCTCACATCCCTGAATTCCGTGCTCAAGATGGTGGAAGAGGTTGGCGGCTTCTCAATGGATATTCCTGATGTTCTGCTGGAGCCATGGCACTTGCCGTGCGCTCTTCAACCAGTATCCAACATCTTCCAATGCTAA.

[0017] As a specific implementation scheme, the nucleotide sequences of its amplification primers are shown in SEQ ID NO.5-6.

[0018] A biological material, which is any one of the following: (1) An expression cassette containing the dragon fruit low-temperature response gene; (2) A recombinant vector containing the dragon fruit low-temperature response gene or the expression cassette described in (1); (3) Recombinant microorganisms containing the dragon fruit low-temperature response gene, the expression cassette in (1) or the recombinant vector in (2); (4) Recombinant cells containing the dragon fruit low-temperature response gene, the expression cassette in (1) or the recombinant vector in (2); This invention also claims protection for the following applications: The application of the dragon fruit low-temperature response protein, the dragon fruit low-temperature response gene, or the biomaterial in improving plant cold resistance.

[0019] The application of the dragon fruit low-temperature response protein, the dragon fruit low-temperature response gene, or the biomaterial in the creation of highly cold-resistant plant varieties.

[0020] The present invention also claims a method for improving the cold resistance of plants or creating highly cold-resistant plant varieties by overexpressing the dragon fruit low-temperature response protein or the dragon fruit low-temperature response gene in the plant.

[0021] Preferably, the plant is Arabidopsis thaliana or tomato.

[0022] Preferably, the dragon fruit low-temperature response gene is cloned into a plant expression vector to obtain a recombinant vector, the recombinant vector is recombined into Agrobacterium to obtain recombinant Agrobacterium, and the plant is genetically transformed using recombinant Agrobacterium, and positive plants are screened to obtain the final product.

[0023] Preferably, the plant expression vector is pPZP6k9.

[0024] Preferably, the Agrobacterium is GV3101.

[0025] More preferably, the plant is a plant of the Brassicaceae, Solanaceae, or Cactaceae families.

[0026] More preferably, the plant is Arabidopsis thaliana, Nicotiana benthamiana, tomato, or dragon fruit.

[0027] More preferably, the nucleotide sequence is as shown in SEQ ID NO. 11-12. Primers are used to detect positive plants.

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention has discovered a novel gene in dragon fruit that responds to low-temperature stress. HubZIP6 , it is bZIPThe gene family member, 408 bp in length, encodes 135 amino acids. The function of the aforementioned dragon fruit cold response gene was verified using Agrobacterium-mediated genetic transformation in Arabidopsis and tomato, revealing overexpression in both plants. HubZIP6 After gene expression, the overexpressing lines suffered significantly less damage from low temperatures than the wild type. The transgenic plants reduced ion osmotic pressure, malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2). .- The accumulation of [a substance] increases the activity of antioxidant enzymes and promotes the expression of related cold-response genes, thereby improving the cold resistance of transgenic plants, indicating [the following]. HubZIP6 It plays a positive regulatory role in the plant's response to low-temperature stress. In the field of agricultural production, HubZIP6 The gene has considerable potential for promotion and application in improving the cold resistance of plants. By using this gene to carry out plant variety improvement work, it is possible to effectively prevent and alleviate the damage of low temperature stress to plants, thereby increasing crop yield and creating more significant economic benefits. Attached Figure Description

[0029] Figure 1 Dragon fruit and Arabidopsis thaliana bZIP An evolutionary tree diagram of genes.

[0030] Figure 2 At a low temperature of 4 ℃, HubZIP6 Gene expression patterns between two dragon fruit varieties with different cold resistance.

[0031] Figure 3 This is an agarose gel electrophoresis image of total RNA extracted from the tender stems of "SCAU-NH2" dragon fruit after low-temperature treatment.

[0032] Figure 4 for HubZIP6 Agarose gel electrophoresis image of gene clone amplification.

[0033] Figure 5 for HubZIP6 Gene subcellular localization map.

[0034] Figure 6 For the transfer HubZIP6 Agarose gel electrophoresis image of PCR amplification product of Arabidopsis thaliana gene (A) and expression level of overexpression lines (B).

[0035] Figure 7 for HubZIP6 Phenotypic results of transgenic Arabidopsis lines (OE#1, OE#2 and OE#5) and WT plant seedlings (one week old) after non-cold acclimatization (NA, -6 ℃, 1 h) and cold acclimatization (CA, -8 ℃, 3 h) low temperature treatment.

[0036] Figure 8 The phenotypes of transgenic lines (OE#1, OE#2 and OE#5) and WT plants after 2 days of low-temperature treatment at 4 ℃ and 6 h of freezing treatment at -6 ℃ are shown (four-week-old plants). Figure 9 The changes in physiological and biochemical indicators of transgenic lines and WT plants after low-temperature treatment.

[0037] Figure 10 The expression changes of cold-response genes in transgenic lines and WT plants after low-temperature treatment.

[0038] Figure 11 For the transfer HubZIP6 Agarose gel electrophoresis image of PCR amplification products of the gene-expressing tomato (A) and the expression level of its overexpression lines (B).

[0039] Figure 12 For the transfer HubZIP6 Phenotypic images of genetically modified tomatoes and wild-type plants before and after low-temperature treatment; A shows the phenotypes of transgenic lines (OE#5, OE#7) and WT plants after 5 days of low-temperature treatment at 4 ℃; B shows the results of NBT and DAB staining of leaves of transgenic lines and WT plants.

[0040] Figure 13 The changes in physiological and biochemical indicators of transgenic lines and WT plants after low-temperature treatment.

[0041] Figure 14 The expression changes of cold-response genes in transgenic lines and WT plants after low-temperature treatment. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0043] Example 1: Dragon Fruit bZIP Screening and cluster analysis of related genes I. Experimental Methods The bZIP conserved domain models PF00170.24, PF03131.20, and PF07716.18 were downloaded from the Pfam database, and all possible bZIP transcription factor sequences were identified in the entire dragon fruit genome (http: / / www.pitayagenomic.com) using HMMER. Further analysis of the Pfam database removed sequences lacking or with incomplete bZIP conserved domains, ultimately identifying 74 bZIP transcription factor sequences from the dragon fruit genome database. bZIP Genes, classified as HubZIP Family genes.

[0044] II. Experimental Results The genes are named sequentially from the first gene on chromosome 1. HubZIP1~HubZIP74 See Table 1.

[0045] Table 1. Identification and renaming of the bZIP gene family in dragon fruit:

[0046] Multiple sequence alignment was performed on 75 Arabidopsis bZIP protein sequences downloaded from TAIR (https: / / www.arabidopsis.org / ) and the dragon fruit bZIP protein sequences in Table 1, with default parameters set. The alignment results were analyzed using MEGAX software to construct a phylogenetic tree. The alignment method was maximum likelihood (ML), with the bootstrap replicates parameter set to 1000. Based on the phylogenetic tree results, the identified dragon fruit bZIP protein family was classified at the subfamily level. The phylogenetic tree results are shown below. Figure 1 As shown.

[0047] Example 2: Dragon Fruit HubZIP 6 Gene expression patterns at low temperatures I. Experimental Methods 1. Experimental Materials Two red-skinned, red-fleshed dragon fruit cuttings (preserved in the Dragon Fruit Germplasm Resource Nursery of the College of Horticulture, South China Agricultural University) are 'SCAU-NH2' (cold-resistant germplasm, collected from Wuming, Guangxi) and 'SCAU-KX2' (cold-sensitive germplasm, collected from Kaixian, Chongqing).

[0048] 2. Low temperature stress treatment The method for low-temperature stress was as follows: using 'SCAU-NH2' and 'SCAU-KX2' dragon fruit cuttings as materials, the cuttings were treated at a temperature of 4℃ in an artificial climate chamber for 0 h, 3 h, 6 h, 12 h, 24 h and 48 h, respectively. The stem segments of the cuttings were then taken, flash-frozen in liquid nitrogen and stored at -80 ℃ for subsequent analysis. Three biological replicates were used.

[0049] 3. Extraction of total RNA from dragon fruit Total RNA was extracted from all samples using the EASYspin Plus Plant RNA Rapid Extraction Kit (RN53) (Aidlab, China) (see instruction manual for specific methods). 2 μL of RNA solution was placed on a nucleic acid analyzer to determine the concentration and purity of the RNA, and its integrity was checked by 1% agarose gel electrophoresis. Samples meeting the requirements were used for the next stage of experiments.

[0050] 4. Transcriptome sequencing Total RNA extracted from the two dragon fruit germplasms after low-temperature treatment was used to construct libraries for transcriptome sequencing. Analysis of the sequencing results revealed that... HubZIP6 (The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.) It is expressed at a low level in the 'SCAU-KX2' transcriptome and at a high level in the 'SCAU-NH2' transcriptome.

[0051] 5. Synthesis of the first strand of cDNA Total RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Japan). Refer to the instruction manual for specific procedures. Store at -20°C for later use. 6. RT-qPCR detection RT-qPCR test HubZIP6 The expression levels of the two dragon fruit germplasms under low-temperature stress at different time points were determined, with three biological replicates for each sample. The specific method was as follows: Using cDNA as a template, dragon fruit HuActin As an internal reference gene, it is used for detection. HubZIP6 Level of expression. Among them, HubZIP6 The upstream and downstream primers for gene quantification are shown in SEQ ID NO.3-4; HuActin The upstream and downstream primers for gene quantification are shown in SEQ ID NO.5-6: Among them, SEQ ID NO.3: ATCGATGCCGTCTCGCAG; SEQ ID NO. 4: AAGCCGCCAACCTCTTCC.

[0052] SEQ ID NO.5:AAAGGCTAACAGGGAGAAAA; SEQ ID NO. 6: GACCACTGGCGTAAAGAGAA.

[0053] RT-qPCR was performed using the RealUniversal Color PreMIX (SYBR Green) (TIANGEN, Beijing) kit on a CFX384-Real-Time system (C1000 Touch Thermal Cycler, USA). After the reaction, RT-qPCR was performed using... Method calculation HubZIP6 Gene expression during different treatment periods.

[0054] II. Experimental Results The results showed that in the 'SCAU-NH2' dragon fruit variety HubZIP6 The expression level was higher at a low temperature of 4 ℃, and the expression level generally showed an increasing trend with the extension of time, while the expression level was lower in the 'SCAU-KX2' dragon fruit variety. Figure 2 ).illustrate HubZIP6 It is a low-temperature response gene.

[0055] Example 3 HubZIP6 Cloning and Vector Construction I. Experimental Methods (1) HubZIP6 amplification Total RNA was extracted from the young stems of 'SCAU-NH2' dragon fruit after low-temperature treatment according to the method in Example 2, and its integrity was detected by electrophoresis on a 1% agarose gel. Then, the first strand of cDNA was synthesized according to the method in Example 2.

[0056] Then, using it as a template, specific primers (upstream and downstream primer sequences are shown in SEQ ID NO.7-8, respectively) were used to... HubZIP6 For PCR amplification, please refer to the instruction manual of ClonExpress Ultra One StepCloning Kit V3 (Vazyme, Nanjing) for the specific reaction system and reaction steps.

[0057] Among them, SEQ ID NO.7: ATGATGCAGGTGAGTTCAGGGT; SEQ ID NO. 8: TTAGCATTGGAAGATGTTGGAT.

[0058] The amplification conditions were: 98 ℃ for 2 min; 98 ℃ for 10 s, 56 ℃ for 15 s, 72 ℃ for 5 min, for a total of 35 cycles; 72 ℃ for 10 min. After the reaction, 2 μL of the amplification product was taken for electrophoresis to check whether the PCR amplification product was correct.

[0059] (2) Recovery of PCR products All PCR amplification products were subjected to agarose gel electrophoresis. The gel was cut under UV light with a scalpel, and the DNA was recovered using the StarPrep Rapid DNA Gel Recovery Kit (see instruction manual for details). After recovery, an appropriate amount of product was taken for agarose gel electrophoresis, and the purity and concentration of the recovered product were determined using a nucleic acid analyzer.

[0060] (3) Connection and transformation of target fragments The purified gel product was mixed with the pEASY-Blunt cloning vector at a molar ratio of 7:1, and ligated at 25 °C for 10 min, following the specific instructions for pEASY. ® -Blunt Cloning Kit Instructions. Add the ligation product to 50 μL of DH5α competent cells, gently swirl to mix, incubate on ice for 30 min, heat shock in a 42 ℃ water bath for 40 s, immediately place on ice for 2 min, then add 600 μL of LB medium on a clean bench, incubate at 37 ℃ with shaking at 200 rpm for 1 h, centrifuge at 4000 rpm for 3 min, reserving 150 μL of supernatant. Resuspend the precipitate using a pipette tip, and then transfer 80 μL to a container containing 100 mg L... -1 Amp + Incubate overnight in an inverted LB agar plate at 37 °C. On the second day, pick a single colony with a sterile toothpick onto a plate containing 100 mg L... -1 Amp + After incubating in 600 μL LB liquid culture medium at 37 ℃ with shaking at 200 rpm for 3–4 h, positive clones were obtained by bacterial PCR. The specific steps are as described in 2× Taq Following the instructions in the Master Mix (Vazyme, Nanjing) user manual, select bacterial cultures with correctly sized amplified bands and send them to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Align the sequenced data with the genome sequence to obtain the ligation sites. HubZIP6 Cloning vector.

[0061] II. Experimental Results The results of agarose gel electrophoresis of total RNA are as follows: Figure 3As shown, the results indicate that the extracted total RNA exhibits two clear bands, 28 S and 18 S, with the 28 S band being approximately twice as bright as the 18 S band. There is no tailing or genomic DNA contamination, indicating that the extracted total RNA is of good quality, with no significant degradation, and can meet the needs of subsequent experiments.

[0062] HubZIP6 The results of gene amplification are as follows Figure 4 As shown, a clear band is present at 408 bp, indicating that the PCR amplification product is correct. (The remaining text appears to be incomplete and possibly contains errors.) HubZIP6 The cloning vector was successfully constructed.

[0063] Example 4 HubZIP6 Subcellular localization analysis I. Experimental Methods 1. Construction of pC18-HubZIP6 subcellular localization recombinant plasmid The pC18-GFP vector was used as the vector for subcellular localization experiments. Hin d III and Bam HI is used as a recombination site, and the specific method is as follows: 1) Prepared according to Example 3 HubZIP6 Using a cDNA cloning vector as a template, the nucleotide sequence is amplified using primers with vector homologous arms (the upstream and downstream sequences of the primers constructed from the pC18-HubZIP6 vector, respectively), as shown in SEQ ID NO. 9–10. HubZIP6 The full-length cDNA sequence of the gene (excluding the stop codon).

[0064] Among them, SEQ ID NO.9: GTCGACGGTATCGATAAGCTTATGATGCAGGTGAGTTCAGGGT; SEQ ID NO. 10: TTTACTCATACTAGTGGATCCGCATTGGAAGATGTTGGAT.

[0065] 2) Then, the amplification product was ligated into the pC18-GFP vector according to the instructions of ClonExpress Ultra One Step Cloning Kit V3 (Vazyme, Nanjing). The PCR ligation product was then transformed into E. coli DH5α competent cells, using the same method as in Example 3, with kanamycin (Kan) as the antibiotic.

[0066] 3) Perform bacterial culture PCR detection using primers with nucleotide sequences as shown in SEQ ID NO. 9-10. Sequencing the PCR-positive strains yields the pC18-HubZIP6 subcellular localization recombinant plasmid.

[0067] 2. Recombinant plasmids were transformed into Agrobacterium tumefaciens using the following transformation method: 1) Remove Agrobacterium GV3101 (pSoup+p19) competent cells from the -80 ℃ freezer and place them on ice for 5 min until the bacterial solution thaws.

[0068] 2) Add 5 µL of recombinant plasmid pC18-HubZIP6 to 50 µL of Agrobacterium competent cells, gently tap the centrifuge tube to mix it, place it on ice for 30 min, freeze it in liquid nitrogen for 5 min, incubate it in a water bath at 37 ℃ for 5 min, and then in an ice bath for 2 min. 3) Add 600 µL of antibiotic-free YEP liquid medium to a clean bench, incubate at 28 °C and 200 rpm with shaking for 3–4 h, centrifuge at 4000 rpm for 5 min, and retain 100 μL of supernatant. Resuspend the precipitate with a pipette tip, and then spread 30 μL onto YEP solid agar plates (containing 100 mg L of antibiotics). -1 Kan and 50 mg L -1 Rif) was incubated upside down in a 28 ℃ incubator for 2–3 days.

[0069] 4) Use a sterile toothpick to pick a single colony in a solution containing 100 mg L -1 Kan and 50 mg L -1 In 600 μL of YEP liquid culture medium, Rif cultured overnight at 28 ℃ with shaking at 200 rpm. The bacterial culture PCR verification followed the same steps as above. Positive Agrobacterium strains with correct verification results were stored for later use.

[0070] 3. Transient expression of Agrobacterium tumefaciens in tobacco leaves Using an empty vector as a control, the cells were transformed into GV3101 (pSoup+p19) competent cells according to method 2 above. The positive bacterial culture was added to 10 mL of a solution containing 100 mg L at a ratio of 1:300. -1 Kan and 50 mg L -1 Rif was cultured in YEP liquid medium at 28°C with shaking at 200 rpm until OD. 600 After reaching 0.6–0.8, resuspend in MAA solution (0.5 μM MES, 0.02 μM AS, 0.5 μM MgCl2, pH 5.7) until OD is reached. 600 =0.2. Inject 1 mL of the solution into the underside of *Nicotiana benthamiana* leaves, simultaneously injecting *Agrobacterium* containing the empty vector pC18 as a control. Allow the bacterial solution to spread throughout the entire leaf. Inject two plants (three leaves per plant) for each treatment and label them. After incubating *Nicotiana benthamiana* at 25 °C for 2-3 days, observe the fluorescence using a laser confocal microscope (ZEISS, Germany) as follows: 1) Take leaves of *Tobacco Benedict* that have been treated with *Agrobacterium* for 2 days, and cut the infiltrated area of ​​the leaf into pieces of about 1 cm. 2 Place a small piece on the water droplet on the slide and moisten it. Carefully cover it with a coverslip using tweezers, being careful not to leave any air bubbles.

[0071] 2) Place the prepared slide under a laser confocal microscope (ZEISS LCM-800, Germany), set the microscope eyepiece to 20×, and observe the leaf epidermal cells under bright field.

[0072] 3) Using the software, select the excitation wavelength of GFP (488 nm) and mCherry (610 nm), scan under different excitation lights, select the area where most cells are on the same plane, adjust the fluorescence field GFP, mCherry and bright field gain to the optimal, perform dual-channel fluorescence imaging, and take pictures.

[0073] II. Experimental Results The results are as follows Figure 5 As shown, in the transformed tobacco leaves, the GFP fluorescence signal of the control vector filled the entire cell, while the RFP fluorescence signal was only displayed in the cell nucleus. However, after pC18-HubZIP6 was transformed into tobacco leaves, the positions of its GFP and RFP fluorescence signals completely overlapped, and both were only displayed in the cell nucleus, indicating that HubZIP6 is located in the cell nucleus.

[0074] Example 5 Agrobacterium tumefaciens-mediated HubZIP6 Arabidopsis genetic transformation I. Experimental Methods 1. Construction of vectors, recombinant plasmids and transformation of Agrobacterium tumefaciens Will HubZIP6 Genes were constructed into the 35S-driven expression vector pPZP6k90. Xba At the I restriction site, the primer nucleotide sequence containing the vector homologous arm is shown in SEQ ID NO. 11-12, and the recombinant vector pPZP6K90-HubZIP6 is prepared. SEQ ID NO.11: CATTCTACAACTACATCTAGAATGATGCAGGTGAGTTCAGGGT; SEQ ID NO. 12: AGCTTGCATGCCAATTCTAGATTAGCATTGGAAGATGTTGGAT.

[0075] The recombinant vector pPZP6K90-HubZIP6 was transformed into Agrobacterium GV3101. After the bacterial culture was detected as positive by PCR, it was stored for later use. The specific operation was the same as in Example 4.

[0076] 2. Agrobacterium tumefaciens-mediated transformation in Arabidopsis thaliana (1) In a clean bench, add 1 mL of the above bacterial solution to 200 mL of YEP medium (containing 100 mg / L of antibiotics). -1 Kan and 50 mg L -1 In Rif), incubate overnight at 28 ℃ and 220 rpm with shaking until OD reaches the specified value. 600 =0.8-1.0. Centrifuge at 4000 rpm for 10 min, collect the bacterial cells, and dilute them in a large petri dish with flower immersion medium [1 / 2 MS + 5% sucrose + 0.03% surfactant (Silwet L-77), pH 5.8]. Adjust OD. 600 =0.6~0.8, to be used.

[0077] (2) Select robust Arabidopsis plants in full bloom, remove the open flowers and pods, lay the Arabidopsis plants to be transformed flat, and immerse the flower buds completely in the Agrobacterium suspension for about 1 minute. Then, remove the culture pot, tilt it, and place it on a large tray to dry the excess liquid on the surface of the Arabidopsis. Cover the treated Arabidopsis with a plastic lid and incubate in the dark for 24 hours. Then, place it under light conditions of 23-25℃ to allow it to grow normally. It can be inoculated again after 1 week. After 3-4 weeks, when the Arabidopsis pods begin to turn yellow, cut them off and place them in a culture dish to dry. When most of the Arabidopsis pods have turned yellow, collect the seeds and put them into 1.5 mL centrifuge tubes (add an appropriate amount of silica gel to keep them dry). After they are completely dry, transfer them to new centrifuge tubes. For short-term storage, it can be placed in a 4 ℃ refrigerator; for long-term storage, it should be placed in a -20 ℃ refrigerator.

[0078] (3) Take a portion of Arabidopsis thaliana seeds (200-300 seeds) on a clean bench and put them into a 1.5 mL sterile centrifuge tube. First, treat the seeds with 70% alcohol twice, 30 s each time; then suspend the seeds in anhydrous ethanol and pour them onto a sterile filter paper; after the anhydrous ethanol has evaporated, sow the seeds evenly on the seed selection medium (1 / 2 MS + 30 g sucrose L). -1 +5~6 g L of agar -1 +Kan 100 mg L -1 (pH 5.8); seal the petri dishes with Parafilm and place them in a 4 ℃ refrigerator for 24 h, then place them in a 23-25 ​​℃ environment with 16 h light / 8 h darkness for 8-10 days before transplanting them into nutrient pots. After culturing in a culture room for 2-3 weeks, further identification can be performed.

[0079] 3. Identification of transgenic plants After the transplanted seedlings have survived, take leaves approximately 2 mm in size and perform PCR amplification using the T5 Direct PCR Kit (Plant) (TSINGKE) to obtain positive plants. Refer to the instruction manual for specific steps. The upstream and downstream primer sequences for PCR are shown in SEQ ID NO. 13-14. SEQ ID NO.13: CGGAGAGGTACGTATTTTTACAAC; SEQ ID NO. 14:TCAACACATGAGCGAAACCCTATA.

[0080] After identifying positive plants, T1 generation seeds were collected by division. The T1 generation seeds were then sterilized and sown in a soil containing 100 mg L... -1 Normally growing seedlings were transplanted onto Kans 1 / 2 MS medium and cultured until seed maturity to harvest T2 generation seeds. Seed sterilization and resistance screening were repeated until resistance-free lines emerged for subsequent experiments.

[0081] Further RT-qPCR was used to detect the presence of [unclear - possibly a specific substance or pattern] in homozygous transgenic plants. HubZIP6 The level of expression.

[0082] After sterilization, the homozygous seeds of the transgenic line were sown in a solution containing 100 mg L -1 On Kan's 1 / 2MS medium, after vernalization at 4 °C for 2 days, the cells were grown under normal conditions for about 10 days, then transplanted into the substrate and cultured for about 2 weeks before undergoing low-temperature treatment.

[0083] 4. Freezing treatment of one-week-old Arabidopsis thaliana One-week-old overexpression line seedlings were subjected to non-cold acclimatization treatment (NA, seedlings were directly frozen at -6℃ for 1 h without 4 ℃ pretreatment) and cold acclimatization treatment (CA, seedlings were pretreated at 4℃ for 48 h and then frozen at -8℃ for 3 h). Wild type was used as control. The phenotype of seedlings before and after treatment was observed and photographed (before treatment, control).

[0084] 5. Cold treatment and freezing treatment of four-week-old Arabidopsis thaliana (1) Processing method Four-week-old plants were subjected to cold treatment (4 ℃ for 48 h) and freezing treatment (-6 ℃ for 6 h) sequentially, as follows: First, the incubator was cooled to 4 ℃, and the potted seedlings were transferred from normal conditions to the 4 ℃ incubator for 2 days of low-temperature acclimatization; then the potted seedlings were placed in a -6 ℃ incubator for 6 hours; finally, they were transferred to a 4 ℃ incubator for 12 hours of dark treatment, and then resumed growth under normal conditions.

[0085] (2) Detection of Arabidopsis thaliana growth status Survival rates were calculated 5 days and 2 weeks after recovery under normal conditions; phenotypic observations, photographs, and determination of cold-resistance-related physiological and biochemical indicators were conducted before low-temperature treatment, after low-temperature treatment, and 5 days and 2 weeks after recovery.

[0086] (3) Detection of relevant physiological and biochemical indicators Arabidopsis leaves were taken before treatment (22℃), after treatment at 4℃ for 48 h, and after treatment at -6℃ for 6 h, and ion leakage was measured.

[0087] Arabidopsis thaliana leaves treated at 4 ℃ for 24 h and 48 h were collected, and malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2) were measured. .- The contents of plant ion permeability, MDA, H2O2, and O2 were determined using a kit (Suzhou Comin Biotechnology, China). .- For the content, please refer to the kit instructions for specific methods.

[0088] 6. Changes in the expression of downstream cold response genes Transgenic lines and wild-type Arabidopsis plants were subjected to low-temperature treatment in a 4 ℃ incubator. Leaves were collected at 0 h, 6 h and 12 h, respectively. The methods for RNA extraction and cDNA synthesis are described in Example 2.

[0089] RT-qPCR was used to analyze transgenic lines and wild-type Arabidopsis thaliana plants. CBF Gene( AtCBF1, AtCBF2 and AtCBF3 ) and cold response genes ( AtRD29A , AtCOR15A , AtCOR47 and AtKIN1 Expression changes in Arabidopsis thaliana before and after low-temperature treatment. AtACTIN2 (AT1G13320) is the internal reference gene, used... The data were analyzed using the following method. All experiments were performed in triplicate. The relevant primers are shown in Table 2 below.

[0090] Table 2:

[0091] II. Experimental Results The identification results of the transgenic plants are as follows: Figure 6 As shown in A, WT is the negative control, and it is not present in wild-type Arabidopsis thaliana. HubZIP6Gene. P1 is the positive control, consisting of the pPZP6K90-HubZIP6 plasmid. Lanes A1-A2 and A4-A10 all show a distinct single band at approximately 450 bp, indicating successful transfer of the target gene into Arabidopsis thaliana. Different lines of transgenic Arabidopsis thaliana... HubZIP6 Gene expression results as follows Figure 6 As shown in B, the transgenic lines OE#1, OE#2, and OE#5 are... HubZIP6 Since the expression level was high, three transgenic lines, HubZIP6-OE#1, HubZIP6-OE#2 and HubZIP6-OE#5, were selected for subsequent low-temperature treatment experiments.

[0092] Results of freezing treatment (-4 ℃) on one-week-old seedlings showed heterologous overexpression HubZIP6 It can significantly improve the frost resistance of Arabidopsis thaliana. Figure 7 The results of revival after freezing treatment of four-week-old plants showed... HubZIP6 Overexpression lines suffered significantly less damage from low temperatures than wild types, and most plants were able to recover growth, bolting, and flowering after frost treatment. Figure 8 Overall, under low-temperature stress, HubZIP6 The survival rate of the overexpression lines was significantly higher than that of the wild-type plants.

[0093] The changes in relevant physiological and biochemical indicators of transgenic lines and WT plants after low-temperature treatment are shown in the figure. Figure 9 Ion permeability is an important indicator of cell membrane damage in plants during environmental stress responses. Ion permeability measurements showed that under normal conditions (22 ℃), ion permeability... HubZIP6 The ion permeability level in the overexpression lines was not significantly different from that in the WT plants, but after cold treatment (4 ℃ for 48 h) and freezing treatment (-6 ℃ for 6 h), HubZIP6 The ion permeability of the overexpression lines was significantly lower than that of the WT plants, indicating that... HubZIP6 Overexpression lines exhibited stronger cold resistance and less damage from low-temperature treatment, while WT plants showed severe cell membrane damage and increased permeability, resulting in greater electrolyte leakage from the cells. Compared to WT, HubZIP6 MDA content, H2O2 and O2 in overexpression lines .- The accumulation of [something] exhibits a similar trend to that of ion permeability. Under normal conditions (22 °C). HubZIP6 MDA, H2O2 and O2 in overexpression lines and WT plants .- The content was low and showed no significant difference; however, when the plants were subjected to low temperature stress, the content gradually increased in all strains, with MDA, H2O2, and O2 showing higher levels in the overexpressing lines. .- The content was significantly lower than that of WT plants.

[0094] Three transgenic lines and wild-type plants under low temperature stress CBF Gene( AtCBF1 , AtCBF2 and AtCBF3 Changes in the expression of ) and its downstream cold response genes are shown in Figure 10 Under normal conditions (22 ℃). AtCBF1 , AtCBF2 and AtCBF3 Expression was relatively low in transgenic lines and WT plants with no significant difference, but after low-temperature treatment (4 ℃ for 6 h), expression in transgenic lines increased significantly. AtCBF1 , AtCBF2 and AtCBF3 The expression level was significantly higher than that in WT plants, and the highest expression level was observed at 12 h. COR Genes are a class of downstream target genes regulated by CBF, such as AtRD29A , AtCOR15A , AtCOR47 and AtKIN1 Under normal conditions, COR The gene expression was relatively low in transgenic lines and WT plants with no significant difference, but was significantly induced after cold treatment (4 ℃ for 6 h). Furthermore, during the cold treatment process, the expression of the gene in transgenic lines... COR The expression levels of these genes were significantly higher than those in the WT plants. These results indicate that under low-temperature stress, HubZIP6 Overexpression can regulate Arabidopsis thaliana AtCBF1 , AtCBF2 and AtCBF3 and its downstream COR Gene expression, in turn, enhances the cold resistance of transgenic plants.

[0095] Example 6 Agrobacterium tumefaciens-mediated HubZIP6 Tomato genetic transformation I. Experimental Methods 1. Construction of vectors, recombinant plasmids and transformation of Agrobacterium tumefaciens Following the method in Example 5, the recombinant vector pPZP6K90-HubZIP6 was transformed into Agrobacterium GV3101, and the bacterial culture was positive by PCR detection.

[0096] 2. Disinfection and sowing of tomato seeds Take an appropriate amount of tomato seeds and place them in a 50 mL centrifuge tube. Disinfect with 75% ethanol for 1 min, then disinfect with 10% sodium hypochlorite solution for 20 min. Discard the liquid and rinse with sterile water 4-5 times. Sow the seeds on 1 / 2 MS medium + 3% sucrose + 0.66% agar (pH=5.8). After treating at 4 ℃ for 24 h, place them in a light incubator for cultivation (day and night temperature 25 ℃ / 20 ℃, photoperiod 16 h / 8 h, light intensity 6000 Lux).

[0097] 3. Pre-culture Cut off the cotyledons of tomato plants cultured for 9–10 days, remove the top and bottom ends, and keep the middle part (if it is too large, it can be divided into two parts). Inoculate the middle part onto the pre-medium (MS + 3% sucrose + 0.66% agar + 1 mg / L ZR + 1 mg / L GBR5, pH=5.8) and pre-culture in the dark at 20 ℃ for 24 h.

[0098] 4. Preparation of Agrobacterium The specific operation is the same as in Example 5. However, the activated cells were resuspended in an infection solution (MS + 2% sucrose + 0.02% KH₂PO₄ + 1 mg / L GBR₅ + 200 mM AS + 0.2 mg / L 2,4-D + 0.1 mg / L KT, pH=5.7), and the OD₂O₅ of the bacterial solution was adjusted. 600 Adjust it to 0.2-0.3.

[0099] 5. Infection of explants The pre-cultured explants were placed in the activated bacterial solution and soaked for 5 minutes. The excess bacterial solution was then removed by blotting on filter paper. The explants were then inoculated into a new pre-culture medium and cultured in the dark at 20 °C for 3 days.

[0100] 6. Differentiation and culture of explants: a. After dark culture, the explants were transferred to callus induction medium (MS + 3% sucrose + 0.66% agar + 2 mg / L ZR + 1 mg / L BG0031 + 175 mg / L Kan, pH=5.8) and cultured at 25 ℃. The callus induction medium was changed every two weeks. b. Once callus tissue has grown from the explants, they are transferred to subculture medium (MS + 3% sucrose + 0.66% agar + 1 mg / L ZR + 1 mg / L BG0031 + 175 mg / L Kan, pH=5.8) and cultured at 25 ℃, with the subculture medium being changed every two weeks.

[0101] 7. Rooting culture of explants a. When the new shoots of the explants grow to about 2 cm, cut them off and inoculate them into rooting medium (1 / 2 MS + 1% sucrose + 0.66% agar + 0.5 mg / L BG0031 + 0.025 mg / L NAA + 0.5 mg / L NT, pH=5.8), and culture at 25 ℃ for 15–25 days; b. Harden off the rooted plants. After 3 days, wash the plants with tap water and transplant them into the substrate for cultivation (keeping them warm and moist during the process). 8. Identification of genetically modified tomato plants Genomic DNA and RNA were extracted from the plant leaves, and PCR and qRT-PCR techniques were used to detect whether the target gene was successfully transferred into the tomato plant genome and expressed. The detection steps for transformed plants and the upstream and downstream primer sequences for PCR are described in Example 5. T0 generation positive plants were cultured to harvest T1 generation seeds, and T1 generation plants were replanted to obtain T2 generation seeds.

[0102] 9. Low-temperature treatment of genetically modified tomato plants One-month-old T2 generation tomato plants were placed in a 4 ℃ incubator (photoperiod of 16 h / 8 h, relative humidity of 60-70%, light intensity of 6000 Lux) for low-temperature treatment, and the phenotype of the transgenic tomato plants was observed after 5 days.

[0103] 10. DAB and NTB staining experiments Leaves were collected before low-temperature treatment (25 ℃) and 5 days after low-temperature treatment. DAB and NBT staining experiments (DAB method and NBT method) were performed using a plant tissue reactive oxygen species detection kit (Coolaber, Beijing). The specific methods were described in the kit instructions.

[0104] 11. Determination of the activity of reactive oxygen species (ROS) scavenging enzymes and proline (Pro) content. Tomato leaves were taken before low-temperature treatment (22 °C) and 48 h after low-temperature treatment to measure ion permeability.

[0105] Tomato leaves were taken before low-temperature treatment (22 °C) and 48 h after low-temperature treatment, and treated with MDA, H2O2, and O2, respectively. .- Assay kit (Suzhou Comin Biotechnology, Co., Ltd., China) for the determination of MDA, H2O2 and O2 .- For the content of [specific ingredient], please refer to the kit instructions for details.

[0106] Before low-temperature treatment (22 °C) and 48 h after low-temperature treatment, the levels of wild-type and [unspecified] plants were measured using a plant superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and proline (Pro) assay kit (Suzhou CominBiotechnology, Co., Ltd., China). HubZIP6 The SOD, POD, CAT activities and Pro content of overexpressed tomato plants were determined by referring to the kit instructions.

[0107] 12. Changes in the expression of downstream cold response genes Wild type and HubZIP6The RNA extraction and cDNA synthesis methods for leaves of overexpressing tomato plants before (25 °C) and after 24 h of low-temperature treatment are described in Example 2.

[0108] RT-qPCR was used to detect wild-type and HubZIP6 Overexpression in tomato plants SlCBF1 , SlCBF2 , SlCBF3 and SlCOR47 Changes in tomato gene expression before and after low-temperature treatment. SlACTIN As an internal reference gene, used The data were analyzed using the following method. All experiments were performed in triplicate. The relevant primers are shown in Table 3 below.

[0109] Table 3:

[0110] II. Experimental Results The identification results of the transgenic plants are shown in [link to relevant documentation]. Figure 11 ,like Figure 11 As shown in A, WT is the negative control, which is absent in wild-type tomatoes. HubZIP6 Gene. P1 is the positive control, consisting of the pPZP6K90-HubZIP6 plasmid. Lanes A4, A5, A7, and A9 all show a distinct single band at approximately 450 bp, indicating that the target gene has been successfully transferred into the tomato genome. Among other things, such as... Figure 11 B in the figure shows that the OE#5 and OE#7 transgenic lines HubZIP6 Since the expression level was high, HubZIP6-OE#5 and HubZIP6-OE#7, two transgenic lines, were selected for subsequent low-temperature treatment experiments.

[0111] After 5 days of low-temperature treatment, the leaves of WT plants showed significant shrinkage and drooping, while the leaves of two transformed plants... HubZIP6 The genetically modified tomato strains have fewer wilted leaves but can still grow normally, indicating that... HubZIP6 Overexpression lines exhibit stronger cold resistance and experience less damage from low-temperature treatment. Figure 12 (A in the middle).

[0112] NBT and DAB staining of leaves from transgenic and wild-type plants before and after low-temperature treatment revealed that the leaves of transgenic plants were lighter in color after staining, indicating that the transgenic plants contained higher levels of H2O2 and O2 compared to the wild-type plants. .- Lower content ( Figure 12 (B in the middle).

[0113] The results of ROS scavenging enzyme activity and Pro content determination in transgenic lines and WT plants showed that ( Figure 13 Under low temperature stress, compared with WT plants, HubZIP6 Overexpression significantly increased the activities of SOD, POD, and CAT in tomato plants, and increased the content of Pro. HubZIP6 Ion permeability, MDA content, H2O2 and O2 in overexpression tomato lines .- The accumulation of these substances shows a similar trend. At normal temperatures (22 ℃), HubZIP6 Ion osmotic pressure, MDA, H2O2, and O2 in overexpressed lines and wild-type (WT) plants .- The contents of all three were low and showed no significant difference; however, after low-temperature stress treatment, the contents of ion permeability, MDA, H2O2, and O2 increased. .- In both transgenic and wild-type plants, the ion permeability, MDA, H2O2, and O2 levels gradually increased with prolonged stress time in transgenic lines. .- The content was significantly lower than that of WT plants, cold response gene SlCBF1 , SlCBF2 , SlCBF3 and SlCOR47 exist HubZIP6 The expression level was significantly increased in overexpressing tomato plants. Figure 14 ).

[0114] The above results indicate that overexpression HubZIP6 The cold resistance of tomato plants can be improved by enhancing the activity of antioxidant enzymes in transgenic plants and promoting the expression of cold response-related genes.

Claims

1. A dragon fruit low-temperature responsive protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. A dragon fruit low-temperature response gene, characterized in that, It encodes the dragon fruit low-temperature response protein as described in claim 1.

3. The dragon fruit low-temperature response gene according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

4. A biomaterial, characterized in that, It can be any of the following: (1) An expression cassette containing the dragon fruit low-temperature response gene as described in claim 2; (2) A recombinant vector containing the dragon fruit low-temperature response gene as described in claim 2 or the expression cassette as described in (1); (3) A recombinant microorganism containing the dragon fruit low-temperature response gene of claim 2, the expression cassette of (1) or the recombinant vector of (2); (4) Recombinant cells containing the dragon fruit low-temperature response gene of claim 2, the expression cassette of (1) or the recombinant vector of (2).

5. The application of the dragon fruit low-temperature response protein of claim 1, the dragon fruit low-temperature response gene of claim 2 or 3, or the biomaterial of claim 4 in improving plant cold resistance.

6. The application of the dragon fruit low-temperature response protein of claim 1, the dragon fruit low-temperature response gene of claim 2 or 3, or the biomaterial of claim 4 in the creation of highly cold-resistant plant varieties.

7. A method for improving the cold resistance of plants or creating highly cold-resistant plant varieties, characterized in that, Overexpression of the dragon fruit low-temperature response protein of claim 1 or the dragon fruit low-temperature response gene of claim 2 in plants.

8. The method according to claim 7, characterized in that, The dragon fruit low-temperature response gene described in claim 2 is cloned into a plant expression vector to obtain a recombinant vector. The recombinant vector is then recombined into Agrobacterium to obtain recombinant Agrobacterium. Plants are genetically transformed using the recombinant Agrobacterium, and positive plants are screened to obtain the final product.

9. The method according to claim 8, characterized in that, The plant expression vector is pPZP6k9.

10. The method according to claim 8, characterized in that, The Agrobacterium species in question is GV3101.

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