A Siberian apricot PsCBF2 gene and its application in cold resistance and delayed flowering

Abstract

The present invention discloses a Siberian apricot PsCBF2 gene and its use in cold resistance and delayed flowering. The coding sequence of the PsCBF2 gene is shown in SEQ ID NO.1, and the protein sequence encoded by it is shown in SEQ ID NO.2. The gene can be used to improve the cold stress response ability of Siberian apricot and Arabidopsis thaliana, promote their vegetative growth, delay flowering, and reduce the proline content in plants under cold stress conditions. Experiments have shown that Arabidopsis thaliana introduced with the PsCBF2 gene exhibits lower ion leakage rate and proline content under cold stress conditions, and has good antifreeze performance. The present invention also discloses a recombinant vector and recombinant engineered bacteria containing the PsCBF2 gene. The recombinant vector and recombinant engineered bacteria containing the PsCBF2 gene can be used in research related to plant cold resistance and delayed flowering.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, specifically a Siberian apricot PsCBF2 gene and its application in cold resistance, promoting vegetative growth and delaying flowering. Background Art

[0002] The Siberian apricot, a kernel-producing apricot, is an important ecological and economically important native tree species in my country's Three Norths region. In these shelterbelt regions, vigorously developing kernel apricots not only improves the ecological environment, prevents soil erosion and desertification, but also yields significant short-term economic benefits. With an oil content of 50-60% and a protein content of approximately 35%, the apricot tree is an excellent woody oil crop and high-quality plant protein. However, due to its early flowering and fruiting period, the Siberian apricot tree is susceptible to late frosts and cold snaps, which can cause freezing of flowers and fruits, leading to reduced yields and total crop failure, severely impacting both production and the development of the industry. Annual yield losses due to freezing during the flowering and fruiting period have become a limiting factor in the development of the kernel apricot industry. Therefore, studying the cold tolerance mechanisms of the Siberian apricot, identifying genes associated with cold tolerance, and improving its cold tolerance through genetic engineering are of great significance.

[0003] CBF transcription factors, also known as dehydration-responsive element (CRT / DRE) binding factors, possess a conserved AP2 / EREBP domain and are a subfamily of the plant AP2 / ERF transcription factor superfamily. CBF proteins can bind to CRT / DRE elements in the promoters of downstream genes, thereby activating the expression of downstream cold-responsive genes (CORs) and improving plant tolerance to freezing. CBF genes play a key role in signal transduction and inducing the expression of numerous downstream stress-response genes. However, the role of PsCBF genes in responding to cold stress, delaying flowering, and promoting vegetative growth in Siberian apricot has not been reported. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a Siberian apricot PsCBF2 gene and its application in cold resistance and delayed flowering. When highly expressed in plants, this gene can enhance the plant's cold stress response ability, promote its vegetative growth, and delay flowering.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] A Siberian apricot PsCBF2 gene, the coding sequence of the Siberian apricot PsCBF2 gene is shown as SEQ ID NO.1, and the protein sequence encoded by the Siberian apricot PsCBF2 gene is shown as SEQ ID NO.2.

[0007] The above-mentioned application of the Siberian apricot PsCBF2 gene is to use the Siberian apricot PsCBF2 gene to improve the cold stress response ability of Siberian apricot and / or Arabidopsis, or to use the Siberian apricot PsCBF2 gene to promote the nutritional growth of Siberian apricot and / or Arabidopsis, or to use the Siberian apricot PsCBF2 gene to delay the flowering period of Siberian apricot and / or Arabidopsis.

[0008] In the above-mentioned application, when the Siberian apricot PsCBF2 gene is used to improve the cold stress response ability of Siberian apricot and / or Arabidopsis, or to promote the vegetative growth of Siberian apricot and / or Arabidopsis, or to delay the flowering period of Siberian apricot and / or Arabidopsis, the expression level of the Siberian apricot PsCBF2 gene in Siberian apricot and / or Arabidopsis is increased. The expression level of the Siberian apricot PsCBF2 gene will increase when subjected to cold stress. In addition, using a plant expression vector containing the PsCBF2 gene coding sequence to transform Arabidopsis can increase the expression level of the PsCBF2 gene in Arabidopsis. Theoretically, the plant expression vector containing the PsCBF2 gene coding sequence can also be used to transform other plants to increase the expression level of the PsCBF2 gene in the transformed plants.

[0009] A recombinant vector containing the Siberian apricot PsCBF2 gene is provided. The recombinant vector is a plant expression vector and contains the coding sequence of the Siberian apricot PsCBF2 gene.

[0010] The original vector of the above-mentioned recombinant vector is pMDC32.

[0011] The recombinant vector is constructed by amplifying the coding sequence of the Siberian apricot PsCBF2 gene using primers with sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. The template DNA used in the amplification is cDNA obtained by reverse transcription of Siberian apricot Z6 flower bud RNA. PCR amplification of the Siberian apricot PsCBF2 gene uses cDNA obtained by reverse transcription of Siberian apricot Z6 flower bud RNA as a template. The PCR amplification yields the coding sequence (CDS) of the PsCBF2 gene. cDNA is obtained by reverse transcription of mRNA directly involved in protein translation. Using cDNA as a PCR template, the coding sequence of the PsCBF2 gene obtained by PCR is accurate and reliable.

[0012] A recombinant engineered bacterium contains the coding sequence of the Siberian apricot PsCBF2 gene.

[0013] A primer set for quantitative PCR of the Siberian apricot PsCBF2 gene comprises primers with sequences set forth in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 7, and SEQ ID NO. 8. The primers with sequences set forth in SEQ ID NO. 7 and SEQ ID NO. 8 are used to amplify 18sRNA during quantitative PCR and serve as internal controls.

[0014] A method for reducing the proline content in a plant under cold stress conditions, comprising transferring a recombinant vector containing the coding sequence of the Siberian apricot PsCBF2 gene into the plant.

[0015] In the above method, the plant is Arabidopsis thaliana; and the original vector of the recombinant vector is pMDC32.

[0016] The technical solution of the present invention achieves the following beneficial technical effects:

[0017] The Siberian apricot PsCBF2 gene, provided by the present invention, significantly improves plant viability and cold resistance under cold stress conditions when highly expressed in plants. This improvement in cold stress resistance is independent of proline-based stress protectants. This discovery provides a new candidate gene for plant cold resistance genetic engineering, potentially enhancing crop adaptability and yield in cold regions.

[0018] 2. By regulating the expression of the PsCBF2 gene, the present invention effectively delays the flowering period of Arabidopsis thaliana. This method can also be used to delay the flowering period of Siberian apricot, thereby preventing damage to the flowering and fruiting stages caused by early spring cold snaps. This technological improvement helps stabilize and increase crop yields and reduce the risk of yield losses due to frost damage.

[0019] 3. The PsCBF2 gene is closely associated with cold stress response, and its expression level increases when induced by low temperatures. This study, through heterologous expression experiments in Arabidopsis thaliana, demonstrated that high expression of the PsCBF2 gene promotes plant vegetative growth, imparting greater environmental adaptability and enabling plants to maintain stable growth despite potential temperature fluctuations or other abiotic stresses.

[0020] 4. The PsCBF2 gene and its encoded protein have broad application prospects in plant genetic engineering and can be used for genetic improvement of a variety of crops. In particular, for important ecological and economic tree species in my country's "Three Norths" region, such as the Siberian apricot, the application of this invention will significantly improve their cold resistance and growth performance, thereby promoting improvements in the local ecological environment and increasing farmers' incomes, with significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Electrophoresis of the products obtained by amplifying Siberian apricot cDNA using primers ATTB1-PsCBF2 and ATTB2-PsCBF2 in the examples of the present invention;

[0022] Figure 2 Figure 2 shows the test results of the PsCBF2 gene expression level in transgenic Arabidopsis thaliana in the embodiment of the present invention;

[0023] Figure 3 Frontal photos of transgenic PsCBF2-positive Arabidopsis thaliana and wild-type Arabidopsis thaliana at 36 days of age in the examples of the present invention;

[0024] Figure 4 Top view photos of transgenic PsCBF2-positive Arabidopsis thaliana and wild-type Arabidopsis thaliana at 36 days of age in the examples of the present invention;

[0025] Figure 5 Statistical results of the average bolting time and average number of rosette leaves of PsCBF2 gene-positive transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana in the embodiment of the present invention;

[0026] Figure 6 Comparison of the growth of PsCBF2 gene-positive Arabidopsis thaliana and wild-type Arabidopsis thaliana before and after cold stress treatment in the embodiment of the present invention;

[0027] Figure 7 The results of measuring changes in PsCBF2 gene expression in vivo in a cold stress experiment in PsCBF2 gene-positive Arabidopsis thaliana and wild-type Arabidopsis thaliana in the embodiments of the present invention;

[0028] Figure 8 The test results of the change of leaf ion leakage rate in the cold stress experiment of PsCBF2 gene positive Arabidopsis thaliana and wild-type Arabidopsis thaliana in the embodiment of the present invention;

[0029] Figure 9 The test results of proline content in the cold stress experiment of PsCBF2 gene-positive Arabidopsis thaliana and wild-type Arabidopsis thaliana in the embodiment of the present invention;

[0030] Figure 10 The expression level of the PsCBF2 gene at different stages of Siberian apricot flower bud development in the embodiment of the present invention;

[0031] Figure 11 A graph comparing the expression level of the PsCBF2 gene in Siberian apricot flower buds and the average daily temperature in the examples of the present invention. DETAILED DESCRIPTION

[0032] Example 1 Cloning of the Siberian Apricot PsCBF2 Gene

[0033] First, extract total RNA from the flower buds of Siberian apricot Z6. Follow the steps below:

[0034] 1) Grind the flower bud tissue sample in liquid nitrogen until it is finely powdered. Add 1 mL of TRIzol reagent per 100 mg of fresh tissue to prepare a homogenate.

[0035] 2) Transfer the homogenate to a 1.5 mL RNase-free EP tube. Place on ice and let stand for 5 minutes.

[0036] 3) Add 200 μL of chloroform to each tube, mix thoroughly, and let stand on ice for 10 minutes to completely dissociate the nucleoprotein complex.

[0037] 4) Centrifuge at 13,000 rpm for 15 min at 4°C. During this time, take a new EP tube, add 500 μL of isopropanol, and pre-cool on ice.

[0038] 5) After centrifugation, transfer the upper aqueous phase (about 500 μL) to the new EP tube.

[0039] 6) Place on ice and allow the alcohol to precipitate for 10 minutes.

[0040] 7) Centrifuge at 13,000 rpm for 10 min.

[0041] 8) Remove the supernatant and wash the RNA pellet once with 1 mL of 75% ethanol.

[0042] 9) Centrifuge at 12000 rpm for 5 minutes.

[0043] 10) Discard the supernatant and air-dry the RNA pellet for 5 minutes.

[0044] 11) Dissolve the RNA in 30-50 μL of DEPC-treated deionized water.

[0045] 12) Perform spectrophotometric analysis to determine sample concentration and purity. RNA extraction is successful when the sample concentration reaches 100 ng / μL or higher and the purity (OD260 / OD280) reaches 1.8-2.0.

[0046] Following the above method, RNA was extracted from Siberian almond flower bud tissue at different stages of development. Furthermore, the extracted RNA was reverse transcribed using HiScript II QRT SuperMix for qPCR (with gDNA wiper), a qPCR cDNA reverse transcription premix (available from Nanjing Novozymes Biotechnology Co., Ltd.), to obtain cDNA from flower bud tissue at different stages.

[0047] First, genomic DNA (gDNA) was removed from RNA extracted from flower bud tissues of Siberian apricot at different stages. The reaction system is shown in Table 1, where the template RNA is the RNA from Siberian apricot flower bud tissues. The reaction temperature was 42°C and the incubation time was 2 minutes. After the reaction was completed, the reaction solution was obtained to remove gDNA.

[0048] Table 1

[0049] Reagents Dosage <![CDATA[RNase-free ddH2O (Nuclease-free water)]]> Dilute to 16 μL 4×gDNA wiper mix 4 μL Template RNA 1 pg to 1 μg

[0050] The reverse transcription reaction system is shown in Table 2. HiScript II qRT SuperMix II was purchased from Nanjing Novozymes Biotechnology Co., Ltd. Reaction conditions: 50°C for 15 min, 85°C for 5 sec, and storage at −20°C.

[0051] Table 2

[0052] Reagents Dosage 5×HiScript II qRT SuperMix II 4 μL Reaction solution after removing gDNA 16μL

[0053] Primers were designed based on the gateway system linker sequence and the PsCBF2 gene sequence of the Siberian apricot F106 reference genome. Using PCR, the cDNA obtained by reverse transcription of the flower bud RNA of the Siberian apricot Z6 was used as a template to completely clone the coding sequence of the Siberian apricot PsCBF2 gene. Simultaneously, by adding attB-flanked linkers to both ends of the primers, attB-flanked linkers were added to both ends of the PsCBF2 gene coding sequence obtained in the PCR product, thereby obtaining a PsCBF2 gene coding sequence with attB-flanked linkers. This PsCBF2 gene coding sequence with attB-flanked linkers can be transferred to an entry vector using a BP reaction. Furthermore, using an LR reaction, the PsCBF2 gene coding sequence on the entry vector can be transferred to a plant expression vector.

[0054] The primer sequences used are:

[0055] ATTB1-PsCBF2 (SEQ ID NO.5):ggggacaagtttgtacaaaaaagcaggctgcATGGACATGTTCTCCGCTCAGC

[0056] ATTB2-PsCBF2(SEQ ID NO.6):ggggaccactttgtacaagaaagctgggtcTCAGATAGAGAAACTCCAC

[0057] Lowercase letters represent entry vector linker sequences, and uppercase letters represent sequences from the reference template (i.e., the sequence of the PsCBF2 gene). The entry vector used by gateway is pDONR207.

[0058] First, use high-fidelity enzyme (Phusion TM High-Fidelity DNA Polymerase, the enzyme and the matching 5XPhusion TM HF Buffer was purchased from Thermo Fisher Scientific) to amplify the target fragment. The PCR reaction system is shown in Table 3.

[0059] During the various stages of flower bud development in Siberian apricot, the expression of PsCBF2 gene occurs in flower bud tissues ( Figure 10 This can be used as evidence), that is, the cDNA obtained after reverse transcription of RNA from flower bud tissues at different stages can be used as a template for PCR to clone the PsCBF2 gene. Figure 10 The cDNA obtained by reverse transcription of RNA from flower bud tissue collected around November 10 was used as a template for PCR cloning of the PsCBF2 gene. At this time, the expression level of the PsCBF2 gene in the flower bud tissue was high, and the cDNA formed by reverse transcription of the flower bud tissue RNA contained more PsCBF2 gene coding sequences.

[0060] Table 3

[0061]

[0062]

[0063] The PCR reaction program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 36 cycles; and total extension at 72°C for 5 min.

[0064] The obtained PCR products were electrophoresed on 1% agarose gel at 140 V for 25 min. After taking photos of the gel using Bio-Rad, the correct bands were cut out and the PCR products were purified using the Omega gel recovery kit (Cat. No. D2000-02). Figure 1 The electrophoresis image shows a PCR product approximately 750 bp long, approximately equal to the sum of the length of the attB-flanked linker sequence and the PsCBF2 coding sequence. The marker on the left is DL2000. The PCR product was sequenced and analyzed for nucleic acid sequence homology using BLASTN against the NBCI database, confirming that the cloned product is a cbf2 homologous gene. The gene is 696 bp long and its coding sequence is as follows:

[0065] PsCBF2 (SEQ ID NO.1)

[0066] ATGGACATGTTCTCCGCTCAGCTTTCTAACTCCCCCGACCAGCCCGAGTCGAGTTCTTTCTCCGACGCCAGCTTCACCACCCTGCCGGCTTCTTCCTCCGACGAAAACGTCATATTGGCGTCGAGCCGGCCGAAGAAGCGCGCTGGGAGGAGGGTTTTCAAGGAGACGAGGCACCCGGTTTACAGGGGGGTGAGGAGAAGGAACAACAACAAGTGGGTGTGTGAGTTGAGAGAGCCAAACAAGAAGAAATCAAGGATTTGGCTTGGAACGTATCCGACTGCTGAGATGGCTGCTCGTGCCCATGACGTGGCGGCATTGGCGTTCAGAGGGAAGCTTGCCTGCATAAACTTTGCTGACTCCGCATGGCGGCTGCCCTTGCCGGCTTCCATGGATACCATGGATATCCGAAGGGCAGCTGCTGAGGCCGCCGAAGGGTTCAGGCCAGCGGAGTTCGGTGGATTATCCAGCTGCAGCAGTGATGAGAAGGAGAAGATTTTTAGCGTGGATGTGGAAAAAAGCAGCAGCAGCTTGTGCTTGTTTTATTTGGATGAGGAGGAAATGTTTGATATGCCAAGGTTGATTGATAACATGGCTCAAGGGCTTCTTCTTTCTCCACCTCAATGTTCAGCTGGCTACTTGAACTGGGATGACATGGAAACTGAAGCTGATGCCAAACTATGGAGTTTCTCTATCTGA

[0067] The sequence of the protein encoded by this gene is as follows:

[0068] PsCBF2 (SEQ ID NO.2)

[0069] MDMFSAQLSNSPDQPESSSFSDASFTTLPASSSDENVILASSRPKKRAGRRVFKETRHPVYRGVRRRNNNKWVCELREPNKKKSRIWLGTYPTAEMAARAHDVAALAFRGKLACIN FADSAWLPLPASMDTMDIRRAAAEAAEGFRPAEFGGLSSCSSDEKEKIFSVDVEKSSSSLCLFYLDEEEMFDMPRLIDNMAQGLLLSPPQCSAGYLNWDDMETEADAKLWSFSI*

[0070] Example 2 Construction of expression vector for Arabidopsis transformation

[0071] The purified PsCBF2 gene coding sequence with attB-flanked linker was combined with the gateway entry vector pDONR207 (pDONR TM BP reaction was performed with the vector and incubated at 25°C for 4 h. The reaction system is shown in Table 4. TM vector, 5X BP Clonase TM Reaction buffer and BP Clonase TM Enzyme mixes were purchased from Thermo Fisher Scientific.

[0072] Table 4

[0073] Reagents volume attB-PCR product (PsCBF2 gene coding sequence with attB-flanked linker) 4 μL <![CDATA[pDONR TM vector (entry vector pDONR207) 2μL <![CDATA[5X BP Clonase TM reaction buffer]]> 4 μL TE Buffer (10mM Tris-HCl, pH 8.0; 1mM EDTA) 6μL <![CDATA[BP Clonase TM enzyme mix]]> 4μL

[0074] After the BP reaction is completed, the obtained entry vector carrying the PsCBF2 gene coding sequence is transformed into E. coli. The specific operation method is as follows:

[0075] (1) The clean bench was exposed to ultraviolet light for 30 minutes, and the temperature of the constant temperature water bath was adjusted to 42°C.

[0076] (2) Take out a tube (100 μL) of competent E. coli from the -70°C ultra-low temperature freezer and melt it in an ice bath.

[0077] (3) Add the plasmid mixture obtained after the BP reaction, shake gently, and place on ice for 30 minutes.

[0078] (4) After gently shaking, place the tube in a 42°C water bath for 1 to 2 minutes for heat shock, then quickly return it to ice and let it stand for 3 to 5 minutes.

[0079] (5) In a clean bench, add 500 μL of LB medium (without antibiotics) to each of the above tubes and mix gently. Then fix them on the spring frame of a shaker and shake at 37°C for 1 hour for recovery culture.

[0080] (6) After the recovery culture, centrifuge at 3000 rpm for 5 minutes, discard the supernatant, blow off the remaining culture medium with a pipette, resuspend the bacteria, and add it dropwise to the surface of a solid LB plate containing gentamicin, spreading it evenly.

[0081] (7) Mark the coated culture dish and place it in a 37°C constant temperature incubator for 30 to 60 minutes until there is no flowing liquid on the surface of the culture medium. Then turn it upside down and place it in a 37°C constant temperature incubator overnight.

[0082] After the incubation period, single colonies were selected for sequencing and maintained. The culture of the correctly sequenced single colony was inoculated into LB liquid medium containing gentamicin and cultured overnight at 37°C and 200 rpm. The plasmid was extracted using the Omega Plasmid Miniprep Kit (Cat. No. D6943-01) to obtain the constructed entry vector carrying the PsCBF2 gene coding sequence. Specific steps are described in the kit instructions.

[0083] The constructed entry vector (pDONR TM -PsCBF2 vector) and expression vector pMDC32 TM Vector (purchased from Thermo Fisher Scientific) was used for LR reaction and incubated at 25°C for 4 h. The reaction system is shown in Table 5. 5X LR Clonase TM Reaction buffer and LR Clonase TM Enzyme mix was purchased from Thermo Fisher Scientific.

[0084] Table 5

[0085] Reagents volume <![CDATA[pDONR TM -PsCBF2 vector (entry vector carrying the PsCBF2 gene coding sequence)]]> 4 μL <![CDATA[pMDC32 TM vector]]> 2μL <![CDATA[5X LR Clonase TM reaction buffer]]> 4 μL TE Buffer (10mM Tris-HCl, pH 8.0; 1mM EDTA) 6μL <![CDATA[LR Clonase TM enzyme mix]]> 4 μL

[0086] After the LR reaction, the pMDC32-PsCBF2 vector is obtained. This vector is transformed into E. coli using the same method as above. After transformation, single colonies are selected and sequenced to verify the successful construction of the vector. Plasmids are extracted from E. coli to obtain the pMDC32-PsCBF2 vector (i.e., a recombinant plant expression vector).

[0087] Transform GV3101 Agrobacterium using the pMDC32-PsCBF2 vector as follows:

[0088] 1) Remove 200 μL of frozen competent Agrobacterium cells, thaw them, add the pMDC32-PsCBF2 vector, gently tap the tube to mix, and place on ice for 20 minutes.

[0089] 2) Place in liquid nitrogen for 5 minutes.

[0090] 3) After removing the cells, incubate in a 37°C water bath for 5 minutes, add 1 mL of LB, and incubate at 28°C, 150 rpm for 3 hours.

[0091] 4) Centrifuge at 6000 rpm for 1 min, collect the cells and spread them on LB plates containing antibiotics (50 mg / mL kanamycin, 50 mg / mL rifampicin).

[0092] 5) Incubate the tube upside down in an incubator at 28°C. Colonies will become visible after about 2 days. This Agrobacterium can be used to transform Arabidopsis thaliana.

[0093] Example 3: Infection of Arabidopsis thaliana by floral dipping

[0094] 1) Grow robust Arabidopsis plants (approximately 5 weeks old) under a long-day (16 h light, 8 h dark) environment.

[0095] 2) Prune off the first 1-5 cm long primary inflorescence (to encourage the growth of more lateral inflorescences). Perform the infiltration transformation within one week after pruning (usually 3-5 days). Water the plant well one day before the infiltration transformation to allow the stomata to fully open during the transformation.

[0096] 3) Prepare the Agrobacterium culture. Typically, Agrobacterium carrying the pMDC32-PsCBF2 vector is cultured in 1 mL of LB medium containing antibiotics (50 mg / mL kanamycin and 50 mg / mL rifampicin). The culture temperature is 28°C, shaken at 150 rpm, and incubated overnight. Add 150 μL of the overnight culture to 150 mL of fresh liquid LB medium. Incubate at 28°C with shaking at 150 rpm for an additional 24 hours.

[0097] 4) Centrifuge the bacterial suspension at 4000 × g for 20 min, then resuspend in 120 mL of infiltration buffer (10% sucrose + 400 μL / L Silwet L-77, thoroughly mixed before dipping the flowers, OD600 = 0.8-1.0). Meanwhile, remove the fruit pods and fully opened flowers from the Arabidopsis plants.

[0098] 5) Immerse the above-ground part of the plant in the bacterial solution for 1 minute and shake it gently.

[0099] 6) Cover the infected plants with plastic wrap to maintain humidity, incubate in the dark for 1 day, then place under normal culture conditions and remove the plastic wrap after 2-3 days. Watering can be done about 1 week after transformation.

[0100] 7) Continue culturing until the plants mature, collect T0 generation seeds and place them in a dry environment for about 1 week, and then screen for transformants.

[0101] Example 4 Identification of PsCBF2 gene-positive strains

[0102] 1) The T0 generation seeds harvested in Example 3 were sterilized and planted in 1 / 2 MS solid medium containing hygromycin (30 mg / mL Hygromycin B) and vernalized for 2 days. The plants after vernalization were cultured under a normal artificial climate, and their growth and phenotypic changes were observed.

[0103] 2) Extract RNA from leaf tissue of the lines into which the PsCBF2 gene has been introduced and reverse transcribe it into cDNA using the same reverse transcription method as in Example 1.

[0104] 3) Using the cDNA obtained by reverse transcription as a template, quantitative PCR (qPCR) was performed to detect whether the PsCBF2 gene was successfully expressed in the transgenic strain. The primers used in the quantitative PCR are shown in Table 6:

[0105] Table 6

[0106] Primer name sequence AtTUB2-F GAGCCTTACAACGCTACTCTGTCTGTC AtTUB2-R ACACCAGACATAGTAGCAGAAATCAAG PsCBF2-F (SEQ ID NO. 3) GGGGTGAGGAGAAGGAACAA PsCBF2-R (SEQ ID NO. 4) CTTCCCTCTGAACGCCAATG

[0107] The quantitative PCR (qPCR) reaction system is shown in Table 7:

[0108] Table 7

[0109]

[0110] The qPCR program is shown in Table 8:

[0111] Table 8

[0112]

[0113] Use 2 -ΔΔt The relative gene expression was calculated by Figure 2 As shown in the figure, the vertical axis represents the relative expression level. As can be seen from the figure, the PsCBF2 gene is highly expressed in the three lines PsCBF2#2, PsCBF2#4 and PsCBF2#8, and these three lines are PsCBF2 gene-positive transgenic lines (i.e., PsCBF2 gene-positive Arabidopsis).

[0114] Example 5 Phenotypic Identification of Transgenic Arabidopsis

[0115] Wild-type and PsCBF2 gene-positive Arabidopsis thaliana plants sown at the same time and growing in the same direction were selected for phenotypic observation. Figure 3 and Figure 4 This is a photograph of 36-day-old Arabidopsis plants. From left to right, they are: WT (wild type), PsCBF2#2, PsCBF2#4, and PsCBF2#8. As can be seen from the image, the wild-type strain bolts (flowers) earlier than the transgenic PsCBF2-positive Arabidopsis. This result is consistent with the results of PsCBF2 gene expression testing, which showed that increased PsCBF2 expression delayed the flowering period of Arabidopsis. In other words, the wild-type strain flowers significantly earlier than the PsCBF2-positive transgenic strain.

[0116] The average bolting time and average number of rosette leaves of transgenic PsCBF2 gene-positive Arabidopsis and wild-type Arabidopsis were statistically analyzed. Figure 5 The left vertical axis in the figure represents the average number of rosette leaves, and the right vertical axis represents the average bolting time. Figure 5 Under normal culture conditions, the average bolting time for wild-type Arabidopsis was 25 days, while the average bolting times for transgenic Arabidopsis expressing PsCBF2#2, PsCBF2#4, and PsCBF2#8 were 33 days, 38 days, and 46 days, respectively. The later the bolting time, the later the flowering period. The average number of rosette leaves in wild-type Arabidopsis was 20, while the average number of rosette leaves in transgenic Arabidopsis expressing PsCBF2#2, PsCBF2#4, and PsCBF2#8 was 30, 32, and 36, respectively. These results suggest that the PsCBF2 gene delays flowering and promotes vegetative growth in Arabidopsis.

[0117] Example 6 Determination of the Freeze Tolerance of PsCBF2 Gene-Positive Arabidopsis

[0118] 1. Plant culture

[0119] T3 seeds of WT and transgenic PsCBF2-positive Arabidopsis thaliana were spotted onto corresponding 1 / 2 MS medium plates with hygromycin b resistance, vernalized for 48 hours, and incubated in a culture room under alternating daylight conditions of 16 hours of light and 8 hours of darkness (long-day conditions) for 7 days. The seedlings were then transplanted into nutrient soil (soil:vermiculite mass ratio = 1:1). During transplanting, five plants were planted in a 5 cm square, and 25 plants were planted in a 10 cm square, arranged in a 5 x 5 pattern. After transplanting, the plants were incubated in an incubator at 22°C, long days, and 60% humidity for 15–20 days.

[0120] 2. Cold stress treatment

[0121] The plants were cooled at a constant rate (1°C / h) from 24°C to the stress temperature (-4°C) for 12 hours. After the cold stress, the plants were warmed to 4°C at a constant rate (1°C / h) for 12 hours and then allowed to recover at 22°C for 3–4 days. The survival rate and ion leakage rate of Arabidopsis plants were analyzed, as well as the expression of antifreeze-related genes, to assess their antifreeze capacity.

[0122] 3. Freeze-tolerance phenotype

[0123] like Figure 6 As shown in the figure, the top row shows Arabidopsis before cold stress treatment, and the bottom row shows Arabidopsis after 3-4 days of recovery. From left to right, they are WT (wild type), PsCBF2#2, PsCBF2#4, and PsCBF2#8. As can be seen from the figure, transgenic Arabidopsis plants expressing the PsCBF2 gene suffered less damage after cold stress and, after recovery, displayed greater vigor and resilience than the wild type. This demonstrates that the PsCBF2 gene plays an important role in improving Arabidopsis's freezing tolerance.

[0124] 4. PsCBF2 gene expression

[0125] The expression levels of PsCBF2 in transgenic lines and wild-type Arabidopsis were determined using qRT-PCR. The sampling site for the determination was leaf tissue, and the determination method and primers were the same as those used in quantitative PCR in Example 4. The determination results are shown in Figure 2. Figure 7 As shown. Figure 7 It can be seen that the expression level of PsCBF2 gene in transgenic PsCBF2 gene-positive Arabidopsis further increased after cold stress treatment, which shows that the expression of PsCBF2 gene is regulated by cold signals and the function of PsCBF2 gene is related to the cold response of plants.

[0126] 5. Electrical Conductivity Measurement in Arabidopsis Antifreeze Experiment

[0127] Ion leakage rate is calculated by measuring electrical conductivity. To measure conductivity, Arabidopsis seedlings (leaves) are placed in a 15 mL centrifuge tube filled with deionized water (5-10 mL) and allowed to equilibrate at room temperature with shaking for 1 hour or soaked for 12 hours. Conductivity S1 is measured. After boiling for 30 minutes, the tubes are allowed to equilibrate at room temperature with shaking for 1 hour, and conductivity S2 is measured. Ion leakage rate = (S1 - S0) / (S2 - S0) × 100%, where S0 is the conductivity of deionized water.

[0128] like Figure 8The figure shows the ion leakage rate test results. As shown in the figure, after cold stress treatment, the ion leakage rate of wild-type Arabidopsis leaves increased significantly, far exceeding that of transgenic PsCBF2-positive Arabidopsis. This indicates that transgenic PsCBF2-positive Arabidopsis is less susceptible to low-temperature damage. High PsCBF2 gene expression improves Arabidopsis's ability to resist low-temperature damage.

[0129] 6. Measurement results of proline content, a physiological indicator of antifreeze

[0130] The accumulation of proline in plant cytoplasm plays an important role in osmotic regulation. When plants encounter adverse stress, plant cells can rapidly synthesize osmotic regulating substances such as proline, which regulates the osmotic balance of plant cells by reducing the water potential of plant cells to enhance stress resistance. The Proline (Pro) Content Assay Kit was used to measure the changes in free proline content in transgenic lines and wild-type Arabidopsis before and after cold stress treatment. The specific steps were the same as the kit instructions. The results are shown in Figure 2. Figure 9 As shown in the figure, after cold stress treatment, the proline content of each strain increased significantly, and the proline content of the transgenic strain was lower than that of the wild type. It is speculated that the transgenic strain has a high overall resistance to cold stress and is less dependent on stress protectants similar to proline.

[0131] The above results indicate that under cold stress conditions, the PsCBF2 gene expression level in transgenic plants was higher than that in wild-type plants, while the proline content was lower. Proline has a strong hydrophilic nature, and an increase in its content helps protect cells from osmotic stress caused by freezing and dehydration. However, transgenic PsCBF2-positive Arabidopsis plants exhibited a higher freeze tolerance than wild-type plants, but a lower proline content. This suggests that transgenic PsCBF2-positive Arabidopsis may harbor a non-proline-dependent cold stress response pathway mediated by the PsCBF2 gene. Furthermore, according to the results of the transgenic Arabidopsis phenotypic identification in Example 5, transgenic Arabidopsis plants expressed a higher level of the PsCBF2 gene and had a greater number of rosette leaves than the wild-type strain, indicating relatively better vegetative growth. That is to say, under cold stress conditions, the cold stress response pathway mediated by the PsCBF2 gene is very likely to promote physiological and biochemical processes related to plant nutritional growth, enabling plants to have more energy reserves and more active metabolic processes, and thus have better adaptability to cold stress environments.

[0132] Example 7 Analysis of the expression pattern of the PsCBF2 gene in Siberian apricot flower buds in response to cold stress

[0133] From September 2022 to April 2023, tissues of 18-year-old Siberian apricot flower buds were collected at irregular intervals, RNA was extracted and reverse transcribed, and the expression of the PsCBF2 gene in the tissues was studied by quantitative PCR. At the same time, the average daily temperature during the experiment was recorded to verify that the PsCBF2 gene was involved in the response mechanism of Siberian apricot to cold stress. The lowest average temperature in a single day during the entire experiment was -17.15℃, and the average temperature during the entire experiment was 6.13℃. The specific sampling time is as follows: Figure 10 The horizontal axis is shown.

[0134] The methods of RNA extraction and reverse transcription, the preparation of the PCR system, and the PCR procedure were the same as those in Example 4 for the identification of PsCBF2 gene-positive strains. The primers used are shown in Table 9, where 18sRNA-F and 18sRNA-R were used to amplify the internal reference gene sequence.

[0135] Table 9

[0136] Primer name sequence 18sRNA-F (SEQ ID NO.7) GTTACTTTTAGGACTCCGCC 18sRNA-R (SEQ ID NO. 8) TTCCTTTAAGTTTCAGCCTTG PsCBF2-F GGGGTGAGGAGAAGGAACAA PsCBF2-R CTTCCCTCTGAACGCCAATG

[0137] like Figure 10 The results of the determination of PsCBF2 gene expression levels at different stages of Siberian apricot flower bud development are shown in the figure. The horizontal axis represents the measurement date, and the vertical axis represents the relative expression level. Figure 11 The figure shows the comparison between the daily average temperature change and the PsCBF2 gene expression level during the experiment. The horizontal axis in the figure represents the date of measurement, the left vertical axis represents the daily average temperature, and the right vertical axis represents the relative expression level. Figure 10 and Figure 11 It can be seen that the PsCBF2 gene is induced by cold signals, and its expression level increases significantly after low temperature stimulation, and the expression trend is opposite to that of temperature change, indicating that PsCBF2 is involved in the cold stress response process of Siberian apricot.

[0138] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the claims of this patent application.

Claims

1. Application of the Siberian apricot PsCBF2 gene, characterized in that: The coding sequence of the Siberian apricot PsCBF2 gene is shown in SEQ ID NO.1; the application is to use the Siberian apricot PsCBF2 gene to improve the cold stress response ability of Siberian apricot and / or Arabidopsis and simultaneously reduce the proline content in Siberian apricot and / or Arabidopsis under cold stress conditions; when the Siberian apricot PsCBF2 gene is used to improve the cold stress response ability of Siberian apricot and / or Arabidopsis and simultaneously reduce the proline content in Siberian apricot and / or Arabidopsis under cold stress conditions, the expression level of the Siberian apricot PsCBF2 gene in Siberian apricot and / or Arabidopsis is increased.

2. A method for reducing the proline content in plants under cold stress conditions, characterized in that The method comprises the steps of transferring a recombinant vector containing the coding sequence of the Siberian apricot PsCBF2 gene as shown in SEQ ID NO.1 into a plant.

3. The method according to claim 2, characterized in that The plant is Arabidopsis thaliana; the original vector of the recombinant vector is pMDC32.

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