Pinus massoniana tandem zinc finger structure pmtzf1 gene, expression protein and application thereof

Abstract

The application discloses a Pinus massoniana tandem zinc finger structure PmTZF1 gene and an expression protein and application thereof, and relates to the technical field of plant genetic engineering.The Pinus massoniana tandem zinc finger structure PmTZF1 gene is disclosed for the first time, a nucleotide sequence of which is shown as SEQ ID NO.1, and an amino acid sequence of which is shown as SEQ ID NO.2.The application constructs an expression vector pBI121-PmTZF1 of the Pinus massoniana tandem zinc finger structure PmTZF1 gene; the constructed expression vector of the Pinus massoniana tandem zinc finger structure PmTZF1 gene is transformed into Arabidopsis; and a transgenic Arabidopsis plant with improved non-biological stress resistance is obtained through cultivation and screening.Compared with a wild type plant, the root of the transgenic strain is significantly longer than that of the wild type, and the transgenic strain blooms and sprouts earlier; under drought stress, the transgenic strain normally blooms and bears fruits, has more branches, and has more silique quantity, length and other properties, which are significantly better than those of the wild type strain.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to a PmTZF1 gene with a tandem zinc finger structure from Masson pine, its expressed protein, and its applications. Background Technology

[0002] Masson pine (Pinus massoniana Lamb.) is a fast-growing and highly resilient tree species, serving as a pioneer for afforestation of barren hills in southern my country. It possesses excellent characteristics such as drought resistance, cold resistance, and tolerance to poor soil conditions. Masson pine plays an irreplaceable role in industrial timber production, forest product processing, forest resource development, and ecological services. However, drought severely restricts the normal growth and development of plants, and the molecular mechanisms by which Masson pine responds to drought are currently unclear. Therefore, identifying drought-resistant genes in Masson pine at the molecular level and elucidating its drought-regulating mechanisms is of great significance for improving its tolerance to drought stress and expanding its cultivation range.

[0003] Under drought conditions, plant root activity decreases, respiration weakens, and the absorption and transport of water and mineral elements by roots are inhibited. Simultaneously, leaf growth, leaf area, stomatal index, stomatal opening and closing, and chlorophyll content decline, severely disrupting photosynthesis and respiration. Under drought stress, ROS levels in plants increase, and membrane lipids are defatted or oxidized to generate MDA. The plant genome encodes a large number of CCCH zinc finger proteins. The CCCH zinc finger protein family (especially tandem CCCH zinc finger proteins (TZFs)) plays a crucial role in plant growth, development, and the regulation of plant tolerance to abiotic and biotic stresses. Drought stress and ABA treatment can significantly induce the expression of various CCCH zinc finger protein genes. CCCH zinc finger proteins enhance plant drought resistance in multiple ways. One pathway is through stomatal regulation, reducing water loss; CCCH zinc finger proteins can directly regulate downstream genes related to drought stress at the transcriptional level, thereby enhancing plant drought resistance. CCCH zinc finger proteins can also enhance plant drought resistance through ABA-mediated signaling pathways or by improving reactive oxygen species scavenging capacity.

[0004] Zinc finger proteins are a class of transcription factors possessing zinc finger domains. Based on differences in zinc finger structure and function, zinc finger proteins can be classified into nine major classes: C2H2, G8, C6, C3HC4, C2HC, C2HC5, C4, C4HC3, and C3H (C represents cysteine, H represents histidine). Among them, CCCH (i.e., C3H) type zinc finger proteins contain 1–6 CCCH type zinc finger motifs. This motif consists of three cysteine ​​residues and one histidine residue. Compared with other types of zinc finger proteins, research on CCCH zinc finger proteins is relatively limited, and mainly focuses on animals. Reports on plant CCCH zinc finger proteins are very limited, and most focus on TZF zinc lipoproteins.

[0005] Genome-level analysis has identified 68 CCCH genes in the Arabidopsis genome. Despite the large family, only a few CCCH zinc finger proteins have had their functions preliminarily investigated: HUA1 is an RNA-binding protein involved in controlling flowering development; FESI can promote the formation of winter acclimatization traits in Arabidopsis through interaction with FRI and FLC; AtSZF1 and AtSZF2 are involved in regulating Arabidopsis' tolerance to salt stress; SOMNUS has been shown to be a component of the phytochrome signaling pathway, negatively regulating light-dependent seed germination; studies on AtTZF1 have revealed that this protein can shuttle between the nucleus and cytoplasm and bind to both DNA and RNA. In addition to studies on zinc finger proteins in Arabidopsis, a CCCH zinc finger transcription factor, GhZFP1, has also been isolated from cotton, which can enhance salt tolerance and disease resistance through interaction with GZ-lRD21A and GZIPR5. Therefore, CCCH zinc finger proteins play an important role in regulating plant growth, development, and stress responses. Summary of the Invention

[0006] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide a PmTZF1 gene with a tandem zinc finger structure from *Pinus massoniana*. Another technical problem this invention aims to solve is to provide the expression protein of the PmTZF1 gene with a tandem zinc finger structure from *Pinus massoniana*. A further technical problem this invention aims to solve is to provide applications for the PmTZF1 gene with a tandem zinc finger structure from *Pinus massoniana*, for obtaining new plant germplasm with improved tolerance to abiotic stresses.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A PmTZF1 gene with a tandem zinc finger structure from Pinus massoniana, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The amino acid sequence of the PmTZF1 gene, which is a tandem zinc finger structure of Pinus massoniana, is shown in SEQ ID NO.2.

[0010] Vectors, recombinant bacteria, or host cells containing the PmTZF1 gene of the tandem zinc finger structure of Pinus massoniana.

[0011] Application of the PmTZF1 gene with tandem zinc finger structure in Masson pine in promoting plant resistance to abiotic stress.

[0012] The abiotic stress is drought.

[0013] The application of the PmTZF1 gene, a tandem zinc finger structure from Pinus massoniana, in promoting plant resistance to abiotic stress includes:

[0014] 1) Construct an expression vector for the PmTZF1 gene with a tandem zinc finger structure from Pinus massoniana;

[0015] 2) The expression vector of the PmTZF1 gene with the constructed tandem zinc finger structure from Pinus massoniana was transformed into Arabidopsis thaliana;

[0016] 3) Cultivate, screen and obtain transgenic Arabidopsis plants with improved resistance to abiotic stress.

[0017] The expression vector is a plant expression vector.

[0018] The plant expression vector is pBI121-PmTZF1.

[0019] Application of the PmTZF1 gene with tandem zinc finger structure in Masson pine to promote plant growth.

[0020] The promotion of plant growth includes promoting flowering and fruiting, increasing the number of branches, increasing the number of siliques, and increasing the length of siliques.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1) This application discloses for the first time the PmTZF1 gene of the tandem zinc finger structure from *Pinus massoniana*, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of which is shown in SEQ ID NO.2. This invention constructs the expression vector pBI121-PmTZF1 of the PmTZF1 gene from *Pinus massoniana*; transforms the constructed expression vector into *Arabidopsis thaliana*; and cultivates, screens, and obtains transgenic *Arabidopsis thaliana* plants with enhanced resistance to abiotic stresses.

[0023] 2) One-month-old wild-type (WT) Arabidopsis thaliana bolted in 26 days and flowered in 30 days; the selected transgenic lines (L4, 6, 10) bolted in 21 days and flowered in 26 days; the roots of the wild-type Arabidopsis thaliana transgenic lines were significantly longer than those of the wild type.

[0024] 3) After 15 consecutive days of drought, the wild-type strains were slow and stunted under drought stress and had difficulty flowering and fruiting normally, while the transgenic strains could flower and fruit normally and had significantly more branches than the wild-type strains. After three days of rehydration, it was found that the transgenic strains had significantly better traits such as the number and length of siliques than the wild-type strains.

[0025] 4) Under drought conditions, wild-type lines (WT) lose water more quickly, require more water, and struggle to maintain normal physiological activities, resulting in stunted vegetative and reproductive growth. In contrast, transgenic lines exhibit improved drought resistance, lose less water under drought treatment, and still maintain normal physiological activities. These results all indicate that the PmTZF1 gene plays a crucial role in plant resistance to drought stress, enabling plants to exhibit optimal growth. Attached Figure Description

[0026] Figure 1 Agarose gel electrophoresis image of PCR for the open reading frame of the PmTZF1 gene;

[0027] Figure 2 The differential expression level of the PmTZF1 gene in tissues of 15-year-old Masson pine is shown in the figure (FC represents female cones, MC represents male cones, C represents immature cones, YS represents young stems, OS represents old stems, N represents needles, and R represents roots).

[0028] Figure 3 Figure showing the change in expression level of the PmTZF1 gene in Masson pine under drought stress;

[0029] Figure 4 Subcellular localization map of the PmTZF1 gene in tobacco leaf cells;

[0030] Figure 5 PCR identification diagram and relative expression level diagram of transgenic Arabidopsis PmTZF1 (1-10 are transgenic lines, p is positive control, WT is negative control);

[0031] Figure 6 Phenotypic comparison diagrams of transgenic Arabidopsis and wild-type Arabidopsis (A: Schematic diagram of root length; B: Root length data diagram; C: Schematic diagram of flowering; D: Bolting stage; E: Flowering stage);

[0032] Figure 7 Phenotypic comparison of transgenic Arabidopsis and wild-type Arabidopsis under drought conditions (A: Arabidopsis growth status after drought and rehydration; B: Silique comparison; C: Plant height; D: Silique length; E: Number of branches; F: Number of siliques; G: Fresh weight of siliques).

[0033] Figure 8 The graph shows the relative expression levels of PmTZF1 in transgenic Arabidopsis and wild-type Arabidopsis under drought conditions.

[0034] Figure 9 This is a graph showing the relative soil moisture content of transgenic Arabidopsis thaliana lines and wild-type Arabidopsis thaliana. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0036] The pine needle material used for RNA extraction in the following examples was obtained from two-year-old pine seedlings planted by the National Key Laboratory of Forest Genetics and Breeding of Nanjing Forestry University.

[0037] The 15-year-old Masson pine material used in the following examples came from Wasan State-owned Forest Farm in Quanjiao County, Chuzhou City.

[0038] Example 1

[0039] 1. Extract total RNA

[0040] Total RNA was extracted from Pinus massoniana seedlings using a polysaccharide and polyphenol plant total RNA extraction kit (Novizan) according to the instructions. The specific steps are as follows:

[0041] Add an appropriate amount of pine needle powder ground with liquid nitrogen, then add 500 μL of preheated Buffer PRL (with 5% β-mercaptoethanol added before use) at 65°C. Immediately vortex vigorously for 30-60 seconds to ensure complete lysis. Incubate in a 65°C water bath for 5 minutes, inverting the container 1-2 times during this period to aid lysis. After lysis, centrifuge at 12000 rpm for 10 minutes. Transfer the supernatant to a new 1.5 mL RNase-free centrifuge tube, add 0.5 times the volume of anhydrous ethanol to the supernatant, and immediately mix by pipetting. Transfer the mixture to column II (lower receiving tube), centrifuge at 12000 rpm for 2 minutes, and discard the filtrate. Place the column in a new receiving tube and add 500 μL of Buffer PRL. Plus, centrifuge at 12000 rpm for 30 seconds and collect the filtrate; add 0.5 times the volume of anhydrous ethanol to the filtrate and immediately mix by pipetting; transfer the mixture to column IV (lower receiving manifold), centrifuge at 12000 rpm for 2 minutes, and discard the filtrate; add 700 μL of Buffer PRW1 to column IV, incubate at room temperature for 1 minute, centrifuge at 12000 rpm for 30 seconds, and discard the filtrate; add 500 μL of Buffer PRW1 to column IV. Add PRW2 (add 48 mL of anhydrous ethanol before use), centrifuge at 12000 rpm for 30 seconds, discard the filtrate, and repeat the operation once; put the empty column back into the collection tube, centrifuge at 12000 rpm for 2 min, and place it in a fume hood with the lid open for 2-5 min to allow the residual ethanol to evaporate completely; transfer the adsorption column to a new 1.5 mL centrifuge tube, add 30-100 μL of ddH2O dropwise to the center of the adsorption column membrane, place at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min to elute the RNA.

[0042] 2. cDNA synthesis

[0043] Using the extracted total RNA as a template, cDNA strands were obtained through reverse transcription. The entire following procedure must be performed on ice. The specific steps are as follows:

[0044] The sample reaction system is as follows: 1 μg total RNA, 1 μL Oligo(dT)18 (0.5 μg / μL), 10 μL 2×TS ReactionMix, 1 μL RT / RI Enzyme Mix, 1 μL gDNA Remover, and RNase H2O to 20 μL. Mix gently by pipetting and centrifuge briefly to the bottom of the PCR tube. React at 42℃ for 30 min, and then at 85℃ for 5 sec to inactivate the enzymes and gDNA Remover in the reaction system to obtain cDNA. Store at -20℃ for later use.

[0045] 3. Cloning the target gene

[0046] Based on the PmTZF1 sequence screened from the transcriptome data of *Pinus massoniana* under drought stress (PRJNA595650) obtained in the laboratory in the previous period, specific primers were designed to clone the PmTZF1 gene fragment using synthesized *Pinus massoniana* cDNA as a template. The primer sequences for cloning the PmTZF1 open reading frame are as follows:

[0047] PmTZF1-F: 5′-ATGTCAAGCGTTTTCTGCAGAACAG-3′,

[0048] PmTZF1-R: 5′-TTACTTAACAAGCTCGTTCACCCA-3′.

[0049] The PCR reaction system (20 μL) consisted of: 10 μL 2×Hieff Gold PCR Master Mix, 1μL Forward primer, 1μL Reverse primer, 1μL cDNA, 7μL ddH2O.

[0050] The PCR reaction program was as follows: denaturation at 98℃ for 10 seconds, annealing at 57℃ for 5 seconds, extension at 72℃ for 10 seconds, for 35 cycles.

[0051] Mix 5 μL of PCR product (the cloned PmTZF1 gene fragment) with 1 μL of 6×Loading Buffer and spot it into the wells of a prepared 1.2% agarose gel. Electrophoresis was performed at 200V for 18 min in 1×TAE buffer. After confirming the electrophoresis was correct, the remaining PCR product was mixed with 6×Loading Buffer and the target fragment was recovered using an agarose gel extraction kit. 4 μL of the recovered product was excised and placed in a 1.5 mL centrifuge tube, and 1 μL of... -Blunt, gently mix with a pipette, react at room temperature for 15 min, then place the centrifuge tube on ice. Add 50 μL of DH5α E. coli competent cells to the ligation product, gently tap to mix, incubate on ice for 25 min, heat shock at 42°C for 45 sec, immediately place on ice for 2 min, then add 900 μL of LB medium, incubate at 200 rpm and 37°C for 1 h; centrifuge at 5000 rpm for 1 min, discard 800 μL of supernatant, mix by pipetting and aspirating, and spread on an agar plate containing Amp. Incubate the plate upside down at 37°C overnight. The next day, pick a single colony from the LB plate and add it to 10 μL of ddH2O water, mix by pipetting and aspirating, and take 2 μL for positive detection. Add 1 mL of LB liquid medium containing Kan to the remaining bacterial culture, and incubate at 200 rpm and 37°C. After bacterial testing, select bacterial cultures with correct electrophoretic bands and send them to the company for sequencing.

[0052] The positive detection reaction system (20 μL) consisted of: 10 μL 2×Rapid Taq Master Mix, 1 μL M13-F, 1 μL LM13-R, 2 μL bacterial culture, and 6 μL ddH2O.

[0053] The positive test reaction procedure is as follows: 95℃ for 3 min; 95℃ for 15 sec; 60℃ for 15 sec; 72℃ for 15 sec, 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0054] PCR results of the target gene clone are as follows Figure 1 As shown in SEQ ID NO.1, the nucleotide sequence of the target gene was determined based on the sequencing results. The total length of the open reading frame of *Pinus massoniana* C3H32 is 1578 bp, and the gene was named PmTZF1. The amino acid sequence of its expressed protein is shown in SEQ ID NO.2.

[0055] Example 2

[0056] 1. Specific expression of PmTZF1 gene in different organs of Pinus massoniana

[0057] RNA was extracted from male and female cones, cones, young stems, old stems, needles, and roots of 15-year-old *Pinus massoniana* and reverse transcribed into cDNA, as shown in Example 1. Real-time quantitative PCR (RT-qPCR) was used to detect the specific expression of the *PmTZF1* gene in different organs of *Pinus massoniana*. Primer sequences are shown below:

[0058] PmTZF1-qF: 5′-TCCACCACTGTCACCATCTGCGTCTC-3′,

[0059] PmTZF1-qR: 5′-GCCTTTGGGCTTGCCAACCCTCTT-3′.

[0060] qRT-PCR reaction system (10 μL): 1 μL cDNA (diluted to 1 / 20 of the initial concentration), 5 μL SYBR GreenMaster Mix, 0.4 μL PmTZF1-qF, 0.4 μL PmTZF1-qR, 3.2 μL ddH2O.

[0061] The reaction procedure was as follows: 95℃ for 2 min; 95℃ for 10 sec; 60℃ for 30 sec; 72℃ for 30 sec; 40 cycles.

[0062] The results are as follows Figure 2 As shown, the expression level in female cones was set to 1. The results showed that PmTZF1 was expressed in all tissues and had tissue specificity. The highest expression level was in leaves, followed by high expression in roots and young stems; while the lowest expression level was in fruits.

[0063] 2. Response of the PmTZF1 gene to drought stress

[0064] Masson pine seedlings were subjected to natural drought treatment. Samples were taken at 0, 3, 7, 12, and 20 days. Total RNA was extracted according to the method described in Example 1. Using the extracted total RNA as a template, cDNA strands were obtained through reverse transcription. Real-time quantitative PCR (RT-qPCR) was used to detect the specific expression of the PmTZF1 gene in Masson pine under drought conditions. Primer sequences are shown below:

[0065] PmTZF1-qF: 5′-TCCACCACTGTCACCATCTGCGTCTC-3′,

[0066] PmTZF1-qR: 5′-GCCTTTGGGCTTGCCAACCCTCTT-3′.

[0067] qRT-PCR reaction system (10 μL): 1 μL cDNA (diluted to 1 / 20 of the initial concentration), 5 μL SYBR GreenMaster Mix, 0.4 μL PmTZF1-qF, 0.4 μL PmTZF1-qR, 3.2 μL ddH2O.

[0068] The reaction procedure was as follows: 95℃ for 2 min; 95℃ for 10 sec; 60℃ for 30 sec; 72℃ for 30 sec; 40 cycles.

[0069] The results are as follows Figure 3 As shown, in the early stage of drought, the expression level of PmTZF1 did not change significantly. However, as the drought intensified, the expression level of PmTZF1 increased significantly after 20 days of dehydration.

[0070] 3. Subcellular localization

[0071] Activate 1 mL of *E. coli* culture containing the pCAMBIA1302 vector, stored at -80℃ in our laboratory. Take 1-4 mL of the overnight culture and extract the plasmid using a plasmid miniprep kit. Based on the ORF sequences of the pCAMBIA1302 vector and PmTZF1 (with the stop codon removed), design primers carrying restriction enzyme sites (NcoI and BgIII) using CE-Design software. The primer sequences are as follows:

[0072] PmTZF1-mgfp5-F: 5'-acgggggactcttgaccatggATGTCAAGCGTTTCTGCAGAACA-3', PmTZF1-mfp5-R: 5'-tctcctttactagtcagatctCTTAACAAGCTCGTTCACCCAAC-3'.

[0073] The 35S::PmTZF1-mGFP5 fusion vector was constructed using homologous recombination.

[0074] Double digestion with NcoI and BgIII was performed. The digestion system (20 μL) was as follows: 1 μg plasmid, 1 μL NcoI, 1 μL BgIII, and ddH2O was added to 20 μL.

[0075] The ligation system was as follows: 0.02 × cloning vector base pairs (ng) of linearized vector, 0.04 × insert fragment base pairs (ng) of insert fragment, 2 μL 5 × CEII Buffer, 1 μL Exnase II, and ddH2O to a final volume of 10 μL. The mixture was incubated in a PCR instrument at 37°C for 25 min, and then immediately placed on ice.

[0076] The ligation product was transformed into DH5α competent E. coli cells, gently mixed, and then sequentially incubated on ice for 25 min, followed by heat shock at 42°C for 45 sec, and immediately placed on ice for 2 min. Then, 900 μL of LB medium was added, and the cells were incubated at 37°C for 1 h at 200 rpm. After centrifugation at 5000 rpm for 1 min, 800 μL of supernatant was discarded, and the mixture was spread onto a Kansat plate. The plate was incubated overnight at 37°C. The next day, a single colony from the LB plate was picked and added to 10 μL of ddH2O water. After mixing, 2 μL was used for positive detection. 1 mL of LB liquid medium containing Kansat was added to the remaining bacterial culture, and the cells were incubated at 37°C at 200 rpm. After bacterial detection, the bacterial cultures with correct electrophoretic bands were sent to the company for sequencing. Positive clones with correct sequencing results were screened, expanded, and recombinant plasmids were extracted.

[0077] The recombinant plasmid was transformed into Agrobacterium competent cells GV3101 as follows: 10 μL of the recombinant plasmid was added to 100 μL of Agrobacterium competent cells in an ice-water mixture. The mixture was gently stirred at the bottom of the tube and then placed on ice, in liquid nitrogen, in a 37°C water bath, and in an ice bath for 5 min each. 700 μL of LB broth was added and the cells were incubated at 28°C with shaking for 2.5 h. 100 μL of the supernatant was then spread onto a medium containing 50 mg·L⁻¹ of iodine. -1 Kan, 25 mg·L -1 On Rif LB agar plates, inverted and incubated at 28°C for 2 days; single colonies with good growth were picked for PCR detection, and positive single colonies were expanded and cultured. 500 μL of overnight activated bacterial suspension (50 mg / L Kan, 25 mg / L Rif) was inoculated into 50 mL of LB liquid medium and incubated at 28°C with shaking at 220 rpm until OD was reached. 600 The concentration was 0.7-0.8. The supernatant was discarded and the bacterial cells were collected. The bacterial precipitate was resuspended in sterile water containing 10mM MgCl2, 10mM MES (pH=5.6) and 200μM acetylsylgenone (AS). Equal volumes of the two target bacterial solutions were mixed with p19 Agrobacterium at a ratio of 1:1. The OD600 value was adjusted to approximately 1.0. The mixture was then allowed to stand at room temperature in the dark for 3-4 hours.

[0078] The Agrobacterium suspension was injected into the tender leaves of 3-week-old wild Nicotiana benthamiana seedlings using a pressure injection method with a 10 mL syringe (needle-free). The upper surface of the leaf was held down by hand, and the Agrobacterium was slowly injected into the interstitial spaces of the leaf tissue through osmotic pressure, avoiding the veins. The injected tobacco leaves were then marked. A suitable amount of water was added to the bottom of the pot to keep it slightly moist. After culturing in the dark for 1 day, the plants were transferred to light for 1 day, followed by microscopic observation under a laser confocal microscope. The 35S::mGFP5 empty vector plasmid was used as a negative control.

[0079] The results are as follows Figure 4 As shown, the fluorescence signal of the 35S::PmTZF1-mGFP5 fusion protein was observed to be localized in the cell nucleus under laser confocal microscopy, and its overlap with the cell nucleus stained with DAPI indicates that PmTZF1 is specifically localized in the cell nucleus.

[0080] Example 3

[0081] 1. Constructing a carrier

[0082] Primers were designed based on the XbaI and BamHI restriction sites on the pBI121 vector and the open reading frame of PmTZF1. The primer sequences are as follows:

[0083] pBI121-PmTZF1-F;

[0084] 5'-gagaacacgggggactctagaATGTCAAGCGTTTCTGCAGAACA-3',

[0085] pBI121-PmTZF1-R:

[0086] 5′-gggaaattcgagctcggatccCTTAACAAGCTCGTTCACCCAAC-3′.

[0087] The vector was double-digested with XbaI and BamHI according to the aforementioned method. The ORF was then ligated to the digested vector to obtain the plant expression vector pBI121-PmTZF1. After sequencing, the recombinant plasmid was extracted from the successfully constructed plant expression vector and transformed into Agrobacterium competent cells GV3101 using the method described in Example 2.

[0088] 2. Sowing and cultivation of Arabidopsis thaliana

[0089] 1) Disinfection of Arabidopsis thaliana seeds: Place an appropriate amount of Arabidopsis thaliana seeds in a 1.5 mL centrifuge tube, add 1 mL of 75% ethanol to the centrifuge tube, invert for 45 seconds, rinse with sterile water, add 1 mL of 20% sodium hypochlorite, invert for 5 minutes, and rinse repeatedly with sterile water 5-6 times.

[0090] 2) Sowing: Use a 1mL pipette to sow the sterilized Arabidopsis thaliana seeds onto 1 / 2MS medium for culture.

[0091] 3) Arabidopsis thaliana culture: Seal the culture medium after sowing Arabidopsis thaliana seeds and culture it in the dark at 4℃ for 2 days. Then place it in an artificial climate incubator to allow it to germinate and grow. After about a week, transfer the Arabidopsis thaliana seedlings that are growing well in the culture medium to nutrient soil (black soil: vermiculite: perlite = 6:2:1) for further cultivation and cover them with plastic wrap. Remove the plastic wrap on the third day.

[0092] 3. Transformation of Arabidopsis thaliana by inflorescence immersion method

[0093] 1) In a clean bench, Agrobacterium tumefaciens containing the pBI121-PmTZF1 recombinant plasmid was streaked onto an LB agar plate containing 50 mg / L Kan and 25 mg / L Rif.

[0094] 2) Select a single colony in good growth condition, add 5 mL of LB liquid medium containing 50 mg / L Kan and 25 mg / L Rif, and incubate overnight at 200 rpm in a constant temperature shaker at 28°C.

[0095] 3) Take 1 mL of the overnight culture and inoculate it into 50 mL of LB liquid medium containing 50 mg / L Kan and 25 mg / L Rif, and shake and culture until OD600 = 0.8.

[0096] 4) Pour the bacterial culture into a 50mL sterile centrifuge tube, centrifuge at 5000rpm for 10min to collect the bacterial cells, and add 50mL of pre-prepared osmotic buffer to suspend the bacterial cell precipitate.

[0097] 5) Select Arabidopsis thaliana plants that have bolted after about 4 weeks of growth, remove the open flower buds, immerse the inflorescence in the inoculum for 30 seconds, and cover with plastic wrap after inoculum is inoculated.

[0098] 6) Incubate in the dark for 18-20 hours, rinse thoroughly with clean water, and then continue incubation in an incubator.

[0099] 7) After 7-10 days, repeat steps 1-6 to reinfect.

[0100] 8) Harvest transgenic T0 generation seeds after the pods of Arabidopsis plants turn yellow. To promote seed maturation, the number of waterings should be appropriately controlled when the seeds are about to mature.

[0101] 4. Screening of transgenic positive plants

[0102] Preliminary resistance screening was performed on 1 / 2 MS (containing Kan) medium. Untransformed negative-positive plants could not grow normally, while positive transgenic plants could grow normally. Normally growing Arabidopsis seedlings were transferred to nutrient soil for further culture, and genomic DNA and RNA were extracted from leaves. The specific steps are as follows: DNA was extracted from the preliminarily screened transgenic Arabidopsis plants using a plant genomic DNA extraction kit (TIANGEN). Refer to the instruction manual for specific steps. The concentration and OD260 / 280 ratio of the extracted Arabidopsis gDNA were detected using a micro spectrophotometer. Then, PCR detection was performed using the universal 35S-F primers and the aforementioned pBI121-PmTZF1-R primers. The primer sequences are shown below:

[0103] 35s-F: 5′-TGAAGATAGTGGAAAAGGAAGGTG-3′;

[0104] PCR reaction system: 100ng DNA template, 1μL each of forward and reverse primers, 10μL of 2×Phanta Max Master Mix (Vazyme), and ddH2O to a final volume of 20μL.

[0105] PCR reaction procedure: 95℃ pre-denaturation for 3 min; 35 amplification cycles including 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 25 s, 72℃ extension for 5 min, and storage at 4℃. Using a 1.2% agarose gel and UV gel imaging system, the size of the PCR product bands was observed and compared to the size of the target fragment inserted in the recombinant plasmid.

[0106] Total RNA was extracted from positive plants for DNA level detection and reverse transcribed into cDNA. The relative expression level of the PmTZF1 gene in transgenic Arabidopsis was detected by qRT-PCR, with Actin2 as the internal reference gene. RNA extraction, cDNA first-strand synthesis, and qRT-PCR system and program settings are as described in Example 1.

[0107] The results are as follows Figure 5 As shown, Arabidopsis thaliana plants successfully infused with the gene were able to amplify bands of the same size as the plasmid amplification products. Figure 5 A); then, the transgenic lines that were successfully identified at the gene level were identified at the transcriptional level. Figure 5 B) The results showed that the expression levels of different transgenic plants differed. Among the 10 transgenic Arabidopsis plants tested, the expression level of Arabidopsis plant numbered L10 was the highest, followed by L4 and L6.

[0108] 5. Phenotypic traits of transgenic Arabidopsis thaliana

[0109] Phenotypic differences between 1-month-old wild-type (WT) Arabidopsis thaliana and selected transgenic lines (L4, 6, 10) are as follows: Figure 6 As shown, the transgenic Arabidopsis thaliana bolts in 21 days and flowers in 26 days; while the wild-type Arabidopsis thaliana bolts in 26 days and flowers in 30 days; the roots of the transgenic line are significantly longer than those of the wild type.

[0110] 6. Comparison of phenotypic traits of transgenic Arabidopsis thaliana under drought stress

[0111] Arabidopsis thaliana transgenic with the PmTZF1 gene and wild Arabidopsis thaliana were planted in small flower pots (three plants per pot). After being thoroughly watered, the plants were allowed to dry naturally for 15 days (without watering during the 15 days) and then placed in a greenhouse at 25°C for cultivation (16 hours of light and 8 hours of darkness).

[0112] The results are as follows Figure 7 As shown, after 15 consecutive days of drought, the wild-type line developed slowly and was stunted under drought stress, making it difficult to flower and bear fruit normally, while the transgenic line could flower and bear fruit normally, with a significantly higher number of branches than the wild type. After three days of rehydration, it was found that the transgenic line had significantly better traits such as the number and length of siliques than the wild-type line.

[0113] 7. Gene expression levels in transgenic Arabidopsis thaliana under drought stress

[0114] The expression level of the PmTZF1 gene in transgenic Arabidopsis thaliana after 15 consecutive days of drought was detected by qRT-PCR, with transgenic Arabidopsis thaliana that had not undergone drought treatment serving as a control.

[0115] The results are as follows Figure 8 As shown, the expression level of PmTZF1 was significantly increased in transgenic Arabidopsis after encountering drought stress.

[0116] 8. Relative soil moisture content of transgenic Arabidopsis thaliana under drought stress

[0117] After 15 consecutive days of drought, the changes in soil moisture content were compared between wild-type Arabidopsis thaliana and PmTZF1 transgenic lines.

[0118] The results are as follows Figure 9 As shown, under drought conditions, the wild-type line (WT) loses water more quickly, requires more water, and struggles to maintain normal physiological activities, resulting in stunted vegetative and reproductive growth. In contrast, the transgenic line exhibits improved drought resistance, loses less water under drought treatment, and can still maintain normal physiological activities. These results indicate that the PmTZF1 gene plays a crucial role in plant drought stress resistance, enabling plants to exhibit good growth.

[0119] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Application of the PmTZF1 gene, a tandem zinc finger structure of *Pinus massoniana* with the nucleotide sequence shown in SEQ ID NO.1, in promoting the drought stress resistance of *Arabidopsis thaliana*.

2. The application according to claim 1, characterized in that, include: 1) Construct an expression vector for the PmTZF1 gene with a tandem zinc finger structure from Pinus massoniana; 2) The expression vector of the PmTZF1 gene with the constructed tandem zinc finger structure from Pinus massoniana was transformed into Arabidopsis thaliana; 3) Cultivate, screen and obtain transgenic Arabidopsis plants with improved drought resistance.

3. The application according to claim 2, characterized in that, The expression vector is a plant expression vector.

4. The application according to claim 3, characterized in that, The plant expression vector is pBI121-PmTZF1.

5. Application of the PmTZF1 gene, with a nucleotide sequence as shown in SEQ ID NO.1, in promoting the growth of Arabidopsis thaliana under drought stress.

6. The application according to claim 5, characterized in that, Promoting Arabidopsis growth includes promoting flowering and fruiting, increasing the number of branches, increasing the number of siliques, increasing the length of siliques, and increasing the length of the root system.

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