CN118773202A - A Masson pine tandem zinc finger structure PmTZF1 gene and its expression protein and application - Google Patents

Abstract

The invention discloses a Masson pine tandem zinc finger structure PmTZF1 gene and its expression protein and application, and relates to the technical field of plant genetic engineering. The Masson pine tandem zinc finger structure PmTZF1 gene disclosed for the first time in the present application has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2. The invention constructs an expression vector pBI121-PmTZF1 of the Masson pine tandem zinc finger structure PmTZF1 gene; transforms the constructed Masson pine tandem zinc finger structure PmTZF1 gene expression vector into Arabidopsis thaliana; cultivates, screens and obtains transgenic Arabidopsis thaliana plants with improved resistance to abiotic stress. Compared with wild-type plants, the roots of the transgenic strains are significantly longer than those of the wild type, and they bloom and bolt in advance; under drought stress, the transgenic strains bloom and bear fruit normally, have a large number of branches, and the number and length of siliques are significantly better than those of the wild-type strains.

Description

A Masson pine tandem zinc finger structure PmTZF1 gene and its expression protein and application

Technical Field

The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a Masson pine tandem zinc finger structure PmTZF1 gene and its expression protein and application.

Background Art

Masson pine (Pinus massoniana Lamb.) grows fast and has strong stress resistance. It is a pioneer tree species for afforestation on barren hills in southern my country. It has excellent characteristics such as drought resistance, cold resistance, and tolerance to barrenness. Masson pine plays an irreplaceable role in industrial timber, forest product processing, forest resource development and ecological service functions. The drought environment severely restricts the normal growth and development of plants. At present, the site environment of Masson pine is becoming increasingly harsh, and its molecular mechanism of responding to drought is still unclear. Therefore, it is of great significance to explore the drought resistance genes of Masson pine at the molecular level and reveal its drought resistance regulation mechanism, in order to improve the tolerance of Masson pine to drought stress and expand the cultivation range of Masson pine.

Under drought conditions, plant root activity decreases, respiration weakens, and the root absorption and transportation of water and mineral elements are inhibited. At the same time, leaf growth, leaf area, stomatal index, stomatal opening and closing, and chlorophyll content decrease, which seriously damages photosynthesis and respiration. Under drought stress, the level of ROS in plants increases, and membrane lipids are delipidated or oxidized to produce MDA. Plant genomes encode a large number of CCCH zinc finger proteins. The CCCH-type zinc finger protein family (especially tandem CCCH zinc finger proteins (TZFs)) plays a key role in plant growth, development, and regulation of plant tolerance to abiotic and biotic stresses. Drought stress and ABA treatment can significantly induce the expression of multiple CCCH zinc finger protein genes. CCCH zinc finger proteins improve plant drought resistance in a variety of ways. One way is to reduce water loss by regulating stomata; 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 ability.

Zinc finger proteins are a type of transcription factor with a zinc finger domain. According to the different zinc finger structure sequences and functions, zinc finger proteins can be divided into 9 categories: C ₂ H ₂ , G ₈ , C ₆ , C ₃ HC ₄ , C ₂ HC , C ₂ HC ₅ , C ₄ , C ₄ HC ₃ , C ₃ H (C represents cysteine and H represents histidine). Among them, CCCH (i.e. C ₃ H) type zinc finger proteins contain 1 to 6 CCCH type zinc finger motifs. This motif consists of three cysteine and one histidine residue. Compared with other types of zinc finger proteins, CCCH zinc finger proteins have been less studied and are mainly concentrated in animals. Reports on plant CCCH zinc finger proteins are very limited and are mostly concentrated on TZF zinc lipoproteins.

Through genomic analysis, 68 CCCH genes have been identified from the Arabidopsis genome. Although the family is very large, only a few CCCH zinc finger proteins have been preliminarily studied: HUA1 is an RNA binding protein involved in controlling flowering development; FESI can promote the formation of Arabidopsis winter acclimatization characteristics by interacting 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 signal transduction pathway, which can negatively regulate light-dependent seed germination; studies on AtTZF1 have found that the protein can shuttle between the nucleus and cytoplasm, and can bind to DNA and RNA. In addition to the study of zinc finger proteins in Arabidopsis, a CCCH zinc finger transcription factor GhZFP1 was also isolated from cotton, which can improve plant salt tolerance and disease resistance by interacting with GZ-lRD21A and GZIPR5. It can be seen that CCCH zinc finger proteins play an important role in regulating plant growth and development and stress response.

Summary of the invention

In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a Masson pine tandem zinc finger structure PmTZF1 gene. Another technical problem to be solved by the present invention is to provide an expression protein of the Masson pine tandem zinc finger structure PmTZF1 gene. Another technical problem to be solved by the present invention is to provide an application of the Masson pine tandem zinc finger structure PmTZF1 gene for obtaining new plant germplasm with improved abiotic stress tolerance.

In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

A Masson pine tandem zinc finger structure PmTZF1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

The amino acid sequence of the expressed protein of the Masson pine tandem zinc finger structure PmTZF1 gene is shown in SEQ ID NO.2.

A vector, recombinant bacteria or host cell containing a Masson pine tandem zinc finger structure PmTZF1 gene.

Application of the tandem zinc finger structure PmTZF1 gene of Masson pine in promoting the plant's ability to improve its resistance to abiotic stress.

The abiotic stress is drought.

The application of the Masson pine tandem zinc finger structure PmTZF1 gene in promoting the plant's ability to resist abiotic stress includes:

1) Construct the expression vector of Masson pine tandem zinc finger structure PmTZF1 gene;

2) Transform the constructed expression vector of the Masson pine tandem zinc finger structure PmTZF1 gene into Arabidopsis thaliana;

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

The expression vector is a plant expression vector.

The plant expression vector is pBI121-PmTZF1.

Application of the Masson pine tandem zinc finger structure PmTZF1 gene in promoting plant growth.

The promoting of plant growth is to promote flowering and fruiting of the plant, promote the increase of branch number, promote the increase of silique number, and promote the increase of silique length.

Compared with the prior art, the present invention has the following beneficial effects:

1) The Masson pine tandem zinc finger structure PmTZF1 gene disclosed for the first time in this application has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2. The present invention constructs an expression vector pBI121-PmTZF1 of the Masson pine tandem zinc finger structure PmTZF1 gene; transforms the constructed Masson pine tandem zinc finger structure PmTZF1 gene expression vector into Arabidopsis thaliana; cultivates, screens and obtains transgenic Arabidopsis thaliana plants with improved resistance to abiotic stress.

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

3) After 15 days of continuous drought, the wild-type strain was stunted and short under drought stress, and it was difficult to bloom and bear fruit normally, while the transgenic strain could bloom and bear fruit normally, and the number of branches was significantly more than that of the wild type; three days after rehydration, it was observed that the number and length of siliques of the transgenic strain were significantly better than those of the wild-type strain.

4) Under drought conditions, the wild-type strain (WT) loses water faster, requires more water, and has difficulty maintaining normal physiological activities, and its vegetative and reproductive growth are slow. However, the transgenic strain has improved drought resistance, loses less water under drought treatment, and can still maintain normal physiological activities. The above results all indicate that the PmTZF1 gene plays a key role in plant resistance to drought stress, and can enable plants to show a good growth state.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is an agarose gel electrophoresis diagram of PCR of the open reading frame of the PmTZF1 gene;

Figure 2 is a diagram of the differential expression of the PmTZF1 gene in 15-year-old Masson pine tissues (FC is female cones, MC is male cones, C is immature cones, YS is young stems, OS is old stems, N is needles, and R is roots);

FIG3 is a diagram showing changes in the expression level of the Masson pine PmTZF1 gene under drought stress;

FIG4 is a diagram showing the subcellular localization of the PmTZF1 gene in tobacco leaf cells;

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

Figure 6 is a phenotypic comparison diagram of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana (A is a schematic diagram of root length; B is a data diagram of root length; C is a schematic diagram of flowering; D is the bolting period; E is the flowering period);

Figure 7 is a phenotypic comparison of transgenic Arabidopsis and wild-type Arabidopsis under drought conditions (A is the growth status of Arabidopsis after drought and rehydration; B is a comparison of siliques; C is plant height; D is silique length; E is the number of branches; F is the number of siliques; G is the fresh weight of siliques);

FIG8 is a graph showing the relative expression levels of PmTZF1 in transgenic Arabidopsis and wild-type Arabidopsis under drought;

FIG. 9 is a graph showing the relative soil water content of transgenic Arabidopsis lines and wild-type Arabidopsis.

DETAILED DESCRIPTION

In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

The Masson pine needle material used for RNA extraction in the following examples was derived from two-year-old Masson pine seedlings planted in the National Key Laboratory of Tree Genetics and Breeding of Nanjing Forestry University.

The 15-year-old Masson pine materials used in the following examples were sourced from the Washan State-owned Forest Farm in Quanjiao County, Chuzhou City.

Example 1

1. Extraction of total RNA

Use the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Novozyme) to extract total RNA from Masson pine seedlings according to the instructions. The specific steps are as follows:

Add appropriate amount of liquid nitrogen-ground Masson pine needle powder, add 500μL Buffer PRL preheated at 65℃ (add 5% β-mercaptoethanol before use), immediately vortex and shake vigorously for 30-60sec to fully lyse, then place in a 65℃ water bath for 5min, invert 1-2 times during the period to help lysis; after lysis, centrifuge at 12000rpm for 10min, transfer the supernatant to a new 1.5mL RNase-free centrifuge tube, add 0.5 times the volume of supernatant anhydrous ethanol, and immediately blow and mix; transfer the above mixture to column II (lower receiving collection tube), centrifuge at 12000rpm for 2min, and discard the filtrate; put the column into a new collection tube, add 500μL Buffer PRL Plus, centrifuge at 12000rpm for 30sec, collect the filtrate; add 0.5 times the volume of anhydrous ethanol to the filtrate, and mix it immediately by blowing; transfer the above mixture to the IV column (lower receiving collection tube), centrifuge at 12000rpm for 2min, and discard the filtrate; add 700μL Buffer PRW1 to the IV column, let it stand at room temperature for 1min, centrifuge at 12000rpm for 30sec, and discard the filtrate; add 500μL Buffer PRW2 to the IV column (add 48mL anhydrous ethanol before use), centrifuge at 12000rpm for 30sec, discard the filtrate, and repeat the operation once; put the empty column back into the collection tube, centrifuge at 12000rpm for 2min, and leave it in the fume hood with the lid open for 2-5min to completely evaporate the residual ethanol; transfer the adsorption column to a new 1.5mL centrifuge tube, and drop 30-100μL ddH ₂ in the center of the adsorption column membrane O, place at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute to elute RNA.

2. cDNA Synthesis

Using the extracted total RNA as a template, the cDNA chain is obtained by reverse transcription. The following entire operation needs to be performed on ice. The specific steps are as follows:

The sample reaction system was as follows: 1 μg total RNA, 1 μL Oligo (dT) 18 (0.5 μg/μL), 1 μL 2×TS Reaction Mix, 1 μL RT/RI Enzyme Mix, 1 μL gDNA Remover, and 20 μL RNase H ₂ O. The mixture was mixed by gently pipetting and centrifuging to the bottom of the PCR tube. The mixture was reacted at 42°C for 30 min and at 85°C for 5 sec to inactivate the enzymes and gDNA Remover in the reaction system, and cDNA was obtained and stored in a refrigerator at -20°C for later use.

3. Cloning of target gene

Based on the PmTZF1 sequence screened from the transcriptome data of Masson pine under drought stress measured in the laboratory earlier (PRJNA595650), specific primers were designed and the PmTZF1 gene fragment was cloned using the synthesized Masson pine cDNA as a template. The primer sequences for cloning the open reading frame of PmTZF1 are as follows:

PmTZF1-F: 5′-ATGTCAAGCGTTTTCTGCAGAACAG-3′,

PmTZF1-R: 5′-TTACTTAACAAGCTCGTTCACCCA-3′.

PCR reaction system (20 μL): 10 μL 2×Hieff Gold PCR Master Mix, 1μL Forward primer, 1μL Reverse primer, 1μL cDNA, 7μL ddH ₂ O.

The PCR reaction program was: denaturation at 98°C for 10 s, annealing at 57°C for 5 s, and extension at 72°C for 10 s, for 35 cycles.

5 μL PCR product (cloned PmTZF1 gene fragment) was mixed with 1 μL 6× Loading Buffer and spotted into the prepared 1.2% agarose gel well. Electrophoresis was performed at 200V for 18 min in 1×TAE buffer. After the electrophoresis detection was correct, the remaining PCR product was mixed with 6× Loading Buffer using an agarose gel recovery kit to recover the target fragment. 4 μL of the gel-cutting recovery product was taken into a 1.5 mL centrifuge tube and 1 μL -Blunt, gently mix with a pipette, react at room temperature for 15 minutes and then place the centrifuge tube on ice. Add 50μL DH5α E. coli competent cells to the ligation product, flick to mix, ice bath for 25 minutes, heat shock at 42℃ for 45 seconds, immediately place on ice for 2 minutes, then add 900μL LB medium, 200rpm, 37℃ recovery for 1h; centrifuge at 5000rpm for 1min, discard 800μL supernatant, blow and mix well, then spread on the plate containing Amp, and invert the plate in a 37℃ incubator for overnight culture; the next day, pick a single colony on the LB plate in 10μL ddH ₂ O water, blow and mix well, take 2μL for positive detection, add 1mL LB liquid medium containing Kan to the remaining bacterial liquid, 200rpm, 37℃ culture. After the bacterial test, select the bacterial liquid with the correct electrophoresis band and send it to the company for sequencing.

The positive detection reaction system (20 μL) was: 10 μL 2×Rapid Taq Master Mix, 1 μL M13-F, 1 μL M13-R, 2 μL bacterial solution, and 6 μL ddH ₂ O.

The positive detection reaction program is: 95℃3min; 95℃15sec; 60℃15sec; 72℃15sec, 35 cycles; 72℃5min; 4℃∞.

The PCR results of the target gene clone are shown in Figure 1. According to the sequencing results, the nucleotide sequence of the target gene is determined as shown in SEQ ID NO.1, the total length of the Masson pine C3H32 open reading frame is 1578 bp, and the gene is named PmTZF1. The amino acid sequence of its expressed protein is shown in SEQ ID NO.2.

Example 2

1. Specific expression of PmTZF1 gene in different organs of Masson pine

RNA was extracted from male cones, female cones, cones, young stems, old stems, needles and root tissues of 15-year-old Masson pine and reverse transcribed into cDNA, and the specific method is shown in Example 1. Real-time fluorescence quantitative PCR (RT-qPCR) technology was used to detect the specific expression of the Masson pine PmTZF1 gene in different organs. The primer sequences are as follows:

PmTZF1-q-F: 5′-TCCACCACTGTCACCATCTGCGTCTC-3′,

PmTZF1-q-R: 5′-GCCTTTGGGCTTGCCAACCCTCTT-3′.

qRT-PCR reaction system (10 μL): 1 μL cDNA (diluted to 1/20 of the initial concentration), 5 μL SYBR Green Master Mix, 0.4 μL PmTZF1-qF, 0.4 μL PmTZF1-qR, 3.2 μL ddH ₂ O.

The reaction procedure was as follows: 95°C for 2 min; 95°C for 10 sec; 60°C for 30 sec; 72°C for 30 sec; 40 cycles.

The results are shown in Figure 2. The expression level in c 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 roots and young stems; and the lowest expression level was in fruits.

2. Response of PmTZF1 gene to drought stress

The Masson pine seedlings were subjected to natural drought treatment, and samples were taken at 0d, 3d, 7d, 12d, and 20d, respectively. After total RNA was extracted according to the method in Example 1, the extracted total RNA was used as a template to obtain cDNA chains by reverse transcription. Real-time fluorescence quantitative PCR (RT-qPCR) technology was used to detect the specific expression of the PmTZF1 gene in Masson pine under drought. The primer sequences are as follows:

PmTZF1-q-F: 5′-TCCACCACTGTCACCATCTGCGTCTC-3′,

PmTZF1-q-R: 5′-GCCTTTGGGCTTGCCAACCCTCTT-3′.

qRT-PCR reaction system (10 μL): 1 μL cDNA (diluted to 1/20 of the initial concentration), 5 μL SYBR Green Master Mix, 0.4 μL PmTZF1-qF, 0.4 μL PmTZF1-qR, 3.2 μL ddH ₂ O.

The reaction procedure was as follows: 95°C for 2 min; 95°C for 10 sec; 60°C for 30 sec; 72°C for 30 sec; 40 cycles.

The results are shown in Figure 3. In the early stage of drought, there was no obvious change in the expression level of PmTZF1. As the drought deepened, the expression level of PmTZF1 increased significantly after 20 days of dehydration.

3. Subcellular localization

Take 1mL of E. coli culture containing pCAMBIA1302 vector stored at -80℃ in our laboratory for activation, take 1-4mL of overnight culture culture, and extract plasmid using plasmid extraction kit. According to the ORF sequence of pCAMBIA1302 vector and PmTZF1 (remove the stop codon), use CE-Design software to design primers with restriction sites (NcoI and BgIII). The primer sequences are as follows:

PmTZF1-mgfp5-F: 5’-acgggggactcttgaccatggATGTCAAGCGTTTCTGCAGAACA-3’, PmTZF1-mfp5-R: 5’-tctcctttactagtcagatctCTTAACAAGCTCGTTCACCCAAC-3’.

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

Double restriction enzyme digestion was performed using NcoI and BgIII. The restriction enzyme digestion system (20 μL) was as follows: 1 ug plasmid, 1 μL NcoI, 1 μL BgIII, and ddH ₂ O was added to 20 μL.

The ligation system was as follows: 0.02× cloning vector base pair number (ng) of linearized vector, 0.04× insert fragment base pair number (ng) of insert fragment, 2 μL 5×CEIIBuffer, 1 μL Exnase II, and ddH ₂ O supplemented to 10 μL. The mixture was placed in a PCR instrument at 37°C for 25 min and immediately placed on ice.

The ligation product was transformed into DH5α E. coli competent cells, flicked to mix, ice bathed for 25 minutes, heat-shocked at 42℃ for 45 seconds, and immediately placed on ice for 2 minutes, followed by adding 900μL LB medium, 200rpm, and 37℃ for 1h; centrifuged at 5000rpm for 1min, discarded 800μL supernatant, mixed by pipetting, and then spread on a plate containing Kan, and the plate was inverted in a 37℃ incubator for overnight culture; the next day, a single colony on the LB plate was picked up in 10μL ddH ₂ O water, mixed by pipetting, and 2μL was taken for positive detection, and 1mL LB liquid medium containing Kan was added to the remaining bacterial liquid, and cultured at 200rpm and 37℃. After the bacterial test, the bacterial liquid with the correct electrophoresis band was selected and sent to the company for sequencing, and the positive clones with the correct sequencing results were screened for expansion and culture, and the recombinant plasmid was extracted.

The recombinant plasmid was transferred 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 bottom of the tube was gently tapped to mix, and the cells were 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 liquid culture medium was added and placed in a 28°C constant temperature shaker for shaking culture for 2.5 h; 100 μL of the supernatant was spread on an LB plate containing 50 mg·L ^-1 Kan and 25 mg·L ^-1 Rif, and the plate was inverted and cultured in a 28°C incubator for 2 days; monoclonal colonies with good growth were selected for PCR detection, and monoclonal colonies with positive detection were expanded and cultured, and 500 μL of overnight activated bacterial solution (50 mg/L Kan, 25 mg/L Rif) was inoculated into 50 mL of LB liquid culture medium, and cultured at 28°C, 220 rpm/min to OD ₆₀₀ value was 0.7-0.8, 5000rpm/min, the supernatant was discarded, and the bacteria were collected; the bacterial precipitate was resuspended with sterile water containing 10mM _MgCl2 , 10mM MES (pH=5.6) and 200μM acetosyringone (AS), and equal amounts of the two target bacterial solutions were mixed with p19 Agrobacterium in a ratio of 1:1, the OD600 value was adjusted to about 1.0, and it was allowed to stand at room temperature in the dark for 3-4h.

The pressure injection method was used, and a 10mL syringe (without a needle) was used to inject the suspended Agrobacterium into the young leaves of wild Nicotiana benthamiana of 3-week-old seedlings. The Agrobacterium was slowly injected into the tissue gaps on the back of the leaves by osmotic pressure, avoiding the veins, and finally the injected tobacco leaves were marked; an appropriate amount of water was poured into the bottom of the pot to keep it slightly moist. After culturing in the dark for 1 day, it was transferred to light culture for 1 day, and then microscopic observation was performed under a laser confocal microscope. The 35S::mGFP5 empty plasmid was used as a negative control.

The results are shown in Figure 4. Under a laser confocal microscope, the fluorescence signal of the 35S::PmTZF1-mGFP5 fusion protein was observed to be localized in the cell nucleus, and the overlap with the DAPI-stained cell nucleus indicated that PmTZF1 was specifically localized in the cell nucleus.

Example 3

1. Construction of vector

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:

pBI121-PmTZF1-F;

5'-gagaacacgggggactctagaATGTCAAGCGTTTCTGCAGAACA-3',

pBI121-PmTZF1-R:

5′-gggaaattcgagctcggatccCTTAACAAGCTCGTTCACCCAAC-3′.

According to the above method, the vector was double-digested with XbaI and BamHI, and the ORF was connected 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 the recombinant plasmid was transferred into the Agrobacterium competent cell GV3101 using the method described in Example 2.

2. Sowing and cultivation of Arabidopsis

1) Disinfection of Arabidopsis seeds: Place an appropriate amount of Arabidopsis seeds in a 1.5 mL centrifuge tube, add 1 mL of 75% ethanol to the centrifuge tube, turn it upside down for 45 seconds, wash it with sterile water, add 1 mL of 20% sodium hypochlorite, turn it upside down for 5 minutes, and wash it repeatedly with sterile water 5-6 times;

2) Sowing: Use a 1 mL pipette to sow the sterilized Arabidopsis seeds on 1/2 MS medium for cultivation.

3) Arabidopsis culture: Seal the culture medium after sowing Arabidopsis seeds, culture them at 4°C in the dark for 2 days, and then place them in an artificial climate incubator for germination and growth. After about a week, transfer the Arabidopsis seedlings that are growing well in the culture medium to nutrient soil (black soil: vermiculite: perlite = 6:2:1) for further culture, and cover them with plastic wrap. Remove the plastic wrap on the third day.

3. Arabidopsis transformation by inflorescence dip method

1) In a clean bench, streak the Agrobacterium culture containing the pBI121-PmTZF1 recombinant plasmid onto an LB plate containing 50 mg/L Kan and 25 mg/L Rif.

2) Pick a single colony with good growth status, add 5 mL of LB liquid culture medium containing 50 mg/L Kan and 25 mg/L Rif, and culture overnight in a constant temperature shaker at 28°C and 200 rpm.

3) Take 1 mL of overnight cultured bacterial solution and inoculate it into 50 mL of LB liquid medium containing 50 mg/L Kan and 25 mg/L Rif, and culture with shaking until OD600 = about 0.8.

4) Pour the bacterial solution into a 50 mL sterile centrifuge tube, centrifuge at 5000 rpm for 10 min to collect the bacteria, and add 50 mL of the pre-prepared infiltration buffer to suspend the bacterial precipitate.

5) Select Arabidopsis plants that have grown for about 4 weeks and have begun to sprout, remove the opened flower buds, immerse the inflorescence in the infection solution for 30 seconds, and cover with plastic wrap after the infection is completed.

6) Incubate in the dark for 18-20 hours, rinse with clean water and continue to culture in the incubator.

7) After 7-10 days, repeat steps 1-6 to infect again.

8) After the seed pods of the Arabidopsis plants turn yellow, the transgenic T0 generation seeds are harvested. To promote seed maturation, the number of watering should be appropriately controlled when the seeds are about to mature.

4. Screening of transgenic positive plants

Preliminary resistance screening was carried out on 1/2MS (containing Kan) medium. Negative plants that were not successfully transformed could not grow normally, while positive transgenic plants could grow normally. Arabidopsis seedlings that could grow normally were moved to nutrient soil for further cultivation, and genomic DNA and RNA were extracted from leaves. The specific implementation steps are as follows: Use a plant genomic DNA extraction kit (TIANGEN) to extract the DNA of the transgenic Arabidopsis plants obtained by preliminary screening. For specific steps, refer to the instructions. Use an ultra-micro spectrophotometer to detect the concentration and OD260/280 ratio of the extracted Arabidopsis gDNA. Then the universal 35s-F and the aforementioned pBI121-PmTZF1-R primers were used for PCR detection. The primer sequences are as follows:

35s-F: 5′-TGAAGATAGTGGAAAAGGAAGGTG-3′;

PCR reaction system: DNA template 100 ng, forward and reverse primers 1 μL each, 2×Phanta Max Master Mix (Vazyme) 10 μL, ddH ₂ O to 20 μL.

PCR reaction program: 95℃ pre-denaturation for 3min; 35 amplification cycles including 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 25s, 72℃ extension for 5min, and storage at 4℃. Use 1.2% agarose gel and UV gel imaging system to observe and compare whether the PCR product band size is consistent with the size of the target fragment inserted in the recombinant plasmid.

The total RNA of the plants with positive DNA level detection was extracted and reverse transcribed into cDNA, and the relative expression of PmTZF1 gene in transgenic Arabidopsis was detected by qRT-PCR, wherein the internal reference gene was Actin2. RNA extraction, first-strand cDNA synthesis, qRT-PCR system and program settings were set as in Example 1.

The results are shown in Figure 5. Arabidopsis thaliana with successfully transferred genes can amplify a band with the same size as the plasmid amplification product (Figure 5A). Then, the transgenic lines successfully identified at the gene level were identified at the transcription level (Figure 5B). The results showed that there were differences in the expression levels of different transgenic plants. Among the 10 transgenic Arabidopsis thaliana tested, the Arabidopsis plant numbered L10 had the highest expression level, followed by L4 and L6.

5. Phenotypic traits of transgenic Arabidopsis

The phenotypic differences between 1-month-old wild-type (WT) Arabidopsis and the selected transgenic lines (L4, 6, 10) are shown in Figure 6. The transgenic lines of Arabidopsis bolted on 21 days and flowered on 26 days, while the wild-type Arabidopsis bolted on 26 days and flowered on 30 days; the roots of the transgenic lines were significantly longer than those of the wild type.

6. Comparison of phenotypic traits of transgenic Arabidopsis under drought stress

The transgenic Arabidopsis thaliana with the PmTZF1 gene and the wild Arabidopsis thaliana were planted in small pots (three plants in one pot), and after being watered thoroughly, they were subjected to natural drought treatment for 15 days (no watering during the 15 days), and then cultured in a greenhouse at 25°C (16 h of light and 8 h of darkness).

The results are shown in Figure 7. After 15 days of continuous drought, the wild-type strain was stunted and short under drought stress, and it was difficult to bloom and bear fruit normally, while the transgenic strain could bloom and bear fruit normally, and the number of branches was significantly more than that of the wild type. After three days of rehydration, it was observed that the number, length and other traits of the transgenic strain were significantly better than those of the wild-type strain.

7. Gene expression in transgenic Arabidopsis thaliana under drought stress

The expression level of PmTZF1 gene in transgenic Arabidopsis thaliana was detected by qRT-PCR after 15 days of continuous drought, and the transgenic Arabidopsis thaliana that was not subjected to drought treatment was used as a control.

The results are shown in Figure 8, and the expression level of PmTZF1 was significantly increased after transgenic Arabidopsis thaliana encountered drought stress.

8. Soil relative water content of transgenic Arabidopsis under drought stress

After 15 days of continuous drought, the changes in soil water content between wild-type Arabidopsis and transgenic PmTZF1 gene lines were compared.

The results are shown in Figure 9. Under drought conditions, the wild-type strain (WT) loses water faster, requires more water, and has difficulty maintaining normal physiological activities, and its vegetative and reproductive growth are slow. However, the transgenic strain has improved drought resistance, loses less water under drought treatment, and can still maintain normal physiological activities. The above results all indicate that the PmTZF1 gene plays a key role in plant low resistance to drought stress, and can enable plants to show a good growth state.

The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims (10)

1. A Pinus massoniana series zinc finger structure PmTZF gene has a nucleotide sequence shown as SEQ ID NO. 1.

2. The expressed protein of the pinus massoniana tandem zinc finger structure PmTZF gene of claim 1, wherein the amino acid sequence of the expressed protein is shown as SEQ ID No. 2.

3. A vector, recombinant bacterium or host cell comprising the pinus massoniana tandem zinc finger structure PmTZF1 gene of claim 1.

4. Use of the pinus massoniana series zinc finger structure PmTZF gene according to claim 1 to promote plant to raise abiotic stress resistance.

5. The use of a pinus massoniana series zinc finger structure PmTZF gene according to claim 4 to promote plants to increase resistance to abiotic stress, wherein the abiotic stress is drought.

6. The use of the pinus massoniana series zinc finger structure PmTZF gene according to claim 4 to promote plants to increase abiotic stress resistance, comprising:

1) Constructing an expression vector of a pinus massoniana tandem zinc finger structure PmTZF gene;

2) Transforming the constructed expression vector of the masson pine serial zinc finger structure PmTZF gene into arabidopsis;

3) And cultivating, screening and obtaining the transgenic Arabidopsis plants with improved abiotic stress resistance.

7. The use of a pinus massoniana series zinc finger structure PmTZF gene according to claim 6 to promote plants to increase abiotic stress resistance, wherein the expression vector is a plant expression vector.

8. The use of a pinus massoniana series zinc finger structure PmTZF1 gene according to claim 7 to promote plant improvement of abiotic stress resistance, wherein the plant expression vector is pBI121-PmTZF1.

9. Use of the pinus massoniana series zinc finger structure PmTZF gene according to claim 1 to promote plant growth.

10. The use of a pinus massoniana series zinc finger structure PmTZF gene according to claim 9 to promote plant growth, wherein the promotion of plant growth is to promote plant flowering and fruiting, promote increase of branch number, promote increase of pod length, promote increase of root length.