CN111118036B - Gene Encoding the PHD3 Transcription Factor of Tamarix brixensis and Its Application - Google Patents

Abstract

The invention relates to a gene encoding a tamarisk PHD3 transcription factor and an application thereof, and belongs to the technical field of plant genetic engineering breeding. In order to solve the problem that it is impossible to use the PHD transcription factor coding gene of Tamarix brix to cultivate excellent salt-tolerant plants, the present invention provides the PHD3 transcription factor coding gene of Tamarix brix, i.e. the cDNA sequence of the ThPHD3 gene of Tamarix brix as shown in SEQ ID No: 1, The amino acid sequence encoded by the cDNA sequence is shown in SEQ ID NO:2. The overexpression and suppression expression vectors of ThPHD3 gene of Tamarix biribilis were constructed, and the transgenic Tamarix biribilis was obtained by transient infection. The test confirmed that the overexpression line had obvious salt tolerance. Both the homologous and heterologous overexpression results of the ThPHD3 gene show that the gene can significantly improve the salt-tolerant ability of the transgenic plant, and can be used to improve the salt-tolerant plant or to breed the salt-tolerant transgenic plant.

Description

Gene Encoding the PHD3 Transcription Factor of Tamarix brixensis and Its Application

technical field

The invention belongs to the technical field of plant genetic engineering and breeding, and in particular relates to a gene encoding tamarix bristles PHD3 transcription factor and an application thereof.

Background technique

During the growth process, plants will be subjected to abiotic stresses such as drought, high salinity, osmosis, high temperature, low temperature and hormone stimulation. Transcription factors are a class of DNA-binding proteins that can specifically interact with the cis-acting elements of eukaryotic gene promoters, and activate or repress transcription through the interaction between them or with other related proteins. Under stress stimulation, some transcription factors are overexpressed, and then the signal is transmitted and amplified, and the expression of corresponding downstream functional genes is regulated, thereby improving the stress resistance of plants. Since transcription factors can regulate the expression of multiple downstream genes, overexpressing a transcription factor in plants is a more effective method and approach for improving plant stress resistance than overexpressing individual functional genes.

Plant homeodomain PHD (Plant homeodomain) transcription factors are a class of zinc finger proteins that widely exist in eukaryotes and play an important role in gene transcription and chromatin state regulation. They contain 1 to 3 typical PHDfinger structures ( Cys4-His-Cys3, C4HC3). The PHD finger domain is composed of about 50-80 amino acids, and cysteine residues and histidine residues combine with two zinc ions to form a typical loop (Loop) structure. Studies have shown that members of the Alfin-like PHD finger transcription factor family in plants respond to drought, high-salt, chilling and ABA stress treatments to varying degrees.

Tamarix hispida is a woody halophyte with excellent stress resistance, which can form natural forests in soils with a salt content of 1%, so it is ideal for studying the mechanism of salt tolerance and cloning salt tolerance genes Material. However, due to the lack of understanding of the salt-tolerant function of the PHD transcription factor of Tamarix brixensis and the lack of understanding of its salt-tolerant mechanism, it is still impossible to use the gene encoding the PHD transcription factor of Tamarix brixensis to breed excellent salt-tolerant plants.

Contents of the invention

In order to solve the problem that it is impossible to use the PHD transcription factor coding gene of Tamarix brix to cultivate excellent salt-tolerant plants, the invention provides the PHD3 transcription factor coding gene of Tamarix brix and its application.

Technical scheme of the present invention:

The cDNA sequence of the gene encoding the PHD3 transcription factor of Tamarix biribilis, that is, the ThPHD3 gene of Tamarix biribilis is shown in SEQ ID No: 1.

Further, the amino acid sequence encoded by the cDNA sequence is shown in SEQ ID NO:2.

A plant overexpression vector containing the Tamarix bristles ThPHD3 gene.

A recombinant genetically engineered bacterium containing the plant overexpression vector.

Further, the recombinant genetically engineered bacterium is constructed by introducing a plant overexpression vector containing Tamarix bristle ThPHD3 gene into a host bacterium, and the host bacterium is Escherichia coli or Agrobacterium.

The application of the ThPHD3 gene of Tamarix brixensis in improving the salt tolerance of plants or breeding transgenic plants with salt tolerance.

Further, the application includes constructing a plant overexpression vector containing Tamarix bristles ThPHD3 gene, transforming the constructed plant overexpression vector into woody plants, and cultivating salt-tolerant transgenic woody plants.

Further, the woody plant is poplar or birch.

Further, the application includes constructing a plant overexpression vector containing Tamarix bristles ThPHD3 gene, transforming the constructed plant overexpression vector into herbaceous plants, and cultivating salt-tolerant transgenic herbaceous plants.

Further, the herbal plant is Arabidopsis thaliana or tobacco.

Beneficial effects of the present invention:

The Tamarix bristles ThPHD3 gene provided by the present invention has a full-length cDNA of 759bp, and the amino acid sequence encoded by the cDNA sequence includes 252 amino acids. The overexpression and suppression expression vectors of the Tamarix bristles ThPHD3 gene are constructed respectively, and the transgenic Tamarix bristles are obtained by transient infection. Experiments such as chemical staining and physiological index determination confirmed that the ThPHD3 gene overexpression strain of Tamarix biribilis has obvious salt tolerance, and it is better than the blank control and the suppressed expression strain.

The T. thaliana _ThPHD3 gene was transferred into the model plant Arabidopsis thaliana. After 100mM NaCl stress, the average germination rate of the transgenic Arabidopsis thaliana seeds was 5.43 times that of the wild type, the root length was 1.72 times that of the wild type, and the fresh weight was 1.72 times that of the wild type. 1.31 times. The results of homologous and heterologous overexpression of ThPHD3 gene have shown that this gene can significantly improve the salt tolerance of transgenic plants. Saline transgenic plants.

Description of drawings

Figure 1 is a prediction map of the conserved region of the ThPHD3 gene;

Figure 2 is a multiple sequence alignment of Tamarix bristles ThPHD3 protein and soybean PHD protein;

Fig. 3 is an analysis diagram of the expression pattern of Tamarix bristles ThPHD3 gene under different abiotic stresses and hormone treatments;

Fig. 4 is an analysis diagram of the expression pattern of ThPHD3 gene in transgenic Tamarix bristles under NaCl stress;

Fig. 5 is the histochemical staining analysis diagram of ThPHD3 gene transfection and control Tamarix bristles under NaCl stress;

Fig. 6 is a measurement and analysis diagram of H ₂ O ₂ content of transgenic Tamarix bristles under NaCl stress;

Fig. 7 is the assay analysis diagram of MDA content of transgenic Tamarix bristles under NaCl stress;

Fig. 8 is the measurement and analysis diagram of the SOD content of transgenic Tamarix bristles under NaCl stress;

Fig. 9 is a measurement and analysis diagram of POD content of transgenic Tamarix bristles under NaCl stress;

Fig. 10 is a measurement and analysis diagram of the proline content of transgenic Tamarix setae under NaCl stress;

Fig. 11 is a comparison chart of observation of ThPHD3 transgenic and wild-type Arabidopsis seed germination phenotypes after salt stress;

Figure 12 is a graph comparing the germination rate of ThPHD3 transgenic and wild-type Arabidopsis after salt stress;

Figure 13 is a comparison chart of root development phenotype observations between ThPHD3 transgenic and wild-type Arabidopsis after salt stress;

Figure 14 is a comparison chart of fresh weight between ThPHD3 transgenic and wild-type Arabidopsis thaliana salt stress;

Figure 15 is a comparison of root length between ThPHD3 transgenic and wild-type Arabidopsis under salt stress;

Fig. 16 is a comparison chart of phenotype observation between ThPHD3 transgenic and wild-type Arabidopsis under salt stress.

Detailed ways

The technical solution of the present invention will be further described below in conjunction with the examples, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered by the present invention within the scope of protection.

Example 1: Cloning of Tamarix bristles ThPHD3 gene

The seeds of Tamarix briatus were sown in the artificial soil prepared with peat soil and sand at a mass ratio of 2:1, placed in a relative humidity of 65-75%, a light intensity of 400 μmol·m ^-2 ·s ^-1 , and an average temperature of 22±2 grown in a greenhouse at °C. After 2 months of growth, the roots were irrigated with 0.3mol·L ^-1 NaHCO ₃ solution for stress treatment, and the leaves and roots were taken at 0h, 12h, 24h and 48h respectively and put into liquid nitrogen for quick freezing for RNA extraction.

The CTAB method was used to extract the RNA of Tamarix seedlings in each stress treatment period, and the extracted RNA samples were sent to Shenzhen Huada Gene Technology Co., Ltd. for the construction and sequencing of the transcriptome library. The RNA sequencing results obtained in each processing period were compared and spliced, and the results after sequencing comparison and splicing were searched using "PHD3 transcription factor" as a keyword, and the searched sequences were further compared and confirmed using BLASTX software, combined with ORF The founder (http://www.ncbi.nlm.nih.gov/gorf.html) program determines whether there is a complete open reading frame. PHD genes with intact ORFs were selected.

According to the characteristics of ThPHD3 gene, specific upstream and downstream primers ThPHD3-F and ThPHD3-R shown in SEQ ID NO: 3 and SEQ ID NO: 4 were designed, and ThPHD3 gene was cloned by RT-PCR using Tamarix cDNA as a template.

RT-PCR reaction system is 20 μL, including template 2 μL, 10×Ex Taq PCR buffer 2.0 μL, ThPHD3-F and ThPHD3-R primers (10 μmol/L) each 1 μL, dNTP Mix (10 mmol/L) 0.4 μL, LA Taq (5U/μL) 0.3 μL, supplemented with ddH ₂ O to 20 μL. The reaction program was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 2 min, and 35 cycles; extension at 72°C for 10 min.

The target gene obtained by gel recovery was ligated to the pMD18-T vector, and then the ligated product was transformed into E. coli competent cells, and single clones were picked with gene primers-ThPHD3-F and ThPHD3-R and such as SEQ ID NO:5 Carry out PCR verification with the vector primer-pMD18-T-F and pMD18-T-R shown in SEQ ID NO: 6, and further sequence verification, obtain the Tamarix brixensis DHP3 transcription factor encoding gene shown in SEQ ID No: 1, be the gene of ThPHD3 Full-length cDNA sequence.

Example 2: Using bioinformatics software and online network resources to perform sequence analysis on the gene encoding the DHP3 transcription factor of Tamarix brix

As shown in SEQ ID No: 1, the coding region of the gene coding for the DHP3 transcription factor of Tamarix biribilis cloned in Example 1 is 759 bp long, encoding 252 amino acids shown in SEQ ID NO: 2. Through BLASTx similarity analysis, it was found that there was a typical PHD domain (595-744bp) at the C-terminal of the gene. Combined with ExPASy-PROSITE analysis and Figure 1, the C-terminus of the ThPHD3 protein sequence contains a conserved PHD domain, which is a typical ZF-PHD (Zinc finger PHD-type profile) protein.

The obtained ThPHD3 gene was calculated and deduced protein molecular weight and theoretical isoelectric point with ProtParam ( http://au.expasy.org/tools/protparam.html ) software. Select soybean PHD family protein and Tamarix bristles ThPHD3 protein for multiple sequence alignment. The selected soybean PHD proteins are: GmPHD1 (ABI97246.1), GmPHD2 (ABI97241.1), GmPHD3 (ABI97242.1), GmPHD4 (ABI97243.1), GmPHD5 (ABI97244.1), GmPHD6 (ABI97245.1).

The results showed that the molecular weight of the protein encoded by the ThPHD3 gene was 28.5, and the theoretical isoelectric point was 5.25. Further comparison of the sequence homology between the encoded protein sequence of the gene and the soybean PHD protein sequence by Clustal software found that, as shown in Figure 2, the N-terminal and C-terminal homology of the Tamarix bristle ThPHD3 protein and the soybean PHD protein were high, containing typical The cys4-His-cys3 conserved domain.

Example 3: Analysis of the expression pattern of ThPHD3 gene under different abiotic stresses and hormone treatments

ThPHD3 transcription factor plays an important role in the regulation of plant stress resistance. A single Tamarix ThPHD3 gene with complete open reading frame was cloned and its sequence was analyzed. In order to study the function of Tamarix ThPHD3 gene, real-time fluorescence quantitative RT-PCR was further used to analyze its different abiotic stresses: high salt-400mmol NaCl, drought-20% PEG and hormone stress: ABA-100μmol, GA ₃ -50μmol treatment conditions The expression mode below. Using the same amount of control (0h), the cDNA of the roots and leaf tissues of Tamarix bristles at 6h, 12h, 24h, 48h and 72h of each treatment as templates, real-time quantitative RT-PCR amplification was carried out to detect the ThPHD3 gene of Tamarix bristles under different stresses. The expression situation under treatment, and β-actin, α-tubulin and β-tubulin genes were used as internal references (3 biological experiments were repeated for each treatment of genes and internal references).

The real-time quantitative PCR reaction system is shown in Table 1:

Table 1

2×SYBR Green Real-time PCR Master mix 10.0μl Upstream primer (10μmol/L) 1.0μl Downstream primer (10μmol/L) 1.0μl cDNA 2.0μl Make up volume with ultrapure water to 20.0μl

Fluorescent real-time quantitative RT-PCR reaction conditions: pre-denaturation at 95°C for 10 minutes; 95°C for 15s, 58°C for 15s, 72°C for 30s; 80°C for 1s; plate reading; 45 cycles; 55°C to 99°C, read at intervals of 0.2°C Plate 1s to plot the melting curve.

Fig. 3 is an analysis diagram of the expression pattern of the ThPHD3 gene of Tamarix brixensis under different abiotic stresses and hormone treatments; the results of real-time quantitative RT-PCR showed that under two kinds of abiotic stresses and two kinds of hormone treatments, the ThPHD3 gene was expressed in the roots of Tamarix tamarix and The expression levels in the leaves were all changed, and they were stress response genes, which may be involved in the stress resistance of Tamarix. The expression of ThPHD3 gene in the roots and leaves of Tamarix changed at least one time point of stress treatment, showing tissue-specific expression. Especially under NaCl stress, the expression of Tamarix root was significantly up-regulated at almost all time points studied, indicating that ThPHD3 gene may be related to salt stress.

Embodiment 4: construction of overexpression vector of Tamarix wiry ThPHD3 gene

According to the sequence characteristics of the plant expression vector pROKⅡ and the gene ThPHD3, BamHI and KpnI restriction endonuclease sites were introduced into the 5' end and 3' end of the ThPHD3 gene respectively, using the Tamarix cDNA as a template, with SEQ ID NO: 7 and SEQ ID NO : The gene-specific upstream and downstream primers ThPHD3-CL-F and ThPHD3-CL-R shown in 8 are primers for PCR amplification of the ThPHD3 gene target fragment with restriction sites.

The PCR reaction system is shown in Table 2:

Table 2

10×Ex Taq PCR buffer 2.0 μL dNTP (10mmol/L) 0.4μL ThPHD3-CL-F (10μmol/L) 1.0 μL ThPHD3-CL-R (10μmol/L) 1.0 μL Tamarix cDNA 0.2μg ExTaq (5U/μL) 0.3μL Make up to volume with dd H₂O 20.0 μL

The PCR reaction program was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 2 min, and 35 cycles; extension at 72°C for 10 min.

The ThPHD3 target gene obtained by PCR was purified and recovered using a gel recovery kit. The recovered ThPHD3 gene fragment and pROKII vector plasmid were respectively digested with BamHI and Kpn I restriction endonucleases, and reacted at 37°C for 4-6 hours. The digestion system is shown in Table 3:

table 3

10×Multi-core buffer 2.0 μL BSA 0.5μL BamHI 1.0 μL KpnI 1.0 μL ThPHD3 gene fragment/pROKII vector plasmid 2μL/0.1-0.2μg dd H₂O up to 20 μL

The digested products were purified and recovered using gel recovery kits. After recovery, the ThPHD3 gene fragment and the pROKII carrier fragment were ligated by T4 DNA ligase, cultured at 16°C for 8-12 hours, and the recombinant vector pROKII-ThPHD3 was constructed. The ligation system is shown in Table 4:

Table 4

10×T4 DNA ligase buffer 1.0 μL T4 DNA ligase 1.0 μL pROKII Vector Large Fragment 2.0 μL ThPHD3 target gene fragment 3.0 μL dd H₂O up to 20 μL

The constructed recombinant vector pROKII-ThPHD3 was transformed into Escherichia coli competent cell TOP10, then spread in LB medium containing 50 mg/L kanamycin, cultured upside down at 37°C for 8-10 hours, and 10 positive cells were picked. Clonal plaque expansion culture. Using gene-specific upstream and downstream primers ThPHD3-CL-F, ThPHD3-CL-R shown in SEQ ID NO: 7, SEQ ID NO: 8 and overexpression shown in SEQ ID NO: 9, SEQ ID NO: 10 The upstream and downstream primers pROKⅡ-F and pROKⅡ-R of the vector pROKII were used for bacterial liquid PCR detection. The reaction system is shown in Table 5:

table 5

10×Taq DNA polymerase buffer 2.0 μL Taq DNA polymerase (5U/μL) 0.3μL dNTP Mix (10mmol/L) 0.4μL ThPHD3-CL-F/pROKII-F (10μmol/L) 1.0 μL ThPHD3-CL-R/pROKII-R (10μmol/L) 1.0μg Denatured bacteria liquid 2μL Make up to volume with dd H2O 20.0 μL

The PCR reaction program was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 2 min and 30 s, and 35 cycles; extension at 72°C for 10 min.

Select 2 clones that can amplify specific target bands for sequencing. The correct sequence is the plant overexpression vector pROKII-ThPHD3. Save the strain and extract the pROKII-ThPHD3 E. coli plasmid for future use.

Example 5: Transformation of Agrobacterium tumefaciens and PCR detection of positive clones

The pROKII-ThPHD3 Escherichia coli plasmid was transformed into EHA105 by liquid nitrogen freeze-thaw method, the specific steps are as follows:

(1) Take 0.1-1 μg (5-10 μL) of pROKII-ThPHD3 plasmid in 50 μL Agrobacterium competent cell EHA105, mix it in ice bath for 30 minutes, put it into liquid nitrogen for quick freezing for 1 minute, and quickly transfer it to a 37°C water bath for 5 minutes , and then stand in an ice bath for 4 minutes;

(2) Add 500 μL of LB liquid medium without any antibiotics, place on a shaker at 28°C, shake at 120rpm and incubate for 2 to 4 hours;

(3) Take 150-300 μL of the above-mentioned culture solution, spread evenly on the LB solid medium containing 50 mg/L Rif and 50 mg/L Kan, and incubate at a constant temperature of 28 °C for 2 days.

Randomly pick 10 positive single clones, inoculate them in LB liquid medium containing 50mg/LKan and 50mg/LRif, culture overnight at 28°C on a shaker at 200rpm. Take 10 μL of bacterial liquid and denature at 98°C for 5 minutes. Using the denatured bacterial liquid as a template, use the gene-specific upstream and downstream primers ThPHD3-CL-F, ThPHD3-CL-R shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 The upstream and downstream primers pROKⅡ-F and pROKⅡ-R of the indicated overexpression vector pROKII were used to detect positive clones by bacterial liquid PCR. 0.8% agarose gel electrophoresis was used to detect, and the pROKII-ThPHD3 Agrobacterium strain with specific target band and correct size was selected and preserved for future use.

Embodiment 6: Construction of suppression expression vector of Tamarix brix ThPHD3 gene

1. Construction of pFGC5941-ThPHD3-Cis vector

Based on the analysis of the multiple cloning sites of the plant interference vector pFGC5941 and the ThPHD3 gene. NcoI and AscI restriction endonuclease sites were introduced at the 5' and 3' ends of the target gene, respectively.

Use the pROKII-ThPHD3 plasmid as a template, and use ThPHD3-5941-cis-F/R shown in SEQ ID NO: 11 and SEQ ID NO: 12 as primers to perform PCR to amplify with NcoI and AscI restriction endonuclease sites Point target gene fragment, PCR system is shown in Table 6:

Table 6

10×Ex Taq PCR buffer 2.0 μL dNTP Mix (10mmol/L) 0.4μL ThPHD3-5941-cis-F (10μmol/L) 1.0 μL ThPHD3-5941-cis-R (10μmol/L) 1.0 μL pROKII-ThPHD3 plasmid 0.2μg ExTaq (5U/μL) 0.3μL Make up to volume with dd H₂O 20.0 μL

The PCR reaction program was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 40 s, and 35 cycles; extension at 72°C for 10 min.

The gel recovery kit was used to recover and purify the PCR product ThPHD3-Cis target fragment. The ThPHD3-Cis target fragment and the plant interference vector pFGC5941 plasmid were double-digested with restriction endonucleases NcoI and AscI. The double-digestion reaction system is shown in Table 7:

Table 7

10×Multi-core buffer 2.0 μL BSA 0.5μL NCOI 1.0 μL AscI 1.0 μL ThPHD3-Cis/pFGC5941 vector 2μL/0.1-0.2μg dd H2O up to 20 μL

After incubating at 37°C for 4 hours, the obtained enzyme-cleaved products were recovered and purified respectively.

The digested products were purified and recovered using gel recovery kits. After recovery, the ThPHD3-Cis gene fragment and the pFGC5941 carrier fragment were ligated by T4 DNA ligase, cultured at 16°C for 8-12 hours, and the recombinant vector pFGC5941-ThPHD3-Cis was constructed. The ligation system is shown in Table 8:

Table 8

10×T4 DNA ligase buffer 1.0 μL T4 DNA ligase 1.0 μL Large fragment of pFGC5941 vector 2.0 μL ThPHD3-Cis target gene fragment 3.0 μL dd H₂O up to 20 μL

The constructed recombinant vector pFGC5941-ThPHD3-Cis was transformed into Escherichia coli competent cell TOP10, then spread in LB medium containing 50 mg/L kanamycin, cultured upside down at 37°C for 8-10 hours, and 10 cells were picked Positive monoclonal plaque expansion culture. Use the gene-specific upstream and downstream primers ThPHD3-5941-cis-F/R shown in SEQ ID NO: 11 and SEQ ID NO: 12 and the upper part of the interference vector pFGC5941 shown in SEQ ID NO: 13 and SEQ ID NO: 14 The downstream primer 5941-Cis-F/-R was used for bacterial liquid PCR detection, and the reaction system is shown in Table 9:

Table 9

10×Taq DNA polymerase buffer 2.0 μL Taq DNA polymerase (5U/μL) 0.3μL dNTP Mix (10mmol/L) 0.4μL ThPHD3-5941-cis-F/5941-Cis-F (10μmol/L) 1.0 μL ThPHD3-5941-cis-R/5941-Cis-R (10μmol/L) 1.0μg Denatured bacteria liquid 2μL Make up to volume with dd H2O 20.0 μL

The PCR reaction program was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 40 s, and 35 cycles; extension at 72°C for 10 min. Use 1% agarose gel electrophoresis to detect, select 2 clones that can amplify specific target bands for sequencing, and the sequenced one is the plant recombinant vector pFGC5941-ThPHD3-Cis, save the strain and extract pFGC5941-ThPHD3-Cis large intestine Bacillus plasmid spare.

2. Construct pFGC5941-ThPHD3-Anti

Use the pROKII-ThPHD3 plasmid as a template, and use ThPHD3-5941-anti-F/R shown in SEQ ID NO: 15 and SEQ ID NO: 16 as primers for PCR to amplify with XbaI and BamHI restriction endonuclease sites Point target gene fragment, PCR system is shown in Table 10:

Table 10

10×Ex Taq PCR buffer 2.0 μL dNTP Mix (10mmol/L) 0.4μL ThPHD3-5941-anti-F (10μmol/L) 1.0 μL ThPHD3-5941-anti-R (10μmol/L) 1.0 μL pROKII-ThPHD3 plasmid 0.2μg Ex Taq (5U/μL) 0.3μL Make up to volume with dd H₂O 20.0 μL

The PCR reaction program was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 40 s, and 35 cycles; extension at 72°C for 10 min.

Purify and recover the PCR product ThPHD3-anti target fragment with gel recovery kit. The ThPHD3-anti target fragment and the recombinant vector pFGC5941-ThPHD3-Cis were double-digested with restriction endonucleases XbaI and BamHI. The double-digestion reaction system is shown in Table 11:

Table 11

10×Multi-core buffer 2.0 μL BSA 0.5μL wxya 1.0 μL BamHI 1.0 μL ThPHD3-anti/pFGC5941-ThPHD3-Cis 2μL/0.1-0.2μg dd H2O up to 20 μL

After incubating at 37°C for 4 hours, the digested products were purified and recovered using gel recovery kits. After recovery, the ThPHD3-anti gene fragment and the recombinant vector pFGC5941-ThPHD3-Cis fragment were ligated by T4 DNA ligase, and cultured at 16°C for 8-12 hours to construct the recombinant vector pFGC5941-ThPHD3. The connection system is shown in Table 12:

Table 12

10×T4 DNA ligase buffer 1.0 μL T4 DNA ligase 1.0 μL Large fragment of pFGC5941-ThPHD3-Cis vector 2.0 μL ThPHD3-anti target gene fragment 3.0 μL dd H₂O up to 20 μL

The constructed recombinant vector pFGC5941-ThPHD3 was transformed into Escherichia coli competent cell TOP10, and then spread in LB medium containing 50 mg/L kanamycin, cultured upside down at 37°C for 8-10 hours, and 10 positive cells were picked. Clonal plaque expansion culture. Use the gene-specific upstream and downstream primers ThPHD3-5941-anti-F/R shown in SEQ ID NO: 15 and SEQ ID NO: 16 and the upper part of the interference vector pFGC5941 shown in SEQ ID NO: 17 and SEQ ID NO: 18 The downstream primer 5941-anti-F/-R was used for bacterial liquid PCR detection, and the reaction system was shown in Table 13:

Table 13

10×Taq DNA polymerase buffer 2.0 μL Taq DNA polymerase (5U/μL) 0.3μL dNTP Mix (10mmol/L) 0.4μL ThPHD3-5941-anti-F/5941-anti-F (10μmol/L) 1.0 μL ThPHD3-5941-anti-R/5941-anti-R (10μmol/L) 1.0μg Denatured bacteria liquid 2μL Make up to volume with dd H2O 20.0 μL

The PCR reaction program was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 40 s, and 35 cycles; extension at 72°C for 10 min. Use 1% agarose gel electrophoresis to detect, select 2 clones that can amplify specific target bands for sequencing, and the sequenced one is the plant recombinant vector pFGC5941-ThPHD3, save the strain and extract the pFGC5941-ThPHD3 E. coli plasmid for future use.

The pFGC5941-ThPHD3 Escherichia coli plasmid was transformed into EHA105 by the liquid nitrogen freeze-thaw method, and the strain with the correct detection result was selected and stored for future use.

Example 7: Acquisition of transiently transformed ThPHD3 gene Tamarix

The constructed overexpression vector pROKII-ThPHD3(OE) and suppressor expression vector pFGC5941-ThPHD3(RNAi) were transformed into Tamarix brixensis by transient transformation technology, so that ThPHD3 gene could be transiently overexpressed or suppressed. At the same time, the empty pROKII vector was transformed into Tamarix brix as a control (Con).

1. Preparation of culture medium

Infection solution: 2mM MES-KOH (pH5.4, adjusted with KOH) + 10mM CaCl ₂ + 120μM AS + 2% sucrose + 270mM mannitol + 20μM 5-Azacytidine + 100uM DTT + 0.02% Tween (PH=5.6);

Washing solution: 40mM CaCl ₂ +120μM AS+3% sucrose+0.5mg/L NAA+2mg/L 6BA+200mg/L DTT;

Co-culture solid medium: MS+0.5mg/L NAA+2mg/L 6BA+150μM AS+200mg/L DTT+7g/L agar+1% sucrose;

Stress medium: MS+150mM NaCl;

Among them, AS, DTT, NAA, 6BA, and Tween cannot be sterilized by high temperature, and should be added after high temperature sterilization.

2. Preparation of infecting engineering bacteria

(1) Inoculate pROKII-ThPHD3(OE), pROKII(Con), pFGC5941-ThPHD3(RNAi) Agrobacteria on LB solid medium containing 50mg/L Rif and 50mg/L Kan respectively, and culture at 28°C for two days;

(2) Pick a single colony and inoculate it in 10ml LB liquid medium containing 50mg/L Rif and 50mg/L Kan, cultivate overnight at 28°C and 200rpm until the OD ₆₀₀ is 0.8, then dilute it 50 times, and then use fresh LB (50mg /L Rif+50mg/L Kan+5mM SPD+20μM 5-Azacytidine+120μM AS+150mg/L DTT) for secondary activation, 28°C, 200rpm shaking culture until the OD ₆₀₀ of the bacterial solution is 0.6-0.7;

(3) Transfer the bacterial liquid to a 50 mL sterilized centrifuge tube, put it into a low-temperature, high-speed, large-capacity centrifuge and centrifuge at 3000 rpm for 10 min, collect the bacterial cells, and adjust the concentration of the bacterial liquid to an OD ₆₀₀ of 0.6 with the infection solution for later use.

3. Instant transformation of Tamarix

(1) Divide Tamarix seedlings evenly into three parts, then quickly transfer to the infection solution that has added bacterial strains pROKII-ThPHD3, pROKII or pFGC5941-ThPHD3, and evacuate in a vacuum pump for 5 minutes;

(2) Soak the material in the infection solution, incubate at 25 degrees and 90 rpm for 1 hour, then add 1/3 volume of new infection solution (sterile fresh solution), and incubate for another 1.5 hours;

(3) Transfer the seedlings to the washing solution and wash them quickly for 1 minute, let the plants rehydrate, the time should not exceed 2 minutes, then dry the water with sterile filter paper, insert the co-cultivation solid medium, and place them in the greenhouse for cultivation.

(4) After 24 hours of co-cultivation, the seedlings were transferred to the stress medium for stress treatment.

In order to analyze the expression of ThPHD3 gene in the transiently overexpressed and suppressed Tamarix bristles, the total RNA of the transiently infected Tamarix bristles was extracted after 150 mM NaCl stress for 12 h, 24 h and 48 h, respectively, reverse-transcribed into cDNA, and the ThPHD3 gene was detected by qRT-PCR. The expression level of ThPHD3 was analyzed, and the results are shown in Figure 4. Compared with Con (control), the expression level of ThPHD3 gene in OE plants was significantly increased, while it was significantly decreased in RNAi suppressor lines. It shows that this example successfully obtained ThPHD3 gene transient transformation of Tamarix, and the expression of ThPHD3 gene can be induced by salt stress treatment.

Example 8: Histochemical staining analysis of transiently transformed ThPHD3 gene Tamarix setae seedlings under NaCl stress

Diaminobenzidine (DAB) can be oxidized by oxygen ions released by hydrogen peroxide in cells to form a brown precipitate, so that the specific site of peroxidase activity can be located. DAB dyeing method can accurately reflect the content of H ₂ O ₂ through the depth of dyeing.

Nitroblue Tetrazolium (NBT) can be oxidized by superoxide ion O ^2- into blue methylhydrazone, and the amount of O ^2- content can be reflected by the depth of the blue color of methylhydrazone.

Evans Blue (Evans Blue) staining can judge whether the plant cell membrane is complete or not by the depth of staining. In normal living cells, the cell membrane structure is complete and can repel Evans blue so that it cannot enter the cell. Cells with inactive or incomplete cell membranes have increased membrane permeability and can be stained blue by Evans blue. And the more the number of cells with loss of activity or incomplete cell membrane, the darker the Evans blue staining result.

Therefore, NBT, DAB and Evans Blue staining can be used to analyze the content of H ₂ O ₂ and O ₂ ^- in plants and the integrity of plant cell membranes.

The specific preparation method of the DAB staining solution in this example is as follows: 50 mg of DAB powder was weighed and dissolved in 50 mL of phosphate buffer (pH 7.0). The specific preparation method of NBT staining solution is: weigh 50mg NBT powder and dissolve it in 50mL phosphate buffer solution (0.1M/L, pH7.0). The specific preparation method of Evans blue staining solution is: weigh 50mg Evans blue and dissolve in 50mL distilled water.

In this example, the transiently transformed Tamarix brixensis plants of the overexpression vector pROKII-ThPHD3 (OE), the transiently transformed Tamarix brixensis plants of the suppressed expression vector pFGC5941-ThPHD3 (RNAi), and the blank (CON) control of the transiently transformed empty pROKII vector were analyzed DAB, NBT and Evans blue staining of Tamarix brixensis plants after 0h and 4h of 150mM NaCl stress, and then compared the active oxygen content and cell membrane damage of the three transgenic plants after adversity stress.

The dyeing method in this example is as follows: respectively place the above-mentioned three kinds of transgenic Tamarix bristles after stressing with 150 mM NaCl for 0 hour and 4 hours in a 50 mL centrifuge tube, then add different dyeing solutions respectively, so that the leaves are completely submerged in the dyeing solution, and place at room temperature At an appropriate time, after DAB staining was left at room temperature for a period of time, it was exposed for 1 hour, and after the staining result was obvious, it was decolorized with a decolorizing solution (glacial acetic acid: alcohol = 1:3).

The staining results are shown in Figure 5. When there is no stress, the staining degrees of the three transient expression lines are basically the same. After 4 hours of stress, the coloring of the three transgenic lines all deepened. However, in DAB staining, the OE strain was lighter, and the RNAi strain was darker. It indicated that the content of H _{2 O 2 in the OE strain was the least after the stress, while the content of H 2 O 2 in the} RNAi strain was more. Similarly, in the NBT staining, the OE strain is lighter, and the RNAi strain is darker, indicating that the overexpression of ThPHD3 can effectively remove the O ₂ ^- content in the cell, while the inhibition of expression is the opposite, leading to an increase in the accumulation of intracellular O ₂ ^- . Further Evans blue staining results also showed that the overexpression of ThPHD3 gene can significantly reduce the damage of cell membrane after NaCl stress. The above results indicated that the overexpression of ThPHD3 gene after salt stress can improve the ROS scavenging ability of plants, thereby reducing the damage of stress to cells and achieving the purpose of resisting salt stress.

Example 9: Analysis of Physiological Indexes of Transiently Transformed ThPHD3 Gene Tamarix brixensis Under NaCl Stress

According to the result of embodiment 7, to the blank (CON) of the tamarisk plant of transiently transformed overexpression vector pROKII-ThPHD3 (OE), the blank (CON) of transiently transformed tamarisk plant of suppressed expression vector pFGC5941-ThPHD3 (RNAi) and transiently transformed empty pROKII vector The physiological indicators of the control Tamarix briatus plants were measured after transient infection and co-cultivation under 150mM NaCl stress for 24h. The contents of H ₂ O ₂ , malondialdehyde (MDA), SOD, POD and proline in the three transgenic Tamarix biribilis were analyzed and compared.

The measurement of H ₂ O ₂ , MDA, SOD, POD and proline content in this example adopts the H ₂ O ₂ content, MDA content, SOD content, POD content and proline content produced by Nanjing Jiancheng Bioengineering Research Institute. Assay kits;

H ₂ O ₂ is an oxidative metabolite in cells. The worse the ability of plants to resist adversity stress, the more serious the damage of cells and the higher the H ₂ O ₂ content. Figure 6 shows that under 150mM NaCl stress, the H ₂ O ₂ content of the transgenic lines increased after 24 hours of stress, but the H ₂ O ₂ content of the ThPHD3 overexpression line was 68.59% of that of Con, and the H ₂ O 2 content of the RNAi plants The _O2 content is 1.2 times that of Con.

MDA is one of the most important products of membrane lipid peroxidation. Under stress conditions, the cells of plants undergo membrane lipid peroxidation and produce malondialdehyde (MDA). MDA is usually used as an indicator of lipid peroxidation, indicating the degree of lipid peroxidation in cell membranes and the strength of plant responses to stress conditions. Figure 7 shows that under salt stress, the change trend of MDA content in three transgenic Tamarix species is similar to that of H ₂ O ₂ content. Compared with the MDA content at 0h, the MDA content of the three transgenic Tamarix bristles increased to varying degrees after stress, and the MDA content of RNAi was the highest, followed by Con, and the lowest in OE. It can be seen that the degree of cell damage and the number of death in the RNAi strain are high, and the degree of cell damage and the number of death in the OE strain are low.

SOD can resist the damage of active oxygen or other peroxide free radicals to the cell membrane system by catalyzing the disproportionation reaction of superoxide anion free radicals, and effectively enhance the stress resistance of plants. POD can catalyze the oxidation of other substrates by H ₂ O ₂ , and is an important protective enzyme for removing oxygen free radical damage in plants, and is closely related to the stress resistance of plants. Figure 8 and Figure 9 show that under salt stress, the contents of SOD and POD in the three transgenic tamarisks all showed an increasing trend, and the contents in OE plants were the highest, followed by Con, and the lowest in RNAi. It can be seen that the overexpression of ThPHD3 gene can effectively increase the content of SOD and POD in cells, and then remove superoxide anion and oxygen free radicals in cells, so as to achieve the purpose of salt tolerance.

Under normal conditions, the free proline content of plants is very low, but when encountering drought, low temperature, saline-alkali and other adversities, free proline will accumulate in large quantities, and the accumulation index is related to the stress resistance of plants. Therefore, proline can be used as a biochemical indicator of plant stress resistance. Using sulfosalicylic acid to extract free proline in plants not only greatly reduces the interference of other amino acids, is quick and easy, and is not limited by the state of the sample (dry or fresh sample). Under acidic conditions, proline reacts with ninhydrin to form a red substance, which has a maximum absorption peak at a wavelength of 520nm after extraction with toluene. The level of proline concentration is directly proportional to its absorbance value within a certain range. Figure 10 shows that consistent with the changes in SOD and POD content, under salt stress, the proline content in the three transgenic tamarisks all showed an increasing trend, and the content in OE plants was the highest, followed by Con, and the lowest in RNAi. Further illustration, overexpression of ThPHD3 gene can enhance the stress resistance of plants.

Example 10: Obtaining and Salt Tolerance Identification of ThPHD3 Transgenic Arabidopsis

The ThPHD3 gene was transformed into the wild-type Arabidopsis Columbia ecotype by Agrobacterium-mediated flower dipping method, and the transgenic homozygous line of the T ₃ generation was obtained after screening. In order to analyze the effect of the ThPHD3 gene on the salt tolerance of Arabidopsis thaliana, the T3 homozygous lines ( _OE13 , OE21) and wild-type seeds of the ThPHD3 gene-transferred Arabidopsis thaliana were sterilized, and then sowed in 1/2MS and containing 100mM NaCl 1/2MS culture medium, cultured vertically in an artificial climate chamber for 10-15 days, and counted the germination rate.

The results shown in Figure 11 and Figure 12 show that under normal growth conditions, there is no difference between the germination rate and phenotype of transgenic Arabidopsis and wild-type Arabidopsis, while under 100mM NaCl stress conditions, the average germination rate of transgenic Arabidopsis lines It is 5.43 times the germination rate of the wild-type strain, indicating that the ThPHD3 gene can enhance the salt tolerance of Arabidopsis seeds.

At the same time, in order to determine the effect of salt stress on the growth of transgenic and wild-type Arabidopsis thaliana, Arabidopsis seedlings grown on normal medium for 3 days were transplanted to 1/2MS and 1/2MS medium containing 100mM NaCl respectively. , put them in an artificial climate chamber and cultivate them vertically for 10-15 days, then count the root length and fresh weight. The results of Fig. 13, Fig. 14 and Fig. 15 show that the average root length of the transgenic lines after salt stress is 1.72 times that of the wild type, and the fresh weight is 1.31 times that of the wild type.

At the same time, salt stress was carried out on the three-week-old transgenic and wild-type Arabidopsis seedlings grown normally. The results in Figure 16 show that under non-stress conditions (CK), the growth status of two different transgenic lines, OE13 and OE21, and WT There was no significant difference; after 7 days of 200mM NaCl stress, the growth status of the two transgenic lines OE13 and OE21 as well as WT changed. The growth status of OE13 and OE21 overexpressed lines of ThPHD3 gene was significantly better than that of WT, indicating that the stress resistance of OE13 and OE21 plants was higher than that of WT.

In summary, through the analysis of transgenic Tamarix and Arabidopsis, the salt-tolerance function of ThPHD3 gene in Tamarix biribilis was comprehensively analyzed from two aspects of homologous and heterologous overexpression of ThPHD3 gene. The salt-tolerant ability of transgenic plants, therefore, the gene has a very important application prospect in the preparation of transgenic plants, especially transgenic forest trees.

For the molecular biology experimental methods not specifically described in the above examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition, J. Sambrook), or follow the kit product instructions to operate.

SEQUENCE LISTING

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Claims (9)

1. Tamarix bristles ThPHD3 gene, characterized in that the cDNA sequence of the Tamarix bristles ThPHD3 gene is shown in SEQ ID No: 1. 2. A plant overexpression vector containing the Tamarix wirsalis ThPHD3 gene according to claim 1. 3. A recombinant genetic engineering bacterium containing the plant overexpression vector according to claim 2. 4. The recombinant genetically engineered bacterium according to claim 3, wherein the plant overexpression vector containing Tamarix bristle ThPHD3 gene is introduced into a host bacterium to construct, and the host bacterium is Escherichia coli or Agrobacterium. 5. The application of the Tamarix wirsalis ThPHD3 gene as claimed in claim 1 in improving plant salt tolerance or breeding salt-tolerant transgenic plants. 6. application as claimed in claim 5, is characterized in that, described application comprises constructing the plant overexpression vector that contains Tamarix bristata ThPHD3 gene, the plant overexpression vector constructed is transformed in the woody plant, cultivates and obtains salt tolerance Transgenic woody plants. 7. The application according to claim 6, wherein the woody plant is poplar or birch. 8. application as claimed in claim 5, is characterized in that, described application comprises constructing the plant overexpression vector that contains Tamarix bristata ThPHD3 gene, the plant overexpression vector constructed is transformed in the herbaceous plant, cultivates and obtains the salt tolerance transgene herb. 9. The application according to claim 8, wherein the herbaceous plant is Arabidopsis thaliana or tobacco.

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