CN107164388B - Wheat salt-tolerant gene TaPEX1 and application thereof - Google Patents

Abstract

The invention discloses a wheat salt-tolerant gene TaPEX1, a plant expression vector pSTART-TaPEX1 containing the gene and application of the gene in salt-tolerant plant cultivation. Experiments prove that the salt tolerance of the obtained transgenic plant is obviously improved by transferring the gene into arabidopsis thaliana or wheat by an agrobacterium tumefaciens mediated method, and the gene has wide application prospect.

Description

Wheat salt-tolerant gene TaPEX1 and application thereof

Technical Field

The invention belongs to the technical field of biological gene engineering, and particularly relates to a wheat salt-tolerant gene (gene TaPEX1) and application thereof.

Background

Salt tolerance seriously affects crop yield, and particularly, with the development of industry, land salinization becomes more and more serious, so that salt stress becomes a social problem concerned globally. China has a large population, and the soil salinization disaster is more serious, so the method becomes an important factor for restricting the economic and social development of China. Therefore, in addition to relieving soil salinization, the cultivation of new varieties of salt-tolerant crops is a very urgent task at present.

The new characters are transferred into the high-biomass plants by utilizing the transgenic technology improved plants, so that the high-efficiency new transgenic plant variety is developed and used for planting in saline-alkali soil, and the technology has wide application prospect.

At present, great progress is made in the research of plant salt tolerance by using genetic engineering technology, a large number of related genes are cloned, and the genes are transferred into plants for the research of salt tolerance mechanism. Some experiments show that the salt tolerance of transgenic plants can be improved by transferring the genes related to salt tolerance in the plants and other organisms into the plants and the transcription and translation products of the genes.

The search shows that some genes capable of obviously improving the salt tolerance of the plants are found, but the reports about the effects of the BAS genes in the salt tolerance process of the plants are less.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a salt-tolerant gene, namely a wheat gene TaPEX1 and application thereof.

The technical scheme of the invention is as follows: the gene TaPEX1 is separated from wheat and then transformed into arabidopsis thaliana to carry out transgene function verification (arabidopsis thaliana transformation screening and salt stress surface analysis) so as to realize the research on the function and salt tolerance mechanism of the BAS gene.

The salt-tolerant gene of wheat is characterized in that: the gene is named as a wheat salt-tolerant gene TaPEX1, and the nucleotide sequence of cDNA of the gene is shown in SEQ ID No. 1.

The invention also provides a plant expression vector pSTART-TaPEX1 containing the wheat salt-tolerant gene TaPEX 1.

The invention relates to application of a wheat salt-tolerant gene TaPEX1 and a plant expression vector pSTART-TaPEX1 containing the wheat salt-tolerant gene TaPEX1 in cultivating salt-tolerant plants, wherein the plants are preferably common wheat or arabidopsis thaliana.

The wheat salt-tolerant gene TaPEX1 and the plant expression vector pSTART-TaPEX1 containing the wheat salt-tolerant gene TaPEX1 can be widely used for cultivating salt-tolerant crop varieties.

The salt-tolerant wheat gene TaPEX1 is introduced into plant cells, and the plants can correspondingly obtain the salt-tolerant capacity. In order to facilitate the selection of transgenic plants or cell lines, a plant expression vector (pSTATR-TaPEX1) containing the gene TaPEX1 can be processed, for example, a selection marker (GUS, etc.) or a marker for antibiotics having resistance (hygromycin, kanamycin, gentamicin, etc.) can be added.

Practically, any vector that can introduce a foreign gene into a plant for expression can be used, and a preferred vector of the present invention is pSTART.

The invention has the beneficial effects that: by utilizing the existing plant genetic engineering technology, the salt-tolerant gene TaPEX1 of wheat is obtained by first cloning, and the gene is transferred into arabidopsis thaliana by an agrobacterium tumefaciens mediated method, and comparative analysis proves that the salt-tolerant capability of a transgenic plant is obviously improved, thereby proving that the gene of the invention has wide application prospect.

Drawings

FIG. 1: amplification result of full-Length cDNA sequence of TaPEX1 Gene

Wherein: m is lambda DNA/(BamH I + Sac I) Marker.

FIG. 2: NaCl and H₂O₂Real-time quantitative PCR analysis of TaPEX1 in wheat under stress

Wherein: panel A is 200mM NaCl treatment; b shows 10mM H₂O₂And (6) processing. SR3-L represents No. 3 leaf of wheat variety Shanshan; SR3-R represents No. 3 root of wheat variety Shanshan-Rong.

FIG. 3: transgenic genome PCR and real-time quantitative PCR detection of Arabidopsis TaPEX1 transgenic plant

Wherein: panel A is a genome PCR electropherogram, which verifies that TaPEX1 is integrated into the Arabidopsis genome; panel B shows the real-time quantitative PCR results, which verify that TaPEX1 is normally expressed in the transgenic lines. OE1 and OE2 are two independent transgenic lines; col-0 is wild type Arabidopsis thaliana; AtActin is an internal reference of an Arabidopsis thaliana Actin gene; genomic PCR was performed to verify that TaPEX1 was integrated into the arabidopsis genome by Genomic PCR; RT-PCR was performed to verify that TaPEX1 was normally expressed in the transgenic lines by RT-PCR. The genomic PCR results showed that a TaPEX1 band could be amplified in the transgenic lines, but not in the wild type, indicating that TaPEX1 was integrated into the Arabidopsis genome. The real-time quantitative PCR result shows that the transcript of TaPEX1 can be detected in the transgenic line, and the expression of TaPEX1 in Arabidopsis is indicated.

FIG. 4: growth status of Arabidopsis seedlings in NaCl-containing Petri dishes

Wherein: a is the plant growth state under the control condition; b, the growth state of the plants under the condition of 100mM NaCl treatment; the graph C is the fresh weight of the overground part; the D picture is the root length. Col-0 is an untransformed Arabidopsis plant; OE1 and OE2 are two independent transgenic lines. The data show that the growth states of all strains are similar under the control condition, while the growth state of the transgenic arabidopsis thaliana transformed with TaPEX1 is better in the culture medium containing NaCl with different concentrations, which shows that TaPEX1 obviously increases the salt tolerance of the plants.

FIG. 5: growth condition of arabidopsis seedlings under NaCl solution irrigation condition

Wherein: a picture is the growth state of seedlings before treatment; b, the growth state of the irrigation water under the contrast condition is shown; the C picture shows the growth state after the NaCl solution is poured. Col-0 is an untransformed Arabidopsis plant; OE1 and OE2 are two independent transgenic lines. The data show that the growth states of all strains are similar under the control condition of irrigation water, while the growth state of transgenic arabidopsis thaliana transformed with TaPEX1 is better in culture media containing NaCl with different concentrations, which shows that TaPEX1 obviously increases the salt tolerance of the plants.

Detailed Description

Example 1 cloning and expression analysis of TaPEX1

1.1 extraction of wheat Total RNA

1. Placing the tissue material into a mortar precooled by liquid nitrogen, and fully grinding the tissue material into powder in the liquid nitrogen;

2. After the liquid nitrogen is volatilized to be dry, immediately transferring the liquid nitrogen into a 2mL centrifuge tube, adding about 1mL of TRIzol extracting solution of Invitrogen company into every 100mg of material, repeatedly sucking and blowing the melted material by using a sample adding gun, violently oscillating and uniformly mixing the sample to fully crack the sample, and standing the sample at room temperature for 5 minutes;

3. Adding 0.2mL of chloroform, violently shaking and uniformly mixing for 15 seconds, and standing for 10 minutes at room temperature;

Centrifuging at 12000rpm for 15min at 4.4 deg.C;

5. carefully sucking out the upper aqueous phase by using a pipette, adding the upper aqueous phase into a new centrifugal tube of 1.5mL, adding 500 mu L of isopropanol (1:1 volume), fully mixing the mixture, and precipitating the mixture at the temperature of-20 ℃ for 30min or overnight;

Centrifuging at 6.4 deg.C and 12000rpm for 10min, and carefully discarding the supernatant;

Washing the RNA precipitate with 1mL of 75% ethanol; centrifuging at 4 deg.C and 8000rpm for 10min, and collecting precipitate;

8. Washing the RNA precipitate once by using 75% ethanol repeatedly;

9. Removing supernatant, air drying RNA precipitate on sterile operating platform for about 10-15min, making RNA be transparent, adding RNase-free water with appropriate volume (30-50 μ L), and dissolving thoroughly (can be stored at-80 deg.C for a long time);

10. an ultraviolet spectrophotometer and 1% Agrose gel electrophoresis are used for detecting the concentration and the quality of RNA.

Note: a) the RNA yield was measured by UV spectrophotometer, and the absorbance at 260nm was 1 OD-40. mu.g/mL. The purity of the RNA, OD of the pure RNA, was determined from the absorbance at 260nm and 280nm₂₆₀/OD₂₈₀The ratio should be close to 2.0 (preferably, the ratio is between 1.9 and 2.1).

b) the quality and size of the RNA was examined by electrophoresis on a 1% Agrose gel. mu.L of RNA was aspirated, 3. mu.L of RNase-free water was added, and 1. mu.L of loading buffer was added and denatured at 65 ℃ for 5 minutes. After electrophoresis, the cells were stained with EB, and 3. mu.L of 1kb DNA Marker was used as a control.

1.2 cDNA reverse transcription

Reverse transcriptase: M-MLV Reverse Transcriptase (Invitrogen).

1.12 μ L reaction System

Denaturation at 2.65 ℃ for 5min, rapid insertion into ice, then sequential addition of:

5×First-Strand Buffer 4μL

0.1M DTT 2μL

RNaseOUT(Invitrogen)1μL

3. Mixing the mixture gently, and reacting at 37 ℃ for 2 min;

4. Adding 1 mu L M-MLV RT, mixing uniformly, and reacting for 50min at 37 ℃;

Inactivation of M-MLV RT by incubation at 5.70 ℃ for 15 min;

6. mu.L of RNase H (Invitrogen) was added thereto and reacted at 37 ℃ for 20 min;

7. Diluted with ultrapure water to the appropriate concentration. As a template for PCR.

1.3 cloning and sequencing of the open reading frame

1. The primer sequence is as follows: designing upstream and downstream primers of the gene according to the sequencing result, and amplifying the open reading frame of the gene.

PCR reaction (50. mu.L):

The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 45sec, renaturation at 55 deg.C for 45sec, extension at 72 deg.C for 1.5min, and circulation for 35 times; extension at 72 ℃ for 7 min.

4. The amplified fragment was recovered, ligated with pMD-18T vector and transformed into E.coli DH10B, and sequencing was performed by Shanghai Invirtron corporation. The results are shown in FIG. 1.

1.4 Gene expression analysis (real-time quantitative PCR)

Extraction of RNA

The seeds of the No. 3 wheat from the mountain thawing germinated normally, and the salt stress (200mM NaCl or 10mM H) begins to be applied when the Hangload culture solution is cultured until the height of the plant is about 10cm (about 3 weeks)₂O₂) And treating for 0, 1, 6, 12, 24 and 48 hours, and taking tender leaves and roots to extract RNA.

2. Reverse Transcription (RT) to generate cDNA

reverse transcription generates cDNA as described above.

PCR reaction and data processing method

(1) PCR was performed using cDNA as a template.

(2) and (3) PCR system:

(3) PCR procedure:

5min at 95 ℃; 10S at 95 ℃, 10S at 60 ℃, 20S at 72 ℃ and 40 cycles; melting curves are made at intervals of 0.5 ℃ at 65-95 ℃; storing at 10 deg.C.

(4) And judging the product unicity and the dissolution temperature by using the melting curve, and calculating the relative content of the target gene in different samples by using a delta Ct algorithm by using the given Ct value. The results are shown in FIG. 2.

example 2 construction of plant expression vectors

Utilizing a plant expression vector pSTART, and selecting XbaI and BamHI to respectively carry out double enzyme digestion on the pSTART and a pMD18-T vector containing a target gene; recovering large carrier fragment and small target gene fragment and using T₄Transforming escherichia coli DH10B competent cells after DNA ligase ligation, and identifying recombinants to obtain the recombinant plasmid with the target geneA plant expression vector.

1. Double digestion, exemplified by the empty vector pSTART and pMD18-T

Extracting pSTART empty vector and pMD18-T plasmid by alkaline lysis method, taking 10 ug each enzyme, the enzyme digestion system is as follows:

the enzyme is cut in a water bath kettle at the constant temperature of 30 ℃ for more than 2 hours. After double digestion, the product of the digestion was subjected to 0.8% agarose gel electrophoresis using 1 XTAE as the electrophoresis buffer. The 14kb large vector fragment of pSTART and the 789bp band of the gene of interest of pMD18-T were excised with a clean blade under an ultraviolet transilluminator and recovered.

Dephosphorizing the large fragment of the recovered vector by digestion of the pSTART plasmid.

3. The digested and dephosphorylated pSTART vector fragment (about 14kb) and the pMD18-T double-digested recovered fragment (789bp) were ligated in a molar ratio of 1:4 at 16 ℃ overnight.

4. The ligation products were transformed into E.coli DH10B competent cells by heat shock method, and the transformants were cultured on LB solid plate containing 50. mu.g/mL Kan at 37 ℃ for about 16 hours.

5. Identification of recombinants

(1) PCR validation of plasmids

Single colonies are selected and respectively inoculated in 5mL LB liquid culture medium containing Kan and subjected to shaking culture at 37 ℃ overnight, plasmids are extracted by an alkaline denaturation method, and PCR amplification is carried out by using gene specific primers.

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 3 min; 30sec at 94 ℃, 30sec at 55 ℃, 1min at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min. The PCR product was identified by electrophoresis on a 1.0% agarose gel.

(2) Plasmid restriction enzyme identification

Extracting the plasmid and carrying out XbaI and BamHI double enzyme digestion, wherein the enzyme digestion system is the same as the above. And (4) carrying out 0.8% agarose gel electrophoresis, detecting whether fragments with expected molecular weight are contained, and verifying the correct construction of the vector.

Example 3 preparation and transformation of Agrobacterium competence

3.1 preparation of Agrobacterium AGL1/EHA105 competence

1. A single colony of Agrobacterium tumefaciens was picked from a YEP plate (containing 50. mu.g/mL rifampicin), inoculated into a YEP liquid medium (containing 50. mu.g/mL rifampicin), cultured at 200rpm/min at 28 ℃ overnight.

2. 2mL of overnight culture was inoculated into 50mL of YEP broth containing the same antibiotic and cultured under the same conditions to OD₆₀₀Up to 0.5.

3. The bacterial liquid is subjected to ice bath for 30min, centrifuged at 4 ℃ and 5000rpm for 10min, and the thalli are collected.

4. The cells were resuspended in 10mL of 0.15mol/L NaCl in an ice bath, and the cells were collected by centrifugation.

5. Resuspended in 1mL of 20mmol/L ice-cooled CaCl₂In the solution, the bacterial suspension was dispensed into 1.5mL Eppendorf tubes at 200. mu.L/tube, and frozen in liquid nitrogen for 1min and stored at-70 ℃ for further use.

3.2 Freeze-thawing method for transformation of Agrobacterium tumefaciens AGL1/EHA105

1. The agrobacterium competent cells were thawed at room temperature, 1 μ g of expression vector plasmid DNA was added, mixed well and ice-cooled for 30 min.

2. Quickly freezing in liquid nitrogen for 1min, and rapidly transferring to 37 deg.C and maintaining for 3 min.

3. Add 800. mu.L of YEP without antibiotics, shake-culture at 28 ℃ for 3 hours.

Cells were collected by centrifugation at 4.7000rpm for 30s, plated on YEP plates containing rifampicin at 50. mu.g/mL and Kan at 50. mu.g/mL, and cultured in the reverse dark at 28 ℃ for 2-3 days.

3.3 PCR identification of the cells

A single colony of 2.4 was picked and transferred to a PCR system as described above (without DNA template) and PCR amplified with gene specific primers. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 3 min; 30sec at 94 ℃, 30sec at 55 ℃, 1min at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min. The PCR product was identified by electrophoresis on a 1.0% agarose gel.

Example 4 transgenic functional validation-Arabidopsis transformation screening

4.1 Arabidopsis thaliana planting

Soaking the seeds in 70% ethanol for 5min in EP tube, washing for 10-15min, washing with sterile water for 4 times, and vernalizing at 4 deg.C for 72 h. 0.5% agarose (cooled to 40 ℃) was added to the sterilized seeds, spread on 1/2MS solid medium and blown dry on a clean bench. Culturing in artificial climate room for about one week, and transplanting. Putting the artificial soil into a culture pot with a proper size, drying at 70 ℃ for more than 2 hours, then putting the culture pot into nutrient solution to ensure that the culture pot fully absorbs water, transplanting seedlings growing on a 1/2MS solid culture medium for 7-10 days into the artificial soil saturated with the nutrient solution, covering with a preservative film, and transferring into an artificial climate chamber for culture. The preservative film is removed after 1 to 2 days. Pouring water (in a tray under the culture pot) every few days.

4.2 Arabidopsis transformation

1. When an arabidopsis thaliana (colombia wild-type) inflorescence was formed, the tip of the inflorescence was cut off to induce the formation of a sequenced inflorescence, and the material was drenched into a nutrient solution before transformation.

2. The day before transformation, 2mL of activated Agrobacterium AGL1 was added to 200mL of YEP medium containing the corresponding antibiotic and cultured overnight to OD₆₀₀＝1.0-1.2。

3. The cells were collected by centrifugation and resuspended in a staining solution (5% sucrose, 0.04% Silwet L-77) to OD₆₀₀＝0.8。

4. Immersing the inflorescence into the dip dyeing solution for 30 seconds, and swinging the inflorescence back and forth during the immersion to form a layer of film on the inflorescence.

5. Covering inflorescences with preservative film, culturing in dark for one day, removing the preservative film, and culturing in a culture room at 19-22 deg.C.

6. the dip dyeing is carried out once again by the same method every 5 to 7 days.

7. The seeds were harvested approximately one month later.

4.3 wheat and Arabidopsis transformation Positive line screening

1. Harvested T₀after seed generation sterilization, the seeds were plated on MS selection medium containing 50. mu.g/mL Kan (same procedure as above).

Vernalization is carried out for 48h at the temperature of 2.4 ℃, and the seeds are moved to a climatic chamber for 7-10 days of growth. And transplanting the resistant plantlets into soil to continue growing.

3. After most buds of the plants have fruit pods, tying up the individual plants by using small ropes so as to collect seeds T from the individual plants₁And (5) seed generation.

And 4, carrying out PCR amplification by taking the genome DNA of the transformed strain as a template and using a gene specific primer to carry out genome PCR so as to identify the positive clone. The results are shown in FIG. 3A.

5.T₁Seed treatment at T₂And selecting a single-insertion independent strain with the resistance ratio of 3:1 from the generation plants.

6. The seeds of T2 generation plants were plated on MS selection medium containing 50. mu.g/mL Kan as described above, all Kan resistant lines were selected, transplanted into a culture pot, and T3 inbred seeds were harvested for subsequent analysis.

7. 1 leaf was removed from T3 strain with pure line, RNA was extracted according to the above method, real-time quantitative PCR analysis was performed, and expression of transgene was identified. The results are shown in FIG. 3B.

Example 5 functional verification of transgenes-analysis of salt tolerant phenotype

5.1 plant salt tolerant phenotype analysis in Petri dishes

1. In a clean bench, seeds of wild type and over-expression strain are subpackaged into 1.5mL of EP tubes, firstly sterilized by 70% alcohol (added with 1 ‰ triton) for 3min by oscillation, secondly sterilized by 70% alcohol for 2min by oscillation, and finally sterilized by absolute ethyl alcohol for 1min by oscillation, and then spread on sterile filter paper.

2. And (3) after the surfaces of the seeds are dried, putting the arabidopsis thaliana seeds on a 1/2MS culture dish, synchronously treating for 48 hours at 4 ℃, taking out the arabidopsis thaliana seeds, moving to 23 ℃, and vertically culturing for 4-5 days under normal illumination conditions.

3. When the root length of the seedling grows to 0.7-1.3cm, selecting wild type and over-expression strain Arabidopsis seedlings with consistent root length and growth state, and respectively transferring to the Arabidopsis seedlings added with NaCl or H with different concentrations₂O₂And blank 1/2MS culture dishes, after 10-12d of culture observation phenotype and photograph.

4. Root length and aerial parts were weighed out using the RootoAnalysis Ver2.5 software. The results are shown in FIGS. 4D and 4C. 5.2 plant salt tolerant phenotype analysis in soil

1. and (3) dropping wild type and over-expressed arabidopsis thaliana seeds into a culture dish containing a 1/2MS culture medium, transferring arabidopsis thaliana to soil for growth when 4 leaves are opened, culturing in the soil for four to five weeks, and beginning to pour saline water when the leaves grow to 7-8.

2. 50mM NaCl saline was poured first for three days, 100mM NaCl for three days, 150mM NaCl for three days, and 200mM NaCl for one week, with the same amount of water poured as a control. During the salt treatment, the survival condition of the plants needs to be observed and the pictures are taken in time. In order to avoid the influence of soil loading of different flowerpots on salt treatment in the experiment, transgenic plants and wild plants can be planted in the same culture pot simultaneously in the experiment. The results are shown in FIG. 5C.

Sequence listing

<110> Shandong university

<120> wheat salt-tolerant gene TaPEX1 and application thereof

<141>2017-06-12

<160> 1

<210> 1

<211>789

<212>cDNA

<213> wheat

<221> salt-tolerant gene TaPEX1 of wheat

<222>（1）…（789）

<400>1

atggcgtgcg ccttctccgc ctccaccgtg tccacggcgg ccgcgctcgt cgcgtccccg 60

aagccagccg gggcgccgca gtgcctgtcg tttccccgcg ccatcgcagg cgccgccgcc 120

aggccttccc gcctcgccgc cgccagctcg aggacggcca gggcccgcag cttcgtcgcc 180

cgcgcctcag cggagtacga cctgccgctg gtggggaaca aagcaccgga cttcgccgcg 240

gaggccgtgt tcgaccagga gttcatcaac gtcaagctat ctgattacat tgggaagaag 300

tatgtgattc ttttcttcta ccctctggac ttcaccttcg tctgcccaac tgagattacg 360

gctttcagcg acagacatga ggagttcgag aagataaaca ctgaaattct tggtgtttca 420

gttgatagtg tgttttccca tcttgcatgg gtgcagacag agaggaaatc tggtggactt 480

ggtgatctga aatatccgct ggtttctgac gtcaccaaat caatctcaaa gtcttttggt 540

gtattgatcc ctgatcaggg aattgctctg agaggattat tcatcattga caaggagggt 600

gtgattcagc attccactat taacaacctt ggtattggcc gtagtgtgga tgagaccttg 660

agaacccttc aggctctgca atacgtccaa gaaaacccag acgaggtctg cccggcggga 720

tggaaacctg gggaaaagtc gatgaagcct gaccccaagg gcagcaagga gtacttcgct 780

gctatctag 789

Claims (1)

1. the application of a wheat salt-tolerant gene TaPEX1 in cultivating salt-tolerant plants, wherein the nucleotide sequence of the wheat salt-tolerant gene TaPEX1 cDNA is shown as SEQ ID No.1, and the plants are Arabidopsis thaliana.