CN105505956B - A kind of application of alfalfa Single-chip microcomputer dioxygenase gene - Google Patents

Abstract

The invention discloses an alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD, the nucleotide sequence of which is shown in SEQ ID NO: 1 in the sequence listing. The invention also discloses a gene for cloning the The nucleotide sequences of the primer pair for the alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD are respectively shown in SEQ ID NO:4 and SEQ ID NO:5 in the sequence listing. The invention also discloses a preparation method of the alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD and its application in improving the vitamin E content and stress resistance in Arabidopsis thaliana. The invention lays a foundation for further research on the function of the alfalfa MsHPPD gene, and the application of the MsHPPD gene to improve the quality of pasture, especially the molecular improvement of the vitamin E content of the pasture.

Description

Application of a Medicago p-Hydroxyphenylpyruvate Dioxygenase Gene

technical field

The invention relates to the field of molecular biology, in particular to an alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD and its preparation method and application.

Background technique

Vitamin E is a fat-soluble vitamin. Natural products are divided into eight types according to the degree of saturation of the hydrophobic tail and the number and position of methyl groups on the aromatic ring, namely a, β, γ, σ-tocopherol and a , β, γ, σ-tocotrienols, among which a-tocopherol has the highest biological activity.

Vitamin E is an essential micronutrient for the human body, but the human body cannot synthesize it by itself and can only be ingested through diet. Studies have shown that vitamin E can improve the immunity of animals. Relevant studies have shown that the meat quality of livestock supplemented with vitamin E is better than that of normal livestock. Moreover, it can improve the quality of meat and milk and prolong the shelf life. Vitamin E has an antioxidant effect. The main function of vitamin E is to remove active oxygen and protect polyunsaturated fatty acids from oxidation. important for flora and fauna. In addition, vitamin E also plays a role in the regulation of plant stress resistance.

Alfalfa is an important leguminous forage, and it is also the most planted forage variety in the country and even in the world. Because of its wide adaptability, high yield, and good quality, it is known as the "king of forage". With the development of my country's animal husbandry, the requirements for the quality of pastures are also increasing. Vitamin E can improve the freshness of animal meat, prolong the shelf life, improve the fertility of animals, and also has anti-oxidation and anti-stress effects. It has a good value in improving the quality of pasture. However, alfalfa is a cross-pollination tetraploid leguminous forage, its genetic transformation is difficult, the growth cycle is long, and the research on gene function is relatively lagging behind. So far, the research on the genes related to vitamin E synthesis in alfalfa is still blank.

Contents of the invention

The object of the present invention is to provide a kind of alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD, the present invention is to further study the function of the alfalfa MsHPPD gene, and the MsHPPD gene improves forage quality, especially the molecular improvement of forage vitamin E content Application lays the groundwork.

Another object of the present invention is to provide a method for preparing alfalfa p-hydroxyphenylpyruvate dioxygenase gene.

Another object of the present invention is to provide an application of the above-mentioned alfalfa p-hydroxyphenylpyruvate dioxygenase gene in improving the vitamin E content and stress resistance regulation in Arabidopsis.

The first technical solution adopted by the present invention is a Medicago sativa p-hydroxyphenylpyruvate dioxygenase gene, the nucleotide sequence of which is shown in SEQ ID NO: 1 in the sequence listing.

The second technical scheme adopted in the present invention is, a kind of preparation method of alfalfa p-hydroxyphenylpyruvate dioxygenase gene, concretely implement according to the following steps:

Step 1, using the Trizol reagent method to extract the total RNA of alfalfa;

Step 2, carry out RACE clone cDNA full length to the alfalfa total RNA extracted in step 1, and carry out gel recovery to PCR; Described RACE primer comprises GSP1 and GSP2, wherein, the sequence of GSP1 is as SEQ ID NO:4 in the sequence listing As shown, specifically: 5'-ATGGCACCGTTGTTTACGCTGGTGGTG-3'; the sequence of GSP2 is shown in SEQ ID NO: 5 in the sequence listing, specifically: 5'-GACGCCGAACTGGCTTTCACCACC-3'.

Step 3, constructing a recombinant plasmid of the PCR product, and transforming Escherichia coli DH5α competent cells to obtain a bacterial solution;

Step 4, carry out PCR detection to the obtained bacterial liquid, select positive bacterial liquid, send to Invitrogen Company to carry out sequencing, and sequence result is spliced, obtains alfalfa p-hydroxyphenylpyruvate dioxygenase gene cDNA full-length sequence, alfalfa to The nucleotide sequence of the hydroxyphenylpyruvate dioxygenase gene is shown in SEQ ID NO: 1 in the sequence listing.

The third technical scheme adopted by the present invention is the application of the above-mentioned alfalfa p-hydroxyphenylpyruvate dioxygenase gene in increasing the vitamin E content and stress resistance regulation of Arabidopsis thaliana.

The beneficial effects of the present invention are: the present invention is based on the research on the biological mechanism of quality formation in alfalfa, on the one hand provides the alfalfa MsHPPD gene and the protein encoded by the gene, on the other hand also provides the alfalfa MsHPPD gene containing The expression vector and cell of the present invention, as well as the method for cultivating Arabidopsis with increased vitamin E content, also provide their applications.

The invention has very important significance for understanding the function of HPPD gene in the regulation of forage quality and stress resistance, and for the molecular improvement of forage quality. The enhanced expression of the alfalfa MsHPPD gene provided by the invention in Arabidopsis can significantly increase the vitamin E content of Arabidopsis. The important application prospect of the present invention lies in: the discovery of this gene provides genetic resources for improving the vitamin E in main forage plants (especially leguminous plants, such as alfalfa). Play an important role in the creation of new genetically modified germplasm.

The present invention will be further described below in conjunction with accompanying drawing.

Description of drawings

Fig. 1 is a structural diagram of the ppBI121 vector inserted with the MsHPPD gene used for the homologous transformation of the MsHPPD gene mediated by Agrobacterium alfalfa;

Fig. 2 is a graph showing the results of comparative analysis of vitamin E content in Arabidopsis thaliana lines (L1, L2, L3) transfected with MsHPPD gene and empty vector control line (CK).

Fig. 3 is the result figure that the content of pro-amino acid is measured in the embodiment of the present invention 4;

Fig. 4 is a graph showing the results of the determination of peroxidase (POD) activity in Example 4 of the present invention.

detailed description

Example 1 Preparation of alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD

The alfalfa MsHPPD gene provided by the invention is screened by the following method:

Through BLAST comparative analysis, primers were designed according to the conserved domain of the cloned p-hydroxyphenylpyruvate dioxygenase gene (Genbank accession number: XM_003617343) in alfalfa (RT-PCR primer: F: 5'-CCACTTTCTCCGCATCCACTTGTTTC-3 ', its nucleotide sequence is shown in SEQ ID NO:2 in the sequence listing; R: 5'-CCTCCAATGTCCTAAATATATCCTCAG-3', its nucleotide sequence is shown in SEQ ID NO:3 in the sequence listing). Amplified from alfalfa (Medicago sativa L.cv.Zhongmu No.1, Zhongmu No. 1, Beijing Zhonghu Dongfang Grass Technology Co., Ltd.), and recovered a fragment with an expected size of 600bp (recovered after running electrophoresis) Fragments), the recovered fragments were ligated into PMD18-T Vector (Takara), the sequencing results were analyzed by BLASTx in NCBI's non-redundant protein database, and a cDNA highly homologous to the published HPPD gene of related species was obtained sequence. The sequence was cloned by 5'RACE and 3'RACE using SMARTer ^TM RACEcDNA Amplification Kit (Clontech, USA), and finally the full-length sequence of the gene was obtained (the nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing) . Among them, the method of gene cloning is a routine experimental operation in the field of molecular biology, and the specific method is as follows:

1. Extraction of total RNA from alfalfa:

The extraction of total RNA from alfalfa adopts the Trizol reagent method, and the specific steps are as follows:

(1) Add about 0.3 g of alfalfa tissue material to about 3000 μl of Trizol reagent, add liquid nitrogen to quickly grind it, and place it in a sterile environment until it becomes homogenized.

(2) Use a pipette to pipette about 1 mL of the homogenate into a 1.5 mL Eppendorf centrifuge tube, vortex vigorously to mix, place at room temperature for 5 min, and centrifuge at 13,000 rpm for 10 min at 4°C.

(3) Take the supernatant, add 200 μL of chloroform, shake vigorously, place at room temperature for 2-3 minutes, and centrifuge at 13,000 rpm for 15 minutes at 4°C.

(4) The solution is divided into three layers from bottom to top: organic phase, protein, and colorless aqueous phase; take the upper layer and transfer it to a new Eppendorf centrifuge tube, add an equal volume of chloroform, and repeat step 3.

(5) Take the supernatant and add an equal volume of isopropanol, mix well, leave at room temperature for about 10 minutes, and centrifuge at 13,000 rpm for 10 minutes at 4°C to obtain a white RNA precipitate.

(6) Discard the supernatant, add 1000 μL of 75% ethanol (prepared with DEPC (diethyl pyrocarbonate, diethyl pyrocarbonate ) water), wash up the precipitate, centrifuge at 13000 rpm for 10 min at 4°C, pour off the ethanol, and leave the precipitate.

(7) Repeat step 6 and dry in a sterile environment. Add 20-50 μl sterile DEPC water to dissolve.

Perform RNA electrophoresis to detect the quality of RNA extraction, and measure the RNA concentration with the IMPLEN micro-nucleic acid and protein analyzer.

2. Full-length cDNA cloned by RACE

Synthesis of RACE-ready cDNA

(1) Preparation buffer: 2.0 μl 5×First-Strand Buffer (First-Strand Buffer), 1.0 μl DTT (20mM), 1.0 μl dNTP Mix (10mM);

(2) Prepare cDNA reaction solution for 5'RACE: 2.75μl RNA, 1.0μl 5'-CDS Primer A, prepare cDNA reaction solution for 3'RACE: 3.75μl RNA, 1.0μl 3'-CDS Primer A ;

(3) Mix the liquid prepared in step (2), centrifuge briefly, incubate at 72°C for 3 minutes, then cool at 42°C for 2 minutes, and collect the reaction liquid by brief centrifugation;

(4) Add 1 μl of SMARTer IIA oligo to the 5'RACE cDNA reaction solution, and briefly centrifuge to collect the liquid;

(5) Prepare 5'RACE and 3'RACE-Ready cDNA reaction solution (Master Mix): 4.0μl buffer obtained in step (1), 0.25μl RNase inhibitor (40U/μl), 1.0μl SMARTScribe reverse transcription Enzyme (100U), mix the above reagents;

(6) Add 5.25 μl of the reaction solution obtained in step (5) to the 3' RACE obtained in step (3) and the 5' RACE reaction solution obtained in step (4), mix gently, and centrifuge briefly to collect the liquid;

(7) Incubate the prepared reaction solution at 42°C for 90 minutes;

(8) Heat at 70°C for 10 minutes to complete the synthesis of Ready-cDNA.

Design of RACE primers

(1) Gene-specific primers (Gene-Specific Primers, GSPs) meet the following conditions: 23-28bp; GC content 50%-70%; Tm value preferably> 70 ° C; provided in the kit) no complementation occurred.

(2) Two GSP primers need to be designed, the reverse primer (GSP1) is used for 5’RACE, and the forward primer (GSP2) is used for 3’RACE.

According to the above principles, design GSP1 (5'-ATGGCACCGTTGTTTACGCTGGTGGTG-3', its nucleotide sequence is shown in SEQ ID NO:4 in the sequence listing) and GSP2 (5'-GACGCCGAACTGGCTTTCACCACC-3', its nucleotide sequence is as follows Shown in SEQ ID NO:5) two primers in the list.

Rapid synthesis of cDNA ends

(1) Prepare enough PCR mixture (Master Mix): add the following reagents to each 50 μl PCR reaction system:

34.5 μl PCR water, 5.0 μl 10×Advantage 2 PCR buffer, 1.0 μl dNTP Mix (10 mM), 1.0 μl 50×Advantage 2 polymerase Mix. Gently mix the liquid, taking care not to generate air bubbles, and collect the liquid by brief centrifugation;

(2) Prepare PCR reaction solution for 5'RACE: 2.5 μl 5'RACE-ready cDNA, 5.0 μl UPM (10×), 1.0 μl GSP1; 41.5 μl PCR mixture prepared in step (1);

Prepare the PCR reaction for 3’RACE: 2.5 μl 3’RACE–ready cDNA, 5.0 μl UPM (10×), 1.0 μl GSP2; 41.5 μl of the PCR mixture prepared in step (1);

(3) RACE amplification: The reaction system is: 94°C for 30 seconds, 72°C for 3 minutes, a total of 5 cycles: 94°C for 30 seconds, 70°C for 3 minutes, 72°C for 3 minutes, a total of 5 cycles; 94°C for 30 seconds , 68°C for 30 seconds, 72°C for 3 minutes, a total of 20 cycles.

Take the PCR product and carry out electrophoresis detection on 1.0% by weight agarose gel, recover the target band, connect the recovered product to the PMD18-T (Takara) carrier with T4 DNA ligase (Takara), and transform Escherichia coli DH5α competent cells .

3. Escherichia coli transformation of recombinant plasmid

(1) Thaw DH5α competent cells on ice, pipette 50 μl, add 5 μl of DNA ligation product, mix well, and then ice-bath for 30 minutes.

(2) Heat shock at 42°C for 60 sec, and immediately ice bath for 3-5 min.

(3) Add 700 μl LB liquid medium, and culture on a constant temperature shaker (150 r/min) at 37° C. for 60 minutes.

(4) Centrifuge at 5000rmp for 3min to collect the bacterial cells.

(5) LB plates (Amp) coated with X-gal (15-bromo-4-chloro-3-indole-β-D-galactoside, 2%) and IPTG (isopropylthio-β- D-galactoside, 50mg/ml), so that it is evenly coated on the surface of the plate, and dried at room temperature.

(6) Take 100 μl of the bacterial solution and evenly spread it on the LB plate containing antibiotic Amp (100 mg/L), and incubate it upside down in the incubator at 37°C overnight; place the plate at 4°C the next day to fully develop the color.

(7) Pick a single white colony and put it in 1ml LB liquid medium (final concentration of Amp 100mg/L), culture at 225r/min 37°C overnight.

Perform PCR detection on the bacterial liquid, select the positive bacterial liquid, and send it to Invitrogen Company for sequencing. Sequence splicing was carried out on the result, and the result showed that the gene had a nucleotide sequence (2064bp) as shown in SEQ ID NO:1 in the sequence listing.

Example 2 Agrobacterium-mediated MsHPPD gene transformation of Arabidopsis plants

1.1. Materials and reagents

1.1.1. Plant material

The tested alfalfa variety was Medicago sativa L.cv.Zhongmu No.1, Zhongmu No.1.

1.1.2. Agrobacterium strains and plasmid vectors

The Agrobacterium strain used is Agrobacterium tumefaciens: LBA4404 (Beijing Tianenze Gene Technology Co., Ltd.)

Agrobacterium culture medium:

Reagent Content (1L) MgSO ₄ ^. 7H ₂ O 1g/L Peptone 10g/L Yeast extract 1g/L sucrose 5g/L Agar (solid medium) 15g/L pH 7.0

121°C high pressure steam sterilization for 20min; carrier: PBI 121

1.2. Experimental method

1.2.1 Insert the MsHPPD gene into the pBI121 vector plasmid DNA. The specific steps are: design the upstream primer containing the XbaI restriction site at the 5' end and the downstream primer of the BamHI restriction site (P1: 5'-GC TCTAGA ATGGGGACAAATTCTAACAACAACAC- 3' (the nucleotide sequence is shown in SEQ ID NO: 6 in the sequence listing), P2: 5'-CG GGATCC TCATGCAGTTCTTCTAGTTTCCAAAG-3' (the nucleotide sequence is shown in SEQ ID NO: 7 in the sequence listing), wherein , the underlined part is the enzyme cutting site; use this primer to amplify the full-length reading frame of MsHPPD from the alfalfa cDNA template, the PBI vector and the full-length fragment of the target gene are digested with XbaI and BamHI to recover the product, and ligated with T4 DNA Enzyme ligation obtains the pBI121 vector inserted with the MsHPPD gene, which contains the CaMV35s promoter, the MsHPPD gene (as shown in SEQ ID NO: 1 in the sequence listing) and a kanamycin resistance selection marker, which can be passed during genetic transformation The transgenic plants initially identified by kanamycin screening.The structure of the plasmid vector containing the target fragment is shown in Figure 1.

1.2.2 Introduce the pBI121 vector inserted with the MsHPPD gene into Agrobacterium tumefaciens LBA4404, the specific steps are as follows:

A. Add about 1 μg of plasmid DNA to 100 μl of Agrobacterium competent cells LBA4404, mix gently, and ice-bath for 30 minutes;

B. Quickly freeze in liquid nitrogen for 1 minute, and immediately place it in a water bath at 37°C for 5 minutes;

C. Add 800μl YEB liquid medium, and culture at 150rpm at 28°C for 4-6h;

D. Bacteria were spread on a YEB selection plate containing 50 mg/L Kanamycin Sulfate and 100 mg/L Streptomycin, and cultured upside down at 28° C. for two days.

E. Pick a single colony, inoculate it in YEB liquid medium (containing 50 mg/L Kan and 100 mg/L Str), and cultivate overnight at 28° C. with shaking.

The plasmid of the Agrobacterium with the MsHPPD gene introduced was extracted and sequenced. The results showed that the nucleotide sequence of the introduced gene was consistent with the sequence shown in SEQ ID NO: 1 in the sequence listing, indicating that the expression vector containing the target gene MsHPPD was successfully constructed.

1.2.3. Culture of Agrobacterium

The Agrobacterium introduced with the MsHPPD gene was plated on a solid medium containing 50 mg/L Kan and 100 mg/L Str, placed in an incubator, and cultured at 28°C. Two days later, a single colony was picked from the plate, inoculated in 20ml YEB liquid medium containing 50mg/L Kan and 100mg/L Str, cultured at 180rpm, 28°C. Use the shaken bacterial solution to scratch the plate and incubate at 28°C. After a single colony grows, store the plate at 4°C.

Pick a single colony on the plate, inoculate it in 20ml of YEB liquid medium containing 50mg/L Kan and 100mg/L Str, and cultivate it on a constant temperature shaker at 28°C and 180rpm. Two days later, take a small amount of bacterial liquid, dilute it into YEB liquid medium containing 50 μg/m1Kan and 100 μg/m1Str at a ratio of 1:50-1:100, and culture it at 28°C for 6-12h to logarithmic growth phase. Collect the bacteria into a centrifuge tube, centrifuge at 4,000rpm for 10 minutes to enrich the bacteria, discard the supernatant, and resuspend the bacteria with about 20ml of modified SH liquid medium without antibiotics, so that the OD ₆₀₀ value of the bacteria is 0.6 -0.8, to be used.

1.2.4. Preparation of Arabidopsis:

After Col ecotype Arabidopsis seeds were sterilized, the seeds were spread on 1/2 MS solid medium culture dishes and placed at 4°C for vernalization for 48 hours.

Put the vernalized seed plates in the Arabidopsis thaliana incubator (24°C 16h/22°C 8h) for cultivation, and after 7 days, move them into flower pots (vermiculite: nutrient soil: perlite is about 1:1:1 ). Cultivate Arabidopsis thaliana according to the above method, and cut off the main stem that has bloomed for about three weeks to suppress the apical dominance. About four weeks or so, the side shoots drawn out bloom in large numbers, and this is the best time to use the infiltration method to transgene.

1.2.5. Agrobacterium cultivation and harvesting

Take 20 μl of the original bacterial solution, add 5ml of YEP (Yeast extract 10g/L, Tryptone 10g/L, Nacl 5g/L, after sterilization, add antibiotics), shake at a constant temperature of 30°C overnight, pour this 5ml of bacterial solution into 250ml of YEP ( Add antibiotics) culture medium, and shake overnight at 30°C. At this time, the concentration of Agrobacterium should reach OD600=1.8. Centrifuge at 4000rpm for 15 minutes, discard the supernatant, and add infiltration medium (1/2MS + 1×MS Trace+1×MS Iron Salt+1×MS Inositol+1×MS Vitamin+MES0.5g/L+Sucrose 5%, pH5.7 Autoclaved). Agrobacteria were resuspended in the infiltration medium at a ratio of 1:1, and the surfactant Silwet77 was added to make the final concentration 0.02% (200 μl per liter).

1.2.6. Infiltration transgene

Cover the Arabidopsis thaliana flowerpot with gauze and tie it tightly with a rubber band to prevent the culture medium from leaking when the flowerpot is turned upside down. Put the flower pot upside down on the infiltration tank with 250ml of bacterial suspension, so that the inflorescence of the plant is immersed in the bacterial solution, and the inflorescence lasts for 2 minutes. Dry. Transformed plants were covered with plastic wrap overnight and then the film was removed. Normal growth, harvest seeds.

1.2.7 Screening of transgenic Arabidopsis plants

Harvested Arabidopsis seeds were disinfected with 70% alcohol for 2 minutes and 5% sodium hypochlorite for 5 minutes. Seeds of Arabidopsis thaliana were sowed in a petri dish containing 50 mg/L Kan in 1/2 MS solid medium, and vernalized at 4°C for 2 days. The Arabidopsis thaliana seedlings that grow normally on the resistant medium are transformed into Arabidopsis thaliana successfully, and the Arabidopsis thaliana seedlings that fail to transform will not grow normally. Transplant Arabidopsis thaliana seedlings that can grow normally into flowerpots, and grow them in an artificial climate chamber. Using the genomic DNA of the transgenic Arabidopsis plant as a template, the primers P ₃ /P ₄ of the nptⅡ gene (the amino acid sequences are shown in SEQ ID NO: 8 and SEQ ID NO: 9 in the sequence listing, respectively), and the primers of the gus gene P ₅ /P ₆ (The amino acid sequences are respectively shown in SEQ ID NO: 10 and SEQ ID NO: 11 in the sequence listing) to detect transgenic plants containing the pBI121-MsHPPD vector. Positive plants were kept (12 plants were obtained in total: L1-L12) and harvested separately. The T ₁ generation was screened with the same antibiotic, and its segregation ratio (3:1) was tested by Chi-square test, and a total of 4 3:1 segregated lines were obtained: L1, L7, L8, L9. These lines were kept and harvested to obtain the T ₂ generation. Homozygotes _were screened from the T2 generation, and the seeds were harvested separately.

Example 3 Evaluation of vitamin E content in Arabidopsis thaliana in pBI121 vector of MsHPPD gene

Agrobacterium was transformed with plasmid pBI121 to obtain recombinant Agrobacterium, and Arabidopsis was transformed with recombinant Agrobacterium to obtain a control plant that was transformed into an empty vector. The method was as in Example 1, and the control plant (CK) that was transformed into an empty vector was obtained as Comparative Example 1.

Evaluate the vitamin E content of the Arabidopsis thaliana of the transfer empty vector obtained in comparative example 1 and the pBI121 carrier Arabidopsis thaliana that the transformation that obtains in embodiment 1 is inserted into MsHPPD gene, specific method is as follows:

Mix 3g of alfalfa leaves, grind them evenly, then sample 0.03-0.05g, add 1mL of methanol:chloroform (2:1/V:V) containing 0.01% of 2,6-di-tert-butyl-p-cresol (BHT), avoid Light standing for 20min. Add 0.33mL chloroform and 0.6mL water, vortex, mix well, centrifuge at 14000g for 10min, take the organic phase (lower layer), and evaporate to dryness. Dissolve it with dichloromethane:methanol (1:5/V:V) to 400μL, take 20μL in the liquid phase bottle and measure it on the machine.

External standard method and quantitative analysis conditions are high-performance liquid chromatography (model: Agilent 1260); double pump system (G1312B); fluorescence detector (G1321B); chromatographic column is LiCHrospher 100Diol 250mm × 4mm, particle size 5 μ m; mobile phase, Methanol: water (97:3/V:V); flow rate 1.2ml·min-1; fluorescence excitation 292nm, emission 330nm. The test results were statistically analyzed. The results are shown in Figure 2: the α-VE (α-vitamin E) content of the transgenic materials (L1, L8, L9) increased by an average of 13 times compared with the control (CK).

Example 4 Salt tolerance evaluation of Arabidopsis thaliana vector pBI121 of MsHPPD gene

Evaluate the co-amino acid content and peroxidase activity in the Arabidopsis thaliana of the transfer empty vector obtained in Comparative Example 1 and the pBI121 vector Arabidopsis plant that is inserted into the pBI121 gene of MsHPPD gene obtained in Example 1, and the specific methods are as follows:

1. Determination of pro-amino acid content

The Arabidopsis plants in Comparative Example 1 and Example 1 were treated with 0mM, 62.5mM and 125mM NaCl for 2 weeks, and 0.2g of each treated Arabidopsis leaves were accurately weighed (each treatment was repeated three times). Grind and extract with 3% sulfosalicylic acid, transfer to test tubes respectively, then add 5ml 3% sulfosalicylic acid solution to each test tube, extract in boiling water bath for 10 min, cool and filter into clean test tubes , the filtrate was adjusted to 5mL with 3% sulfosalicylic acid; draw 2ml of the extract into another clean test tube with a glass stopper, add 2ml of glacial acetic acid and 2ml of acidic ninhydrin reagent, heat in a boiling water bath for 30min; cool Then add 4ml of toluene, shake for 30s, and let it stand for 2h; use a straw to draw the upper proline red toluene solution into the cuvette, use toluene as a blank control, and compare the color at a wavelength of 520nm on a spectrophotometer to obtain the absorbance value. Calculate the concentration of proline in 2ml of the sample solution according to the standard curve, and then convert the content of proline in the sample (μg proline/gFW). Calculated as follows:

Proline content (μg.g-1)=c×(v/a)/w

c is the proline content μg obtained from the standard curve, v is the total volume of the extract in ml, a is the volume of the assay solution in ml, and w is the sample mass in g. The test results were statistically analyzed. The results are shown in Figure 3: under untreated conditions, the proline content of the transgenic material was slightly lower than that of the control, and after salt treatment, the proline content of the transgenic material (L1, L7, L9) was significantly higher than that of the control (CK) , and the higher the salt concentration, the greater the difference in proline content.

2. Determination of peroxidase (POD) activity

With 0mM, 62.5mM, 125mM and 187.5mM NaCl process the Arabidopsis plants in Comparative Example 1 and Example 1 for 2 weeks, take by weighing 1g of Arabidopsis leaves of different treatments, add 20mM KH2PO4 solution 5ml, in a mortar Grind into a homogenous slurry, centrifuge at 33000r/min for 10min, transfer the supernatant to a 25m1 volumetric flask, extract the residue once with 5ml KH2PO4 solution, combine the supernatant, constant volume, mix well, and store in a cool place for later use. Take 2 cuvettes with a light path of 1cm, add 3ml of the mixed reaction mixture and 1ml of KH ₂ PO ₄ solution to one of them as a reference solution; add 3ml of the reaction mixture and 1ml of enzyme solution to the other, and immediately Timed and placed in spectrophotometer. Measure the optical density at 470nm, read every 1min, measure continuously for 30min, re-calibrate with the control before each measurement.

The graph is plotted with time as the abscissa and optical density as the ordinate. In the early stage of the reaction, the activity of catalase increases linearly with the reaction time, and when it reaches the maximum value, there is a turning point in the correlation curve, and the turning point depends on the temperature sooner or later. In the early part of the curve, find a part of an approximate straight line, and the start time t1 and optical density A1 of the straight line, the end time t2 and optical density A2 of the straight line. The peroxidase activity unit (μ) was calculated as 0.01 change in A470 per minute, and its activity and specific activity were calculated.

Enzyme activity (0.01A470·min ^-1 )=A2-A1/(t2-t1)×0.01×D.

Enzyme specific activity (u·g-1) = enzyme activity (μ)/sample weight (g).

A2-A1: Absorbance change, t2-t1: Time change, D: Dilution multiple, that is, the total amount of extracted enzyme solution is the multiple of the enzyme solution in the reaction system. U: enzyme activity unit (ie 0.01A470·min-1). The test results were statistically analyzed. The results are shown in Figure 4: the POD activity in the transgenic materials (L1, L8, L9) was significantly higher than that in the control (CK) before and after treatment, and the POD activity increased more significantly after salt treatment. Studies have proved that both proline content and peroxidase are substances significantly related to the stress resistance of plants. Proline can be used as an osmotic adjustment substance in plants, and its content will increase significantly under plant adversity stress, which can improve the osmotic adjustment ability of plants, thereby improving the stress resistance of plants. Peroxidase is one of the important protective enzyme systems of plants. The level of peroxidase activity and reaction speed determine the stress resistance of plants. The higher the activity, the stronger the stress resistance. From the above test results, it shows that the stress resistance of the transgenic materials, especially the salt tolerance, is significantly higher than that of the control plants.

This gene encodes p-hydroxyphenylpyruvate dioxygenase in the vitamin E synthesis pathway in alfalfa. After transforming Arabidopsis thaliana, the content of vitamin E in Arabidopsis thaliana was increased. In addition, the transgenic lines increased the content of pro-amino acid and the activity of peroxidase POD under the condition of salt stress, thus improving the salt tolerance. This gene enriches the study of vitamin E biosynthetic pathway in alfalfa.

The above-mentioned embodiments are only descriptions of preferred implementations of the present invention, and are not intended to limit the scope of the present invention. Variations and improvements should fall within the scope of protection defined by the claims of the present invention.

Claims (1)

1. An application of the alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD, characterized in that: the nucleotide sequence of the alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD is as SEQ ID in the sequence listing Shown in NO: 1; the application of the alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD in the regulation of Arabidopsis thaliana.