CN103756971A - Method for preparing blackberry cell suspension culture solution - Google Patents

Abstract

The invention discloses a method for preparing a blackberry cell suspension culture solution. In the preparation method, a γ-TMT (γ-tocopherolmethyl-transferase, γ-tocopherol methyl-transferase) gene expression vector is transferred into blackberry explants through Agrobacterium In somatic cells, the suspension culture fluid is extracted after cell suspension culture; wherein the gamma-TMT gene expression vector contains polynucleotide fragments encoding gamma-TMT. The invention can effectively obtain the suspension culture liquid rich in α-tocopherol, and opens up a new way of bioengineering for industrial production of highly active natural vitamin E.

Description

A kind of preparation method of blackberry cell suspension culture fluid

technical field

The present invention relates to a preparation method of blackberry cell suspension culture fluid, in particular to a preparation method of suspension culture fluid rich in α-tocopherol.

Background technique

Vitamin E is a fat-soluble strong antioxidant. It is only synthesized in plants and other photosynthetic organisms in nature. It is one of the micronutrients that humans and animals must ingest from food. When it is deficient, it is easy to cause atrophy and damage to the reproductive system. , so it is also called "tocopherol". Vitamin E has a variety of physiological functions, has always been valued, and is widely used in food, cosmetics, medicine and animal feed products.

Vitamin E consists of four tocopherols (α-, β-, γ-, δ-tocopherols) and four tocotrienols (α-, β-, γ-, δ-tocotrienols), of which α - Tocopherol has the highest activity, because α-tocopherol binding protein (α-TTP) in human and animal tissues can preferentially bind α-tocopherol. However, in the seeds of most oil crops such as rapeseed, peanuts, and soybeans that are natural sources of vitamin E, the main components of _VE are γ-tocopherol and δ-tocopherol, while the content of α-tocopherol with higher activity is lower , even the proportion of α-tocopherol in synthetic vitamin E is not large.

Blackberry is a small berry fruit tree, its fruit is rich in linoleic acid, anthocyanin, vitamin E, γ-aminobutyric acid, superoxide dismutase (SOD) and other nutritional and health ingredients, and its seeds contain vitamin E The content (198.0 mg/100 g) is much higher than that of sunflower seeds (59.0 mg/100 g) and grape seeds (55.4 g/100 g), and the content of γ-tocopherol in vitamin E is 41-109 mg/100g, The α-tocopherol content is 9.7-37.8mg/100g, and the δ-tocopherol is less.

Obtaining natural products with target activities through the cultivation of specific plant tissues has been proven to be an effective way to obtain natural active substances. Cell suspension culture is the core technology of this method. It has the advantages of controllable production conditions of metabolites, and can obtain metabolites exceeding the yield of the whole plant by changing the culture conditions and selecting an excellent culture system. Production of highly active tocopherols by vitamin E-rich safflower, wheat germ, and sunflower cell cultures has been reported. The γ-TMT gene mainly regulates the conversion of γ-tocopherol into α-tocopherol. It has been reported that the overexpression of γ-TMT gene in Arabidopsis seeds and soybean seeds can increase the content of vitamin E in seeds and the content of α-tocopherol in them. - The proportion of tocopherol is greatly increased.

Taking advantage of the rich content of vitamin E (especially α-tocopherol) in blackberry seeds, plus the regulation of γ-TMT gene, vitamin E can be prepared by cell suspension culture, which can open up a new way for industrial production of highly active natural vitamin E.

Contents of the invention

The object of the present invention is to provide a method for preparing a suspension culture liquid rich in α-tocopherol in order to solve the defects in the prior art.

In order to achieve the above object, the present invention provides a method for preparing a transgenic blackberry somatic cell suspension culture medium, in which γ-TMT (γ-tocopherol methyl-transferase, γ-tocopherol methyltransferase) is mediated by Agrobacterium The gene expression vector is transferred into blackberry explant cells, and the suspension culture solution is obtained after cell suspension culture; wherein the gamma-TMT gene expression vector contains polynucleotide fragments encoding gamma-TMT.

The above-mentioned cell suspension culture adopts MS medium, pH 6.0-7.0 (preferably 6.0), which contains sucrose 25-30g/L (preferably 30g/L), 2,4-D 1.0-1.5mg/L (preferably 1.0 mg/L ), hydrolyzed casein 200-300mg/L (preferably 200 mg/L); the inoculum size is 1-2g/50mL (preferably 2g/50mL); dark culture for 15-25 days; culture temperature and shaker speed adopt cell suspension culture Common conditions, the preferred culture temperature is 25±0.2°C, and the preferred shaking table speed is 110r/min.

In the blackberry somatic cell that is used for cell suspension culture in the present invention, introduced gamma-TMT gene, the cloning of this gene, vector construction and import method are as follows:

(1) Extraction of Arabidopsis total RNA.

(2) γ-TMT gene cloning: using the first cDNA strand obtained by reverse transcription of Arabidopsis total RNA as a template, using primers (TMT-F: 5'-CTTGGATCCATGAAAGCAACTCTAGCAGC-3' and TMT-R: 5' -CGGGATCC GAGTGGCTTCTGGCAAGTGAT-3') PCR amplification, gel electrophoresis of the amplified product, comparison with Marker and sequencing confirmation, the full-length fragment of the γ-TMT gene was obtained.

(3) Gene expression vector construction: After connecting the polynucleotide fragment encoding γ-TMT with the pMD19-T vector, transform Escherichia coli competent cells, screen out transformants, perform PCR amplification, and extract the γ-TMT-encoding polynucleotide fragment. The cloning vector pMD-TMT of the TMT gene; the pMD-TMT vector and the pBI121 vector were digested by Bam H I, connected with T4 ligase and transformed into E. coli competent cells, and extracted to obtain the recombinant plasmid pBI121-TMT, which is γ-TMT Gene expression vector.

(4) Genetic transformation: pBI121-TMT was introduced into Agrobacterium tumefaciens EHA105, screened on YEB plates containing kanamycin (Kan) and rifampicin (Rif), and the γ-TMT positive-sense gene engineering for genetic transformation was obtained Streak culture on the YEB solid plate to culture the γ-TMT positive-sense gene transformation engineering strain, after extraction and identification, add it to the YEB culture medium, shake it at 28°C and 250 rpm for 18-24h, and collect the bacteria under sterile conditions , resuspended in MS solution to make OD ₆₀₀ =0.3-0.4, and prepare the engineered bacteria solution for infection and transformation; immerse the pre-cultured blackberry callus (embryogenic callus) into the engineered bacteria solution, gently Shake to make it fully contact with the bacteria, soak for 5-10 minutes, pour off the bacterial solution, transfer to the screening medium and culture for 7 days, and carry out PCR and RT-PCR molecular identification on the callus samples after infection , where the positive callus indicates that the γ-TMT gene has been introduced and can be used for suspension culture.

The method for cell suspension culture among the present invention is as follows:

(1) Establishment of suspension culture system: use MS culture medium, use 25-30g/L sucrose as carbon source, pH 6.0-7.0, add 1.0-1.5mg/L 2,4-D and 200-300mg/L hydrolyzed casein protein; the inoculum amount of blackberry callus transfected with γ-TMT gene is 1-2g/50mL; the culture temperature is 25±2°C; the shaker speed is 110r/min; dark culture is 15-25 days.

(2) Cell suspension culture: pick the γ-TMT gene-transferred callus in a sterile operating table, crush it with tweezers, put it into the culture medium, and carry out cell suspension culture on a rotary shaker with constant temperature and speed.

Compared with the prior art, the present invention has the following advantages: the present invention clones the γ-TMT gene that catalyzes the production of α-tocopherol with low activity γ-tocopherol from Arabidopsis thaliana, constructs the γ-TMT gene expression vector, and Through Agrobacterium-mediated transfer of the gene into suitable blackberry explant cells, after screening, a cell line with high α-tocopherol content and stable properties was obtained, and cell suspension culture was used to obtain a cell line rich in α-tocopherol Suspension culture fluid of this method opens up a new way of bioengineering for industrialized production of highly active natural vitamin E.

The invention obtains a suspension culture system with maximum biomass by screening the suspension culture conditions of the blackberry callus transfected with the gamma-TMT gene. The α-tocopherol content in blackberry seeds accounts for about 24.3% of the total tocopherol content, and the α-tocopherol content in the suspension culture liquid prepared by the present invention increases to 85.7%.

Description of drawings

Figure 1 is the verification electrophoresis of the gene expression vector pBI121-TMT;

Fig. 2 is the electrophoresis figure of PCR detection of the blackberry callus of transferring γ-TMT gene;

Fig. 3 is the RT-PCR detection electrophoresis of blackberry callus transfected with γ-TMT gene.

In Figure 1, lane 1: Mark DL 2000bp, lane 2: target gene band 1047bp, lane 3: 35s forward primer and target gene direction primer combination amplified band;

In Figure 2, lane 1: Mark DL 2000bp, lane 2: blank sample, lane 3-8: blackberry callus samples transfected with γ-TMT gene;

In Figure 3, lane 1: Mark DL 2000bp, lane 2: blank sample, lane 3-8: blackberry callus samples transfected with γ-TMT gene.

Detailed ways

1 Materials, reagents and instruments

1.1 Materials

Arabidopsis (Columbia type), Escherichia coli ( Escherichia coli ) DH5ɑ, expression vector pBI121 plasmid (an improved high copy number plasmid vector, the plasmid has T-DNA that can be integrated into the plant genome, CaMV 35S promoter, and neomycin phosphotransferase (nPtII) gene).

Bright yellow, granular, loosely textured embryogenic callus from seeds of blackberry cultivar 'Kiowa'.

1.2 Reagents

pUM-T simple vector, reverse transcription kit, LA Taq DNA polymerase, Ex Taq DNA polymerase, T4 DNA ligase, and various restriction enzymes were all purchased from TaKaRa Company; Plant RNA Reagent kit and agarose Gel DNA recovery kit was purchased from Tian Gen Company; other reagents were of domestic analytical grade.

1.3 Medium

(1) Basic culture medium

LB medium: peptone 10g/L+yeast extract 10g/L+NaCl5g/L, adjust the pH to 7.2 with NaOH.

LB solid medium: LB medium + 15 g/L agar.

YEB medium: peptone 10 g/L + yeast extract 10 g/L + sucrose 5 g/L + MgSO ₄ 0.493 g/L, adjust the pH to 7.2 with NaOH.

YEB solid medium: YEB medium + 15 g/L agar.

(2) Escherichia coli culture medium

Streak solid medium: LB solid medium + 100 μL/100 mL kanamycin.

Liquid medium: LB medium + 100μL/100mL kanamycin.

(3) Agrobacterium culture medium

Streak solid medium: YEB solid medium + 100 μL/100 mL kanamycin + 50 μL/100 mL rifampicin.

Liquid medium: YEB liquid medium + 100μL/100mL kanamycin + 50μL/100mL rifampicin.

(4) Screening medium

Solid medium: LB solid medium + 100μL/100mL kanamycin + 50μL/100mL rifampicin.

1.4 Instruments

The main equipment includes MultiGene Thermal Cycler (TC9600-G-230V) PCR amplification instrument (Labnet, USA), Tanon-2500 automatic digital gel image analysis system (Tianneng, Shanghai), HZ-2011KA microcomputer constant temperature oscillator (Hualida, Taicang, Jiangsu), TGL-16LM high-speed refrigerated centrifuge (Xingke, Changsha, Hunan), DYCP-31DN horizontal electrophoresis tank (Liuyi, Beijing), TU-1810 ultraviolet spectrophotometer (General Analysis General ,Beijing).

2 methods

2.1 Cloning of Arabidopsis γ-tocopheryl methyltransferase (γ-TMT) gene

2.1.1 PCR primer design

Search NCBI GenBank and find Arabidopsis γ-TMT gene mRNA (GenBank accession number NM_105171.3). Primers TMT-F: 5'-CTTggatccATGAAAGCAACTCTAGCAGC-3'; TMT-R: 5'-CGggatccGAGTGGCTTCTGGCAAGTGAT-3' were designed according to the mRNA sequence, and both upstream and downstream contained BamHI restriction sites.

2.1.2 Arabidopsis RNA extraction and cDNA synthesis

(1) Extraction of total RNA (Plant RNA Reagent kit method)

1) Take the whole plant of Arabidopsis thaliana and chop it up, stir it evenly with a glass rod or a strong homogenizer, grind it with a mortar in liquid nitrogen, add 1 mL Lysis Solution RL and 38 μL Precipitant A for every 50-100 mg tissue homogenate. The tissue sample volume should not exceed 10% of the RL volume.

2) Shake the homogenate sample vigorously, and incubate at 15-30°C for 5 minutes to completely decompose the ribosomes.

3) Centrifuge at 12,000rpm for 10min at 4°C, carefully remove the supernatant and transfer it to a new RNase-free centrifuge tube.

4) Add 0.2mL chloroform for every 1mL RL, tightly cap the sample tube, shake vigorously for 15 sec, and incubate at room temperature for 3 min.

5) Centrifuge at 12,000rpm at 4°C for 10 minutes, the sample will be divided into three layers, namely the lower organic phase, the middle layer and the upper aqueous phase, and the RNA exists in the aqueous phase. The capacity of the aqueous phase layer is approximately 60% of the volume of the added RL, and the aqueous phase is transferred to a new tube.

6) Add 1 volume of 70% ethanol, invert and mix well, and transfer the resulting solution and possible precipitates into the adsorption column RA (the adsorption column is set in the collection tube).

7) Centrifuge at 10,000rpm for 45 sec, discard the waste liquid, and put the adsorption column back into the collection tube.

8) Add 500 μL protein-removing solution RE, centrifuge at 12,000 rpm for 45 sec, and discard the waste liquid.

9) Add 700 μL of rinse solution RD, centrifuge at 10,000 rpm for 15 sec, and discard the waste liquid.

10) Add 700 μL of washing solution RW, centrifuge at 12,000 rpm for 60 sec, and discard the waste liquid.

11) Optional step: Add 500μL of Rinse Solution RW, centrifuge at 12,000rpm for 60 sec, and discard the waste.

12) Put the adsorption column RA back into the empty collection tube, centrifuge at 12,000rpm for 2min, and remove the rinsing solution as much as possible, so as to avoid the residual ethanol in the rinsing solution from inhibiting downstream reactions.

13) Take out the adsorption column RA, put it into an RNase free centrifuge tube, add 50-80 μL RNase free water to the middle part of the adsorption membrane according to the expected RNA yield (heating in a 65-70°C water bath in advance is better), and place at room temperature for 2 minutes , centrifuge at 12,000 rpm for 1 min. If more RNA is needed, add the obtained solution to the centrifugal adsorption column again, and centrifuge for 1 min, or add 30 μL RNase free water, centrifuge for 1 min, and combine the two eluates.

14) The extracted Arabidopsis total RNA was detected by 1% agarose gel electrophoresis, and the bands were clear and bright, indicating that the RNA had good purity and integrity and could meet the requirements of RT-PCR (as shown in Figure 1).

(2) cDNA synthesis

cDNA preparation 2μg RNA and Oligo18(T) primer were used for cDNA first-strand synthesis, and the steps were carried out according to the kit instructions.

1) Prepare reverse transcription reaction solution with the following components: 0.2-2μg Total RNA or Poly(A)RNA, 1μL Oligo dT or Random Primer (50μM), 1μL dNTP Mixture (10mM each), Up to 14μL Rnase Free H ₂ O.

2) Carry out the denaturation and annealing reaction on the PCR instrument according to the following conditions: 65°C for 5 minutes, and then place it on ice for rapid cooling. the

3) Add 4μL 5×first-strand Buffer, 1μL M-MuL V Reverse Transcriptase (200U/μL), 1μL Rnase Inhibitor, Total 20μL to the above denatured and annealed reaction solution.

4) Carry out the reverse transcription reaction on the PCR instrument according to the following conditions: 42-50°C, 45-60min; 70°C, 10min. Immediately after cooling on ice, the obtained cDNA can be used directly for subsequent reactions such as PCR, or can be frozen at -20°C for future use.

2.1.3 Amplification of full-length γ-TMT cDNA

Using the Arabidopsis cDNA as a template, PCR amplification was performed with the specific primers designed in 2.1.1. A 20 μL reaction system was used for PCR amplification, including 2 μL 10× buffer, 0.2 μL 10 mmol/L dNTPs, 2 μL 10 pmol/L primers, 0.2 μL 5 U/μL Taq enzyme, 1 μL cDNA template, and 14.6 μL ddH ₂ O. The PCR reaction program was: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 60 sec, and extension at 72°C for 10 min after 35 cycles. The amplified products were separated by 1.0% agarose gel electrophoresis, and there was a specific band at about 1000 bp compared with the Marker (as shown in Figure 2).

2.1.4 Recovery, ligation and transformation of the full-length target fragment of γ-TMT cDNA

(1) Target fragment recovery

1) Under the long-wave ultraviolet lamp, use a clean blade to cut off the DNA band to be recovered, and try to cut off the gel without DNA. The smaller the gel volume, the better.

2) Put the excised gel containing the DNA band into a 1.5mL centrifuge tube and weigh (weigh an empty 1.5mL centrifuge tube first, then put in the gel block and weigh again, subtract the weight twice, to get the weight of the gel).

3) Add three volumes of sol/binding solution DB (if the gel weighs 0.1g, its volume can be regarded as 100μL, then add 300μL of sol solution).

4) Place in a water bath at 56°C for 10 minutes (or until the glue is completely dissolved). Vortex every 2-3 minutes to help speed up dissolution.

5) Add 150 μL of isopropanol per 100 mg of the initial gel weight, shake and mix.

6) Add the solution obtained in the previous step to the adsorption column AC (the adsorption column is set in the collection tube), centrifuge at 12,000rpm for 30-60sec, and discard the waste liquid in the collection tube.

7) Add 700 μL of washing solution WB, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

8) Add 500 μL of washing solution WB, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

9) Put the adsorption column AC back into the empty collection tube, centrifuge at 12,000rpm for 2min, and remove the rinsing solution as much as possible, so as to avoid the residual ethanol in the rinsing solution from inhibiting downstream reactions.

10) Take out the adsorption column AC, put it into a clean centrifuge tube, add 50μL of elution buffer EB to the middle part of the adsorption membrane (it is better to heat it in a water bath at 65-70℃ in advance), and place it at room temperature for 2min, 12,000rpm Centrifuge for 1 min. If a larger amount of DNA is needed, the obtained solution can be added to the centrifuge tube adsorption column again, and centrifuged for 1 min.

(2) Target segment connection

1) Melt the pUM-T simple vector carrier on ice, and briefly centrifuge the centrifuge tube containing the carrier to make the liquid sticking to the tube wall sink to the bottom of the tube.

2) According to the content of the connection reaction system below, add various components into a sterile centrifuge tube. The molar ratio of the carrier and the fragment recovered from the agarose gel is controlled at 1:3-1:8. Taking the 1000bp fragment as an example, the concentration should be In the range of 50-125ng, the positive rate increases with the increase of fragment concentration. Ligation reaction system: X μL fresh PCR fragment, 1 μL pUM-T (about 40ng/μL), 5 μL 2×T4DNA Rapid Ligation Buffer, 1 μL T4DNA Ligase, sterile deionized water to make up to 10 μL.

3) Blow gently with a sterile pipette tip to mix the contents (if bubbles are generated, centrifuge briefly), place the ligation system at 22-25°C for 10-15min, and the ligation time is 10min when the ligated fragment is less than 1.5kb. When the connection fragment is larger than 1.5kb, the connection time is 15 minutes. After the reaction, the centrifuge tubes were placed on ice for transformation.

(3) Gene transformation

1) Melt the competent cells of the E. coli DH5ɑ plasmid in ice water, add the above ligation product to 50-100 μL of the competent cells, and mix quickly and gently. Stand in an ice-water bath for 30 minutes.

2) Put the competent cells in a 42°C water bath, heat shock for 60-90sec, take them out quickly, and place them in ice water for 1-2min.

3) Add 500 μL of 37°C preheated (without antibiotics) LB medium to the centrifuge tube, shake at 150 rpm and 37°C for 45-60 minutes to express the relevant resistance marker genes on the plasmid.

4) Centrifuge the bacterial solution in the centrifuge tube, discard part of the supernatant, keep about 200 μL, mix with the pelleted cells, add to the LB solid medium that already contains 100 μL/100mL kanamycin and 100 μL/100mL ampicillin (Because this carrier has a very high white spot rate, the bacterial solution can be directly coated on the ampicillin-resistant plate, and blue-white spot screening is not required), use a sterile elbow glass rod to gently spread the cells evenly, if you want For more transformants, centrifuge the bacterial solution at low speed, remove most of the supernatant, and then spread all the suspended bacteria on the ampicillin-resistant plate. After the surface of the plate is dry, invert the plate and incubate at 37°C for 12-16hr.

(4) Plasmid extraction

1) Take 1.5-4.5mL of overnight cultured bacteria solution, centrifuge at 9,000 rpm for 30sec, discard the supernatant, collect the bacteria, and drain the supernatant as much as possible. After processing more than 1.5mL of bacterial liquid, discard the supernatant, add more bacterial liquid into the same 1.5mL tube, and repeat step 1) until enough bacterial cells are collected.

2) Resuspend the bacterial pellet with 250 μL solution P1, and vortex until completely suspended.

3) Add 250 μL of solution P2, gently turn it up and down 6-10 times to fully lyse the bacteria until the solution becomes clear.

4) Add 400 μL of solution P3, immediately flip up and down gently 6-10 times, and place at room temperature for 5 minutes. Centrifuge at room temperature at 13,000 rpm for 10 min, and carefully remove the supernatant.

5) Place the adsorption column on the collection tube, add the supernatant obtained in the previous step to the adsorption column AC (the adsorption column is set in the collection tube, if the solution is too much, add in two times), centrifuge at 13,000rpm for 10min, and discard the filtrate.

6) Add 500μL of washing solution WB, centrifuge at 13,000rpm for 30-60sec, and discard the filtrate.

7) Repeat step 6), centrifuge at 13,000rpm for 30-60sec, and discard the filtrate. Centrifuge the empty column at 13,000 rpm for 2 min. Place at room temperature for 3-5min to remove residual ethanol.

8) Take out the adsorption column AC, put it into a clean centrifuge tube, add 60-100 μL of elution buffer EB to the middle of the adsorption membrane (it is better to heat it in a water bath at 65-70°C in advance), and place it at room temperature for 1 min , centrifuge at 13,000 rpm for 1 min to elute the DNA, which can be used immediately for downstream molecular biology experiments or stored at -20°C.

After resistance screening and PCR amplification product electrophoresis detection, the positive clones were selected for sequencing. The sequencing results (SEQ ID NO: 1) showed that the amplified fragment size was 1047bp, and the ORF region presumed to encode 350 amino acids, which was similar to the gamma gene registered in GenBank. - The homology of the TMT gene sequence is 99.9%, only 1 (A-G) is different in 1047 bases, and the amino acid sequence is almost identical (S-N), not in its main functional domain. This shows that the cloned gene is γ-TMT gene, which can be used for the construction of subsequent expression vectors.

2.2 Construction of γ-TMT cDNA plant expression vector

Sequencing verified that the correct bacterial samples continued to amplify and extract plasmids. The recombinant plasmid pUM-TMT was digested with Bam H I, and the digested product was recovered by tapping the rubber; at the same time, the pBI121 plasmid was digested with Bam H I, and the digested product was recovered by tapping the rubber. The digestion reaction system is as follows: 5 μL plasmid, 2.5 μL Buffer, 1 μL Bam HI, 40.5 μL ddH ₂ O, the total volume is 50 μL.

The two digestion products were ligated with T4 ligase. The ligation product was transformed into Escherichia coli competent cells E.coli DH5ɑ. Spread the plate, pick the white spot shake bacteria, and use the bacteria sample as a template for PCR amplification. The bacterial sample that can expand the target band is positive. The above positive bacteria were shaken, and the extracted plasmid was digested and identified, as shown in Figure 3, the expected target band appeared as a result, indicating that the plant fusion expression vector of the γ-TMT gene was obtained. The successfully recombined vector was named pBI121-TMT.

2.3 Agrobacterium transformation of pBI121-TMT

The positive vector plasmid pBI121-TMT was extracted, transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method, and cultured overnight at 28°C. Pick the plaque, shake the bacteria, and use the bacteria sample as a template for PCR amplification. The bacterial sample that can expand the target band is retained as the final retained bacterial sample for subsequent infection of the callus.

(1) Preparation of Agrobacterium tumefaciens competent cells

1) Pick Agrobacterium tumefaciens EHA105 stored at -70°C in YEB solid medium (containing rifampicin 50mg/L), streak the plate, and culture at 28°C.

2) Pick a single colony and inoculate it in 3 mL of YEB liquid medium (containing 50 mg/L rifampicin), shake at 220 rpm and culture overnight at 28 °C.

3) Inoculate 1 mL of the overnight culture into 50 mL of YEB (containing 50 mg/L rifampicin) liquid medium, and culture with shaking at 28°C until the OD600 is 0.5.

4) Take 2mL of bacterial liquid, centrifuge at 13000rpm for 30sec, and discard the supernatant.

5) Add 1000 μL of 20mM CaCl ₂ to fully suspend the Agrobacterium cells, and ice-bath for 30min.

6) Centrifuge at 13,000 rpm for 30 sec, discard the supernatant, place on ice, add 500 μL of pre-cooled 20 mMCaCl ₂ to fully suspend cells, store in ice bath, use within 24 hr, or freeze in liquid nitrogen for 1 min, and store at -70 °C for later use.

(2) Plasmid DNA into Agrobacterium

1) Add 2~6 μL of plasmid (pBI121) DNA to 200 μL of competent cells, ice-bath for 5 minutes, and freeze in liquid nitrogen for 5 minutes. the

2) Quickly transfer to a 37°C water bath and heat shock for 5 minutes.

3) Add 1mL YEB liquid medium and culture with slow shaking at 28°C for 2~4hr.

4) Centrifuge at 3000rpm for 4min, remove a part of the supernatant, and take 200μL of the bacterial solution and spread it on a YEB plate containing 50μg/mL kanamycin and 50μg/mL rifampicin. the

5) Leave it for about 0.5hr. After the water is dry, incubate at 28°C for about 36-48hr until colonies grow.

2.4 Agrobacterium transformation of blackberry callus

The strains required for the experiment were taken from the -70°C freezer, streaked and cultured on the YEB solid plate, and single colonies grown on the YEB solid plate were picked for micro-extraction and identification. Add the correctly identified bacterial solution to the YEB liquid medium, shake and culture at 250 rpm for 18-24 hours at 28°C, collect the bacterial cells under sterile conditions, resuspend in MS solution to make OD600=0.3-0.4, and use for preparation Infected and transformed engineered bacterial fluid.

The blackberry seed embryogenic callus (bright yellow, granular, loose texture) pre-cultured (MS+ 2,4-D 1.0 mg/L, 25°C, 7d) was immersed in the engineering bacteria solution, and shaken gently to make it Fully contact with the bacteria, soak for 5-10min, remove the bacterial solution, transfer to the co-culture medium (MS+ 2,4-D 1.0 mg/L +AS 100μmol/L) for 3 days, and then transfer to the antibacterial medium (MS+ 2,4-D 1.0 mg/L +AS 100 μmol/L+Cef 500 mg/L) was cultured for 7 days, and then transferred to selective medium containing antibiotics and bacteriostatin (MS+ 2,4-D 1.0 mg/L +Kan 10 mg/L+Cef 500 mg/L) for resistance screening, and the positive calli were transferred and identified.

Molecular identification of the transgenic callus. Four of the samples were found to be positive by PCR and RT-PCR detection, as shown in Figure 2 and Figure 3, indicating that the γ-TMT gene-transformed callus that can be used for suspension culture was successfully obtained.

2.5 Identification of transgenic callus

(1) Minimized extraction of genomic DNA from transgenic callus (CTAB method)

1) Add 500 μL 2×CTAB to a 1.5 mL centrifuge tube, preheat at 65°C, and add 20 μL β-mercaptoethanol before use.

2) Take 1-2 g of young callus, put it into a mortar pre-cooled by liquid nitrogen, add liquid nitrogen and grind it to powder, transfer the powder to a preheated centrifuge tube with a clean sterilized stainless steel spoon , with a total volume of 1 mL, mix well and place in a 65°C water bath for 45-60 min, rotating the test tube occasionally during this period.

3) Add an equal volume of chloroform/isoamyl alcohol, mix gently by inversion, place on ice for 5 min, and centrifuge at 10,000 rpm for 10 min at room temperature.

4) Transfer the supernatant to another new tube. Repeat operation step 3), preferably twice.

5) Add 2 times the volume of absolute ethanol or 0.7 times the volume of isopropanol, a flocculent precipitate appears, place it on ice for 10 minutes, centrifuge at 12000rpm at room temperature for 10-15 minutes, and recover the DNA precipitate.

6) Wash the precipitate twice with 70% ethanol, centrifuge at 1000 rpm for 1 min, dry it in an ultra-clean table, and dissolve it in an appropriate amount of sterilized water.

7) Check its integrity by agarose gel electrophoresis.

(2) PCR identification of transgenic callus

The reaction system and reaction conditions for PCR identification are the same as 2.1.2.

(3) RT-PCR identification of transgenic callus

The callus RNA that was successfully identified by PCR was extracted, cDNA was obtained after reverse transcription, and PCR amplification was performed according to the reaction conditions in 2.1.2.

2.6 Cell suspension culture of transgenic callus

Pick the embryogenic callus tissue (selected from the bright yellow, granular, and loose embryogenic callus tissue produced by the aseptic seedling seeds of the blackberry variety 'Kiowa') in a sterile operating table, crush it with tweezers, and take a certain mass The embryogenic callus was put into a 250mL Erlenmeyer flask prefilled with 50mL culture medium, and the cell suspension culture was carried out on a rotary DKY-Ⅱ type shaker with constant temperature and speed regulation.

2.6.1 Effect of medium type on suspension culture of ‘Kiowa’ callus

The calli of ‘Kiowa’ were inoculated in different types of liquid media (MS, B5, N6) for suspension culture. Hormone 2,4-D 1.0 mg/L was added to the medium, and observation and statistics were carried out every 7 days. 6 bottles were inoculated for each treatment, and each was repeated 3 times.

2.6.2 Effect of sugar species on suspension culture of ‘Kowa’ callus

The calli of 'Kiowa' were respectively inoculated in MS liquid medium supplemented with 25g/L sucrose, maltose and glucose for suspension culture. Hormone 2,4-D 1.0 mg/L was added to the medium, and observation and statistics were carried out every 7 days. 6 bottles were inoculated for each treatment, and each was repeated 3 times.

2.6.3 The effect of sucrose concentration on the suspension culture of ‘Kowa’ callus

The calli of 'Kiowa' were inoculated in MS liquid medium supplemented with different concentrations of sucrose (10g/L, 20g/L, 25g/L, 30g/L, 40g/L, 50g/L, 60g/L) Carry out suspension culture. Hormone 2,4-D 1.0 mg/L was added to the medium, and observation and statistics were carried out every 7 days. 7 bottles were inoculated for each treatment, and each was repeated 3 times.

2.6.4 Effect of pH value on suspension culture of ‘Kowa’ callus

The calli of ‘Kiowa’ were inoculated in MS liquid medium with different pH values (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) for suspension culture. Hormone 2,4-D 1.0 mg/L was added to the medium, and observation and statistics were carried out every 7 days. 6 bottles were inoculated for each treatment, and each was repeated 3 times.

2.6.5 Effects of hormone types on suspension culture of ‘Kowa’ callus

The calli of 'Kiowa' were inoculated in MS liquid medium supplemented with 1.0 mg/L concentration of 2,4-D, NAA, 6-BA, KT, and TDZ for suspension culture, and observations were made every 7 d. statistics. 6 bottles were inoculated for each treatment, and each was repeated 3 times.

2.6.6 Effect of 2,4-D concentration on suspension culture of ‘Kowa’ callus

The calli of 'Kiowa' were inoculated with different concentrations of 2,4-D (0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L, 3.0 mg/L, 5.0 mg/L) Suspension culture was carried out in MS liquid medium, and observation and statistics were carried out every 7 days. 6 bottles were inoculated for each treatment, and each was repeated 3 times.

2.6.7 Effect of inoculum volume on 'Kiowa' callus suspension culture

The calli of 'Kiowa' were inoculated in MS liquid medium according to different inoculum amounts (0.5g, 1g, 2g, 3g, 5g, 10g) for suspension culture. Hormone 2,4-D 1.0 mg/L was added to the medium, and observation and statistics were carried out every 7 days. 6 bottles were inoculated for each treatment, and each was repeated 3 times.

2.6.8 Effect of hydrolyzed casein concentration on suspension culture of ‘Kowa’ callus

The calli of 'Kiowa' were inoculated in MS liquid with different concentrations of hydrolyzed casein (CH) (0 mg/L, 200mg/L, 300mg/L, 400 mg/L, 500mg/L, 600mg/L) Suspension culture in culture medium. Hormone 2,4-D 1.0 mg/L was added to the medium, and observation and statistics were carried out every 7 days. 6 bottles were inoculated for each treatment, and each was repeated 3 times.

2.6.9 Effect of light time on suspension culture of ‘Kowa’ callus

The calli of ‘Kiowa’ were inoculated in MS liquid medium supplemented with 1.0 mg/L 2,4-D for suspension culture, and the light treatment time was 0h and 12h, respectively. Observation and statistics were carried out every 7 days. 6 bottles were inoculated for each treatment, and each was repeated 3 times.

Except for the experimental items, the basic culture conditions for all the above experiments are: the sucrose concentration in MS liquid medium is 25g/L, the pH is 5.8-6.0, the culture temperature is 25±2℃, the light and dark culture are 12h each, and the rotation speed is 110r/min ; The inoculum size is 2g/50mL.

2.6.10 Establishment of suspension culture system

Considering various factors comprehensively, the optimal conditions for suspension culture of blackberry 'Kiowa' transgenic callus are: MS as basic liquid medium, 25-30g/L sucrose as carbon source, pH 6.0-7.0, additional 1.0-1.5mg/L L 2,4-D and 200-300 mg/L hydrolyzed casein, the inoculum size is 1-2g/50mL, cultured in dark. The optimal culture system is: using MS as the basic liquid medium, 30g/L sucrose as the carbon source, pH 6.0, adding 1.0 mg/L 2,4-D and 200 mg/L hydrolyzed casein, the inoculum size is 2g/50mL , temperature 25.2°C, shaker speed 110r/min, culture in dark for 25 days, pay attention to observe and detect the shape and number of suspended cells during culture.

3 results

Taking the embryogenic callus produced from aseptic seedling seeds of blackberry variety 'Kowa' as an example, the content of α-tocopherol in the total tocopherol in the non-transgenic wild sample was 24.3%, and the transgenic callus obtained by using the above optimal culture system The α-tocopherol content of the total tocopherols in the wounded tissue suspension culture was 85.7%.

<110> Institute of Botany, Chinese Academy of Sciences, Jiangsu Province

<120> A kind of preparation method of blackberry cell suspension culture fluid

<160> 1

<170> PatentIn Version 3.3

<210> 1

<211> 1047

<212> PRT

<213> Arabidopsis thaliana

<220>

<400> SEQ ID NO: 1

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the

Claims (9)

1. a preparation method for blackberry, blueberry cell suspending culture solution, is characterized in that: this preparation method proceeds to γ-TMT expression vector in blackberry, blueberry explant somatocyte by agriculture bacillus mediated, obtains described suspending nutrient solution after cell suspension culture; The polynucleotide passage that comprises the γ-TMT that encodes in described γ-TMT expression vector.

2. preparation method according to claim 1, is characterized in that: described cell suspension culture adopts MS nutrient solution, pH 6.0-7.0, and wherein containing sucrose 25-30g/L, 2,4-D 1.0-1.5mg/L, caseinhydrolysate 200-300mg/L; Inoculum size is 1-2g/50mL; The dark 15-25 days that cultivates.

3. preparation method according to claim 2, is characterized in that: described cell suspension culture adopts MS substratum, pH 6.0, and wherein containing sucrose 30g/L, 2,4-D 1.0mg/L, caseinhydrolysate 200mg/L; Inoculum size is 2g/50mL; Culture temperature is 25 ± 0.2 ℃; Shaking speed is 110r/min.

4. according to the arbitrary described preparation method of claims 1 to 3, it is characterized in that: described γ-TMT expression vector adopts plant expression vector.

5. preparation method according to claim 4, is characterized in that: described γ-TMT expression vector adopts PBI121 carrier.

6. according to the arbitrary described preparation method of claims 1 to 3, it is characterized in that: the polynucleotide passage of the coding γ-TMT comprising in described γ-TMT expression vector is prepared by the following method:

(1) extract the total RNA of Arabidopis thaliana;

(2) cDNA obtaining with the total RNA reverse transcription of Arabidopis thaliana, utilize primer TMT-F:5 '-CTTGGATCCATGAAAGCAACTCTAGCAGC-3 ', TMT-R:5 '-CGGGATCCGAGTGGCTTCTGGCAAGTGAT-3 ' to carry out pcr amplification, obtain the polynucleotide passage of described coding γ-TMT.

7. preparation method according to claim 6, is characterized in that: described γ-TMT expression vector is prepared by following steps:

(1) utilize pMD19-T carrier to connect after the polynucleotide passage of described coding γ-TMT, transform competent escherichia coli cell, filter out transformant, carry out pcr amplification, extract and obtain containing the cloning vector pMD-TMT of gene of γ-TMT of encoding;

(2) get cloning vector pMD-TMT and the employing of pBI121 carrier that step (1) obtains bamafter H I enzyme is cut, after T4 ligase enzyme connects, transform competent escherichia coli cell, extract and obtain recombinant plasmid pBI121-TMT; Described recombinant plasmid pBI121-TMT is described γ-TMT expression vector.

8. according to the arbitrary described preparation method of claims 1 to 3, it is characterized in that: described agriculture bacillus mediated employing freeze-thaw method transforms agrobacterium tumefaciens EHA105.

9. according to the arbitrary described preparation method of claims 1 to 3, it is characterized in that: the embryo callus that described blackberry, blueberry explant somatocyte adopts blackberry, blueberry seed to produce.

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