CN100350046C - Method for increasing content of astragalus methglycoside through exogenous gene transfer technology - Google Patents

Abstract

The present invention discloses a method for increasing content of astragalus methglycoside through an exogenous gene transfer technology. After the present invention reforms a transparent fibrillation bacterium hemoglobin gene (a vgb gene), an enzyme cutting technology is combined with a PCR technique for inserting the vgb gene into a plasmid pBI121 for obtaining a middle expression carrier pBI121-vgb which uses a 35s-CaMV strong promoter as a starting element; the vgb gene is transferred into colibacillus DH 5 alpha; a triparental hybrid method (pRK2013, DH 5 alpha and LBA9402) is adopted for obtaining rhizogenes agrobacterium LBA9402-vgb of the transferred vgb gene; the content of astragalus methglycoside is higher than that of a transgenic hairy root by 5 times by measurement.

Description

Enhancing the content of astragaloside IV by transgenic exogenous gene technology

Technical field:

The invention belongs to the technical field of traditional Chinese medicine. Specifically, it relates to a technology of exogenous gene transfer to increase the content of astragaloside IV.

Background technique:

Astragalus membranaceus or A.mongholicus is one of the important varieties of Chinese herbal medicines in my country. It has the functions of tonifying and solidifying the exterior, diuresis and detoxification, promoting muscle and reducing sores, diuresis and detumescence, and is the main component of many traditional Chinese medicine compounds, patent medicines and health care products. With the strengthening of people's growing health care concept, the demand for Astragalus membranaceus will increase year by year; but at present, the wild resources of Astragalus membranaceus are decreasing year by year, the quality of cultivated varieties is declining, and they are facing problems such as pesticide pollution and heavy metal residues. , Export brings certain difficulties. Therefore, the application of modern biotechnology to increase the output of Astragalus membranaceus, improve its quality, and explore feasible ways of industrial production are of great significance to the production of Astragalus membranaceus. Astragaloside Ⅳ is the content detection index stipulated in the Chinese Pharmacopoeia. If an artificial astragaloside product with high content of astragaloside Ⅳ can be found, it will have a good development and application prospect.

Since the 1980s, molecular biology techniques, especially genetic engineering techniques, have been successfully applied to the production, quality and The improvement of identification has injected new vitality into the production of traditional Chinese medicine. The hairy roots induced by Agrobacterium rhizogenesis have the advantages of rapid growth, no need to add exogenous hormones, metabolic pathways of parent plants, stable genetics, and easy expansion and cultivation. If the content of active ingredients can be increased, It will be beneficial to realize industrialized production.

Vitreoscilla Haemoglobin (VHb for short) is a kind of soluble hemoglobin produced in Vitreoscitlas (Vitreoscitlasp.). can survive well. Scientists cloned the Vitreoscilla Haemoglobin gene (Vgb gene for short) based on the reported VHb amino acid sequence. Because the vgb gene regulation mechanism is very conservative in obligate and facultative aerobic bacteria, it has been expressed in a variety of microorganisms, such as Escherichia coli, Pseudomonas, Streptomyces, Erwinia, etc.; this expression can improve Enzyme production, total protein production and antibiotic production. At present, the vgb gene is rarely used in the field of plants, and most of them are concentrated in the field of agriculture. For example, scientists from Sweden and Switzerland applied Vitiligo hyaline hemoglobin to the plant field for the first time. They expressed VHb in tobacco and obtained transgenic tobacco. The seeds of the transgenic tobacco germinated earlier, grew faster, and the chlorophyll content of the plant increased; biological Alkaline levels also changed, with significantly more nicotine and less neonicotinoid. In addition, the transgenic vgb cabbage can increase the waterlogging resistance. But so far, there is no application of this technology in Chinese herbal medicine. Most Chinese medicinal materials in my country use rhizomes as medicines. Because the roots are below the ground, it is much more difficult to obtain oxygen than the parts above the ground, so it is more necessary to solve the problem of hypoxia resistance.

Invention content:

The technical problem to be solved by the present invention is to combine the hairy root technology with genetic engineering to obtain the vgb gene transgenic astragalus hairy root with short growth period, high yield and high content of astragaloside IV.

The invention provides a method for improving the content of astragaloside IV through exogenous gene transfer technology.

In the present invention, after the hemoglobin gene of Vitiligo hyaline is transformed, the vgb gene is inserted into the plasmid pBI121 by enzyme cutting and PCR technology, and the intermediate expression vector pBI121-vgb with the 35s-CaMV strong promoter as the promoter element is obtained, and transformed into Escherichia coli DH5α. Agrobacterium rhizogenes LBA9402-vgb transmuted with vgb gene was obtained by triparental hybridization method (pRK2013, DH5α and LBA9402). LBA9402-vgb was used to infect Astragalus aseptic seedlings, and the molecular identification proved that the transgenic vgb gene Astragalus hairy roots. The growth of transgenic vgb gene Astragalus hairy roots and the content of astragaloside IV were measured, and the results showed that the content of astragaloside IV in transgenic vgb gene Astragalus hairy roots was 5 times higher than that of non-transgenic Astragalus hairy roots, and higher than that of the commercial medicinal material Astragalus membranaceus The content is more than 10 times; compared with the same period, the dry weight of the hairy root of astragalus with vgb gene is increased by 13% to 25% compared with that of the hairy root of astragalus without transgene.

The transgenic hairy root of astragalus membranaceus obtained by the technique of the invention can increase the yield and the content of the active ingredient astragaloside IV. Compared with the same period, the dry weight of hairy roots of Astragalus transgenic with vgb gene increased by 13%-25% compared with that of non-transgenic Astragalus hairy roots; the contents of astragaloside IV in lines 36 and 52 were 20.674mg/g and 20.999mg/g respectively, It is 6.2 times that of non-transgenic Astragalus hairy root (the content of astragaloside IV is 3.334mg/g), and it is 11.6 times that of Shanxi membranous viburnum (the content of astragaloside IV is 1.774mg/g). As the content of astragaloside IV in medicinal materials from different origins is different, refer to the regulations of the Chinese Pharmacopoeia [1]: astragaloside IV should not be less than 0.4mg/g, and the astragaloside IV in Astragalus membranaceus from different sources collected and determined by Shanghai Traditional Chinese Medicine Standardization Center Content: 0.5mg/g～1.9mg/g, it can be inferred that the hairy root of Astragalus transgenic vgb gene has a good ability to synthesize astragaloside IV.

Astragaloside IV is the content detection index stipulated in the Chinese Pharmacopoeia, and has good cardiovascular and cerebrovascular pharmacological activities. Astragaloside IV can improve the survival rate of myocarditis mice, reduce collagen synthesis and myocardial cell apoptosis, and is safe and effective; its anti-apoptotic effect plays an important role in delaying or reversing myocardial fibrosis in viral myocarditis. Studies have proved that astragaloside IV can reduce the permeability of the blood-brain barrier, and is a brain tissue protective agent. Astragaloside IV can reduce the contraction of the isolated rabbit aorta ring caused by KCl and CaCl ₂ and the spontaneous right atrium of the isolated guinea pig. At the same time, the increase of cerebral microvascular permeability caused by histamine can be inhibited by astragaloside IV. These all show that astragaloside IV has a good market application prospect. The transgenic astragaloside hairy root obtained by the transgenic vgb gene of the present invention can not only increase the yield, but also greatly increase the content of astragaloside IV, which can be used for the industrialization of astragaloside IV. Production provides high-quality raw materials and has good development prospects.

Description of drawings:

Figure 1: PCR identification diagram of plasmid pBI 121-vgb and Agrobacterium rhizogenes LBA9402-vgb.

Figure 2: PCR identification diagram of hairy root of Astragalus membranaceus transgenic with vgb gene.

Fig. 3: RT-PCR identification diagram of hairy root of Astragalus membranaceus transgenic with vgb gene.

Figure 4: Determination of the content of astragaloside IV by HPLC-ELSD;

Figure 4.1: Structural diagram of astragaloside IV;

Figure 4.2: HPLC chart of astragaloside IV standard;

Figure 4.3: HPLC chart of astragaloside IV determination of the sample (transformed vgb gene Astragalus hairy root VHb52).

Example 1

1) Extraction of plasmid pBI 121

Preparation of reaction reagents: LB liquid medium; solution 1 (50mM glucose, 25mM Tris-HCl, 10mM EDTA, pH 8.0); solution 2 (0.2M NaOH, 1% SDS); solution 3 (5M KAc 60ml, HAc 11.5ml, H ₂ O 28.5ml); phenol:chloroform:isoamyl alcohol (25:24:1) mixture; RNase (20 μg/ml); absolute ethanol; 70% ethanol.

Operating procedures: Take 2ml of LB liquid medium containing antibiotics, add it to a 15ml well-ventilated test tube, then pick a single colony from the transformation plate, culture at 37°C, 300rpm overnight; take 1.5ml of culture, 4°C, 12000rpm , centrifuge for 0.5min, discard the supernatant, and make the pellet as dry as possible; add 100μl solution 1 to resuspend the bacteria, and shake vigorously; add 200μl solution 2, cover tightly, quickly invert and mix, and place on ice; add 150μl ice to pre-cool 3, cover tightly, mix by inverting, place on ice for 3-5min; centrifuge at 12000rpm, 4°C for 5min, transfer the supernatant; add an equal volume of phenol: chloroform: isoamyl alcohol mixture, shake and mix, 4 ℃, 12000rpm, centrifuge for 10min, transfer the supernatant; add 2 times the volume of ethanol to the supernatant, and place at room temperature for 2 minutes; 12000rpm, 4℃, centrifuge for 5min; discard the supernatant, precipitate; add 1ml 70% ethanol to wash, 4℃ , 12000rpm, centrifuge for 10min, and air-dry the pellet for 10min; add 50μl ddH ₂ O containing trypsinase to dissolve the DNA, and store it at -20°C until use.

2) Double digestion (XbaI-SacI)

10 μl of 10× double digestion buffer, 3 μl of XbaI (10U/μl), 3 μl of SacI (10U/μl), 10 μg of DNA, ddH ₂ O

Water bath at 37°C for 1 hour, remove endonuclease at 75°C; take 5 μl for identification by electrophoresis, extract and purify the rest with phenol:chloroform:isoamyl alcohol (25:24:1), add 1/10 volume of 3M NaAc, add 2 volumes Precipitate DNA with ethanol; let stand for 10 minutes, centrifuge at 12000rpm, 4°C for 10 minutes, wash the precipitate twice with 70% ethanol, dry for about 10 minutes, add appropriate amount of sterile water to dissolve, and connect.

3) Preparation of Competent Bacteria

Escherichia coli DH5α was taken out from the -70°C low-temperature refrigerator, inoculated on LB plates, and cultured overnight at 37°C for activation. Pick a single colony from the activation plate, insert it into 5 ml of LB liquid medium without antibiotics, and cultivate overnight at 37° C. with shaking (250 rpm). The next day, they were transferred to fresh LB liquid medium at a ratio of 1% (V/V), and cultured with shaking at 37°C until OD ₆₀₀ =0.3 (0.3-0.6 is also acceptable). Under sterile conditions, transfer 50-100ml of the culture solution into two pre-cooled sterile centrifuge tubes (Falcon 2070 tubes), and let stand on ice for 10 minutes. Centrifuge at 4000rpm for 10min at 4°C and discard the supernatant. Inverted to drain the culture medium. Add 10ml of pre-cooled 0.1M CaCl ₂ solution, resuspend the bacteria, and ice-bath for 30min. Centrifuge at 4000rpm for 10min at 4°C and discard the supernatant. Add 2ml of pre-cooled 0.1M CaCl ₂ solution to resuspend the bacteria, which are competent cells.

4) Primer design

The following primers were designed according to the published vgb gene sequence, and the expected fragment length was 451bp.

Forward: 5′-AAG GAT CCA TGT TAG ACC AGC AAA CC-3′

Reverse: 5′-ACAAGC TTATTCAAC CGC TTG AGC-3′

5) Ligation of plasmid and insert

1 μl of 10× ligation buffer, the DNA fragment obtained by double digestion and purification of the published vgb gene, the molar ratio of the insert fragment and the vector pBI 121 is about 2:1, T4 DNA ligase (5U/μl) 1 μl, ddH ₂ O, 16°C water bath overnight. The linking solution was used for transformation.

6) PCR reaction

Template 1 μl, primer 1 (5 μM) 2 μl, primer 2 (5 μM) 2 μl, 10× buffer 2 μl, MgCl ₂ (25 mM) 1.6 μl, dNTPs (10 mM) 0.5 μl, Taq (3U/μl) 0.3 μl, ddH ₂ O

PCR cycle conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; final extension at 72°C for 5 min. After the reaction was completed, 4 μl of the reaction product was loaded on 1.0% agarose gel electrophoresis.

6) Transformation and clone screening

Take 100 μl of competent cells, add 4 μl of the connection solution obtained in step 5), after 30 minutes in ice bath, heat shock at 42°C for 1.5 minutes, and cool on ice for 1-2 minutes; add 800 μl of SOC medium, and incubate at 37°C, 180rpm for 1 hr; 100 μl of the culture was spread on an LB plate containing 50 μg/ml kanamycin, cultivated overnight in a constant temperature incubator at 37°C, and E. coli carrying the intermediate vector pBI121-vgb was screened by PCR. In the figure M: 100bp DNA Marker; 1: Negative control/plasmid pBI 121; 2: Escherichia coli containing vgb gene expression vector pBI 121-vgb; 3: Agrobacterium rhizogenes LBA9402-vgb containing vgb gene

7) Three-parent hybridization method to transfer vgb gene into Agrobacterium rhizogenes

Inoculate Agrobacterium rhizogenes LBA9402 on YMB solid medium containing 100 μg/ml rifampicin, and culture at 28°C until a single colony grows. Pick a single colony and inoculate it in YMB liquid medium with 100 μg/ml rifampicin, at 28°C, 250 rpm, and cultivate for about 40 hours. Agrobacterium was inoculated in YMB liquid medium at an inoculation ratio of 1%, and cultivated at 28° C. and 250 rpm until the OD600 was 0.5. Inoculate the Escherichia coli pRK2013 containing the helper plasmid and the Escherichia coli carrying the intermediate vector pBI121-vgb on solid LB medium containing 50 μg/ml kanamycin, culture overnight at 37°C and 300 rpm, and when single colonies grow, pick them separately Bacteria were inoculated in liquid LB medium containing 50 μg/ml kanamycin, and cultured overnight at 37°C and 300 rpm. Inoculate the assisting bacteria and the bacteria containing the intermediate carrier in LB liquid medium at an inoculation ratio of 1%, and cultivate them at 37°C and 300 rpm until the OD600 is 0.5. After mixing the three bacteria in equal volumes, take 50 μl and inoculate them on sterile filter paper with a diameter of 2 cm, place them on YMB solid medium, and incubate at 25°C for one day. Rinse the filter paper with an appropriate amount of sterile water, collect the washed out bacterial liquid, and evenly spread it on the YMB solid medium containing 100 μg/ml rifampicin and 50 μg/ml kanamycin. After culturing at 25°C for about four days, single colony. Pick a single colony for PCR identification, inoculate positive colonies in YMB liquid medium containing 100 μg/ml rifampicin and 50 μg/ml kanamycin, cultivate overnight at 28°C, 250 rpm, and then conduct PCR identification on the plasmid DNA extracted from the bacterial liquid , the confirmed positive bacteria (LBA9402-vgb), the results of PCR identification are shown in Figure 1.

8) Transgenic vgb gene Agrobacterium rhizogenes LBA9402-vgb infects Astragalus aseptic seedlings to induce hairy roots

Inoculate Agrobacterium rhizogenes LBA9402-vgb on YMB solid medium containing 100 μg/ml rifampicin and 50 μg/ml kanamycin, and culture at 28°C until a single colony grows. Pick a single colony and inoculate it in YMB liquid medium with 100 μg/ml rifampicin and 50 μg/ml kanamycin at 28° C., 250 rpm, and cultivate for about 40 hours. Agrobacterium was inoculated in YMB liquid medium at an inoculation ratio of 1%, and cultivated overnight at 28° C. and 250 rpm. Add appropriate amount of acetosyringone to a final concentration of 50 μM, and culture for another day. Under aseptic conditions, the stems and leaves of aseptic Astragalus membranaceus seedlings were cut out, and the incisions were inoculated with Agrobacterium rhizogenes LBA9402-vgb bacteria solution (OD600=0.5), then transferred to MSOH solid medium, and cultured at 25°C in the dark for 2 sky. After the explants were washed 4-5 times with sterile water, they were transferred to solid MSOH medium containing 500 mg/L Claforan, and cultured at 25°C in the dark to induce hairy roots.

9) Extraction of hairy root DNA

Total DNA was extracted by CTAB method. Take 500-600mg of fresh material, grind it into a fine powder with liquid nitrogen, then quickly transfer it to a 1.5ml centrifuge tube; add 600μl 2% CTAB extract (preheated at 60°C), mix well, and place in a 60°C water bath 1hr, mixed gently several times in the middle; centrifuge at 12000rpm for 10min, take the supernatant, add an equal volume of water-saturated phenol: chloroform: isoamyl alcohol (25:24:1), mix well, centrifuge at 12000rpm at 4°C for 10min, and repeat 2 times until there is no white precipitate between the two liquids; take the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge at 12000rpm at 4°C for 10min; take the supernatant and add 1/10 NaAc and Equal volume of isopropanol, mix well, and place at -20°C for 1 hr; centrifuge at 5000 rpm at 4°C for 10 min, discard the supernatant, and rinse the precipitate twice with 70% ethanol; after the precipitate is naturally dried in air, dissolve it in 30 μl TE.

10) PCR identification of characteristic primers of vgb gene in hairy roots of Astragalus membranaceus

The genomic DNA of the transgenic Astragalus membranaceus hairy root was used as a template for PCR detection, and the PCR primers and conditions were the same as steps 4 and 6. The PCR results are shown in Figure 2, and it was preliminarily verified that the target gene vgb had been integrated into the hairy roots of Astragalus membranaceus transgenic with the vgb gene. In the figure: M: 100bp DNA Marker; 1: Negative control/Astragalus aseptic seedling; 2: Positive control/LBA9402-vgb; 3: Negative control/Non-transgenic Astragalus hairy root; 4: VHb10 transgenic Astragalus hairy root ;5: Astragalus hairy root transgenic line VHb36 with vgb gene; 6: Astragalus hairy root transgenic line VHb52 with vgb gene; 7: Astragalus hairy root transgenic line VHb74 with vgb gene

11) Total RNA extraction (one-step method)

Preparation of reaction reagents: denaturing solution (26mM sodium citrate pH 4.0, 0.5% sodium lauryl sarcosinate, 0.125M 2-mercaptoethanol, 4M guanidine isothiocyanate); NaAc (2M, pH 4.7); phenol : chloroform: isoamyl alcohol (25:24:1); 70% ethanol. All reagents are prepared with RNase-free water treated with 0.1% DEPC water, and all glass containers are RNase-free.

Operating procedures: Take an appropriate amount of fresh plant tissue and place it in a ceramic mortar, add liquid nitrogen and quickly grind it to a fine powder, transfer it to a 1.5ml centrifuge tube containing 650μl denaturing solution and mix well; add 65μl 2M NaAc (pH 4.0), and invert repeatedly Mix well; then add 650 μl phenol: chloroform: isoamyl alcohol, mix well, and ice-bath for 15 min; 4°C, 12000 rpm, centrifuge for 20 min; transfer the supernatant to a new 1.5 ml centrifuge tube, add an equal volume of isopropanol, mix After homogenization, place at -20°C for 30 minutes; centrifuge at 4°C, 12,000 rpm for 10 minutes to precipitate RNA; discard the supernatant, dissolve RNA in 0.5ml denaturing solution, and heat at 65°C to dissolve as soon as possible; add an equal volume of phenol: chloroform: isoamyl alcohol, Extract the mixed solution once again, that is, repeat the previous steps; after centrifugation, discard the supernatant, add 1ml of 75% pre-cooled ethanol, rinse the precipitate, centrifuge as above, discard the supernatant; after air drying, add 20μl deionized water to dissolve the RNA , spectrophotometry and gel electrophoresis to detect the concentration and purity, and the remaining part was stored at -20°C for later use.

12) reverse transcription cDNA

Oligo(dT) (37.5μM) 2μl, total RNA 2μg, ddH ₂ O 10.5μl, react at 70°C for 10min, put on ice immediately, 5×RT buffer 4μl, RNasin (40U/μl) 0.5μl, 10mM dNTPs 1μl, M -MLV reverse transcriptase (200U/μl) 1 μl, react at 42°C for 1.5h, and react at 70°C for 10min to inactivate the reverse transcriptase.

13) RT-PCR of the hairy root of Astragalus transgene vgb

Extraction of plant total RNA is carried out according to step 11, and reverse transcription is carried out according to step 12 for synthesizing the first strand of cDNA.

In the figure M: 1kb DNA Marker; 1: Negative control/Astragalus membranaceus sterile seedling; 2: Negative control/Non-transgenic Astragalus hairy root; 3: VHb36 transgenic Astragalus hairy root line; 4: Vgb transgenic Astragalus hairy root Root line VHb52; 5: Transgenic vgb gene Astragalus hairy root line VHb74; 6: Positive control/LBA9402-vgb

14) Determination of growth of hairy root of Astragalus membranaceus transgenic vgb gene

Inoculate non-transgenic Astragalus hairy roots and transgenic Astragalus hairy roots into liquid medium, collect materials on the 15th day, weigh fresh weight; freeze-dry, weigh dry weight, and calculate growth multiples. The results showed that the hairy root of astragalus transgenic with vgb gene grew faster than the hairy root of astragalus without transgene at the same period, and the dry weight increased by 13% to 25% compared with the hairy root of astragalus without transgene.

Table 1 Growth determination of hairy roots of Astragalus membranaceus transgenic with vgb gene and non-transgenic Astragalus membranaceus (n=3) sample Growth multiple dry weight(g) Non-transgenic Astragalus hairy root transmutation vgb gene Astragalus hairy root VHb36 to vgb gene Astragalus hairy root VHb52 to vgb gene Astragalus hairy root VHb74 11.8±0.52213.717±1.51016.044±3.56515.945±1.622 1.191±0.0651.428±0.0251.354±0.0651.492±0.020

15) Determination of the content of astragaloside IV by HPLC-ELSD

Sample extraction and preparation

The sample was pulverized and passed through an 80-mesh sieve. Weighed 3 parts of each sample, each 0.300g, added 10ml of 80% methanol, soaked overnight, ultrasonically extracted 3 times at 80°C for 30min each time, filtered with suction, and combined the filtrates. Evaporate the filtrate to dryness, add ddH ₂ O 5ml to dissolve the residue while hot, transfer to a separatory funnel, extract 3 times with 5ml of water-saturated n-butanol, combine the n-butanol solution, wash once with 0.15% NaOH, collect the n-butanol solution, Evaporate n-butanol to dryness, dissolve the residue with methanol and dilute to 2ml, shake well, and filter through a 0.45μm microporous membrane to obtain the test solution.

Chromatographic conditions

Chromatographic column: Inertsil ODS-3 (4.6*250mm, 5μm); mobile phase: acetonitrile-water (35:65); flow rate: 1.0ml/min; column temperature: room temperature; ELSD parameters: evaporation temperature: 100°C, nebulization Temperature: 80°C, outlet temperature: 60°C; carrier gas flow rate: 1.2ml/min.

Drawing of standard curve

Accurately weigh 2.31 mg of astragaloside IV reference substance, dissolve it in methanol and dilute to 2 ml, and filter through a 0.45 μm microporous membrane. Sequentially draw filtrate 1, 2, 5, 10, 15, 20 μl and analyze according to the above chromatographic conditions respectively, and measure the peak area. Take the logarithmic value of the reference substance content (μg) as the abscissa (X), and the logarithmic value of the peak area (A) of the reference substance as the ordinate (Y), draw a standard curve. And the regression equation, correlation coefficient and linear range are: Y=1.5856X+0.5769, r=0.9995, 1.155～23.100μg.

Precision experiment

Precisely draw 10 μl of astragaloside IV reference substance solution, and repeat the sample injection determination 5 times, the result is astragaloside IV RSD=1.114% (n=5).

Stability test

The same sample was injected at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours respectively, the content was calculated, and the intraday stability was determined. The result was RSD=0.79%.

Repeat experiment

Weigh 5 parts of the same sample, each 0.300g, extract and measure the content of astragaloside IV according to the method under item 3.2.3, the calculated average content of astragaloside IV is 17.352 mg/g, and the RSD is 2.30%.

Recovery experiment

Precisely weighed 5 samples of non-transgenic Astragalus hairy root samples with known astragaloside IV content, 0.300g each, and accurately added standard astragaloside IV, extracted and determined the content of astragaloside IV with the same method, and the average recovery rate was 96.72% , RSD=2.04%.

Determination of samples

Weigh Shanxi-produced commercial Astragalus membranaceus medicinal material, non-transgenic Astragalus hairy root, transgenic vgb gene Astragalus hairy root powder, 3 parts of each, 0.300 g per part, prepare the test solution in the same way, and determine the test solution under the same chromatographic conditions. The content of astragaloside IV in the liquid was calculated by the external standard method to calculate the peak area.

The measurement results

The results are shown in Table 2. The content of astragaloside IV in the hairy root of Astragalus transgenic vgb gene is generally very high. For example, the content of astragaloside IV in lines 36 and 52 is 20.674 mg/g and 20.999 mg/g, which is significantly higher than that of the non-transgenic Astragalus hairy root (astragaloside Ⅳ content 3.334mg/g) and Shanxi membranous viburnum herb (astragaloside Ⅳ content 1.774mg/g).

Table 2 Determination of astragaloside IV content in hairy roots of Astragalus transgenic vgb gene and non-transgenic Astragalus root (n=3) sample Astragaloside IV content (mg/g) Shanxi-produced commercial medicinal material Astragalus membranaceus non-transgenic Astragalus hairy root transformed with vgb gene Astragalus hairy root VHb36 transformed with vgb gene Astragalus hairy root VHb52 transformed with vgb gene Astragalus hairy root VHb74 1.774±0.0243.334±0.05020.674±0.39220.999±0.36917.115±0.171

Claims (1)

1, a kind of trans-exogenous gene engineering improves the method for Astragaloside content, it is characterized in that this method comprises the following steps:

1) extraction of plasmid pBI 121

The preparation of reaction reagent: LB liquid nutrient medium; Solution 1 is 50mM glucose, 25mM Tris-HCl, and the 10mM ethylenediamine tetraacetic acid (EDTA), pH 8.0; Solution 2 is 0.2M NaOH, 1% sodium lauryl sulphate; Solution 3 is 5M KAc 60ml, HAc 11.5ml, aseptic two ionized water 28.5ml that boil off; Phenol: chloroform: 25: 24: 1 mixed solutions of primary isoamyl alcohol; Rnase 20 μ g/ml; Dehydrated alcohol; 70% ethanol;

Schedule of operation: get 2ml and contain antibiotic LB liquid nutrient medium, add in the good test tube of the ventilation of 15ml, choose a single bacterium colony, 37 ℃, 300rpm, overnight incubation from transforming flat board then; Get the 1.5ml culture, 4 ℃, 12000rpm, centrifugal 0.5min removes supernatant liquor, and precipitation adds 100 μ l solution 1 bacterium that suspends again, concuss; Add 200 μ l solution 2, cover tight lid, put upside down mixing fast, put on ice; Add the solution 3 of 150 μ l ice precooling, cover tight lid, put upside down mixing, put 3～5min on ice; 12000rpm, 4 ℃, centrifugal 5min, supernatant shifts; Add isopyknic phenol: chloroform: the primary isoamyl alcohol mixed solution, the vibration mixing, 4 ℃, 12000rpm, centrifugal 10min shifts supernatant liquor; Add 2 times of volume ethanol in supernatant, room temperature was placed 2 minutes; 12000rpm, 4 ℃, centrifugal 5min; Abandon supernatant, precipitation; Add 1ml 70% washing with alcohol, 4 ℃, 12000rpm, centrifugal 10min, precipitation dry air 10min; The aseptic two ionized waters dissolving thymus nucleic acids that boil off that add 50 μ l qiagen rnase enzymes, be stored in-20 ℃ stand-by;

2) double digestion:

10 * double digestion damping fluid, 10 μ l, restriction endonuclease XbaI 10U/ μ l 3 μ l, restriction endonuclease SacI 10U/ μ l 3 μ l, thymus nucleic acid 10 μ g, aseptic two ionized waters that boil off

37 ℃ of water-bath 1hr take out 75 ℃ of deactivation restriction endonucleases; Get 5 μ l electrophoresis and identify, rest part phenol: chloroform: behind 25: 24: 1 abstraction purifications of primary isoamyl alcohol, add the 3M NaAc of 1/10 volume, 2 times of volume of ethanol bulk deoxidation Yeast Nucleic Acid; After leaving standstill 10min, 12000rpm, 4 ℃, centrifugal 10min, precipitation is dried about 10min with after twice of 70% washing with alcohol, adds an amount of sterilized water and dissolves, and connects;

3) preparation of competence bacterium:

Take out bacillus coli DH 5 alpha from-70 ℃ of cryogenic refrigerators, be inoculated in the LB flat board, 37 ℃ of overnight incubation activate, from activating picking list bacterium colony on the plate, inserting 5ml does not contain in the antibiotic LB liquid nutrient medium, 37 ℃ of jolting overnight incubation 250rpm, change in fresh LB liquid nutrient medium by 1% volume ratio next day, and 37 ℃ of joltings are cultured to OD600=0.3～0.6, under aseptic condition, 50～100ml nutrient solution is changed in the aseptic centrifuge tube of two precoolings, leave standstill 10min on ice, 4 ℃, the centrifugal 10min of 4000rpm, abandoning supernatant is inverted and is flow to end nutrient solution, adds the 0.1M CaCl of 10ml precooling ₂Solution, the resuspension bacterium, ice bath 30min, 4 ℃, the centrifugal 10min of 4000rpm, abandoning supernatant adds the 0.1M CaCl of 2ml precooling ₂Solution, the resuspension bacterium is competent cell;

4) design of primers:

According to Vitreoscilla hemoglobin gene sequences Design primer, estimate fragment length 451bp;

Forward: 5 '-AAG GAT CCA TGT TAG ACC AGC AAA CC-3 '

Oppositely: 5 '-ACA AGC TTA TTC AAC CGC TTG AGC-3 '

5) plasmid and segmental connection of insertion

10 * connection damping fluid, 1 μ l, the thymus nucleic acid segment that Vitreoscilla hemoglobin gene obtains through the double digestion purifying, the molar ratio that inserts fragment and carrier pBI 121 is 2: 1, T4 deoxyribonucleic acid ligase 5U/ μ l 1 μ l, aseptic two ionized water that boils off, 16 ℃ of water-baths are spent the night, and connect liquid and are used for transforming;

6) polymerase chain reaction

Template 1 μ l, forward primer 5 μ M 2 μ l, reverse primer 5 μ M 2 μ l, 10 * damping fluid, 2 μ l, MgCl ₂25mM 1.6 μ l, picodna ribonucleoside triphosphote 10mM 0.5 μ l, deoxyribonucleic acid polymerase 3U/ μ l 0.3 μ l, aseptic two ionized waters that boil off;

Polymerase chain reaction cycling condition: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 30 circulations; Last 72 ℃ are extended 5min, after reaction finishes, get 4 μ l reaction product and are splined on 1.0% agarose gel electrophoresis;

7) conversion and colony screening:

Get 100 μ l competent cells, add the connection liquid that 4 μ l step 5) obtain, behind the ice bath 30min, 42 ℃ of heat shock 1.5min, cooled on ice 1～2min; Add SOC substratum 800 μ l, in 37 ℃, 180rpm cultivates 1hr; Get culture 100 μ l and be laid on the LB flat board that contains 50 μ g/ml kantlex, 37 ℃ of constant incubator incubated overnight, the intestinal bacteria that intermediate carrier pBI121-vgb is carried in the polymerase chain reaction method screening;

8) triparental mating changes Vitreoscilla hemoglobin gene over to Agrobacterium rhizogenes:

Inoculation Agrobacterium rhizogenes LBA9402 is on the YMB solid medium that contains 100 μ g/ml Rifampins, 28 ℃ are cultured to single bacterium colony and grow, choose single colony inoculation in the YMB liquid nutrient medium that adds 100 μ g/ml Rifampins, 28 ℃, 250rpm cultivates 40hr, inoculative proportion by 1% is inoculated in Agrobacterium in the YMB liquid nutrient medium, 28 ℃, it is 0.5 that 250rpm is cultured to OD600, inoculation contains the intestinal bacteria pRK2013 that assists plasmid and the intestinal bacteria of carrying intermediate carrier pBI121-vgb in containing on the 50 μ g/ml kantlex solid LB substratum, 37 ℃, the 300rpm overnight incubation, after single bacterium colony grows, choosing single bacterium respectively is inoculated in and contains in the 50 μ g/ml kantlex liquid LB substratum, 37 ℃, the 300rpm overnight incubation, inoculative proportion by 1% will be assisted bacterium and be contained the microbionation of intermediate carrier in the LB liquid nutrient medium, 37 ℃, it is 0.5 that 300rpm is cultured to OD600, behind three kinds of bacterium equal-volume mixings, get on the aseptic filter paper that 50 μ l are inoculated in diameter 2cm and place on the YMB solid medium, cultivated one day for 25 ℃, with an amount of aseptic water washing filter paper, the bacterium liquid that collection washes out, evenly coat and contain 100 μ g/ml Rifampins, on the YMB solid medium of 50 μ g/ml kantlex, cultivate for 25 ℃ and occur single bacterium colony after four days, choose single bacterium colony and carry out the polymerase chain reaction evaluation, positive bacterium colony is inoculated in and contains 100 μ g/ml Rifampins, the YMB liquid nutrient medium of 50 μ g/ml kantlex, 28 ℃, 250rpm, overnight incubation is extracted plasmid deoxyribonucleic acid to bacterium liquid again and is carried out polymerase chain reaction evaluation, the positive bacteria LBA9402-vgb that determines;

9) changeing Vitreoscilla hemoglobin gene Agrobacterium rhizogenes LBA9402-vgb infects Radix Astragali aseptic seedling and induces hairly root

Inoculation Agrobacterium rhizogenes LBA9402-vgb is on the YMB solid medium that contains 100 μ g/ml Rifampins and 50 μ g/ml kantlex, 28 ℃ are cultured to single bacterium colony and grow, choose single colony inoculation in the YMB liquid nutrient medium that adds 100 μ g/ml Rifampins and 50 μ g/ml kantlex, 28 ℃, 250rpm, cultivate 40hr, inoculative proportion by 1% is inoculated in Agrobacterium in the YMB liquid nutrient medium, 28 ℃, the 250rpm overnight incubation, add an amount of Syringylethanone to final concentration 50 μ M, cultivated again one day, and under the aseptic condition, cut the stem and the blade of Radix Astragali aseptic seedling, at incision inoculation Agrobacterium rhizogenes LBA9402-vgb bacterium liquid OD600=0.5, be transferred to the MSOH solid medium then, 25 ℃, dark condition was cultivated 2 days down, behind the sterilized water washing explant 4～5 times, be transferred to the solid MSOH substratum that contains the 500mg/L Cefotaxime, 25 ℃, dark condition is cultivated down and is induced the generation hairly root.

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