CN118028354A - Genetic transformation method for hairy roots of cynanchum glaucescens and application of genetic transformation method - Google Patents

Abstract

The invention provides a genetic transformation method of hairy roots of cynanchum glaucescens and application thereof. Specifically, the method comprises the steps of placing the cynanchum atratum explant in an aggressive solution containing agrobacterium rhizogenes, and carrying out ultrasonic and vibration culture to promote the cynanchum atratum to generate a large number of hairy roots so as to realize rapid propagation; and the Ri plasmid of agrobacterium rhizogenes is utilized to transfer the exogenous gene into the genome of the cynanchum wilfordii, so that the exogenous gene is introduced, and further, the target product, such as secondary metabolite alkaloid, is expressed in the cynanchum wilfordii, and the target product can be produced in a large quantity and rapidly by taking the hairy root as a bioreactor.

Description

Genetic transformation method for hairy roots of cynanchum glaucescens and application of genetic transformation method

Technical Field

The invention belongs to the field of plant genetic transformation, and particularly relates to a method for genetic transformation of hairy roots of cynanchum glaucescens and application thereof.

Background

The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Cynanchum willow Cynanchum stauntonii (Decne.) Schltr. ExLevl. Belongs to the family of Asclepiadaceae medicinal plants. Is mainly distributed in the provinces of Gansu, anhui, jiangsu, zhejiang, hunan, jiangxi, fujian, guangdong, guangxi and Guizhou. A valley wetland, a beside water or in water growing at a low altitude. The whole plant is used for medicine, and has the effects of clearing heat and detoxicating, and reducing qi and phlegm; it is used for radically treating pulmonary diseases, infantile malnutrition, common cold, cough, chronic bronchitis, etc. The existing common medicinal parts are the roots of the rhizoma cynanchi bungei which are sun-dried, and the roots of the rhizoma cynanchi bungei and the medicinal materials such as ginseng contain rich saponin components. In theory, the biosynthesis way of the cynanchum salicifolium saponin is modified by utilizing genetic engineering, metabolic engineering and synthetic biology means, so that the synthesis efficiency of the cynanchum salicifolium saponin can be improved, and the production cost can be saved. However, the current technology is limited by the development of genetic transformation technology, and the transformation of willow leaves by means of genetic engineering and the like is not smooth.

The establishment of hairy root transformation system is achieved by means of a gram-negative bacterium of the genus Agrobacterium of the family Rhizobiaceae, agrobacterium rhizogenes (Agrobacterium rhizogenes). When agrobacterium rhizogenes infects plants, the T-DNA fragment on Ri plasmid is inserted into the genome of the plants, and after the root system of the plants is infested by the agrobacterium rhizogenes, adventitious roots with multiple branches, called hairy roots, are formed at wounds. Hairy roots are also called hairy roots, have the characteristics of multiple branches, losing geotropism and the like, and also have the advantages of hormone autonomy, short growth cycle, stable inheritance, strong accumulation capacity of secondary metabolic substances and the like. Therefore, the hairy root genetic transformation system is an important technical means for researching gene functions and constructing efficient directional biosynthesis pharmacodynamic monomers by utilizing synthetic biology, and is particularly suitable for Chinese medicinal materials taking roots as medicines.

Some traditional Chinese herbal medicine genetic transformation systems established by agrobacterium rhizogenes, such as salvia miltiorrhiza, dandelion, polygala tenuifolia, gynostemma pentaphylla, morinda officinalis and the like, exist in the prior art, but genetic transformation of plants is influenced by genotype, genetic diversity and plant specificity, so far, a universal hairy root genetic transformation method does not exist, and the method is especially aimed at the cynanchum otophyllum. At present, the culture period of the cynanchum wilfordii is long, and how to establish a high-efficiency and stable hairy root genetic transformation system is not clear, which influences the research, development and utilization of the cynanchum wilfordii.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a genetic transformation method of cynanchum bungei, which adopts agrobacterium rhizogenes and proper explants to promote the cynanchum bungei to generate a large number of hairy roots so as to realize rapid propagation; and the Ri plasmid of agrobacterium rhizogenes is utilized to transfer the exogenous gene into the genome of the cynanchum wilfordii, so that the exogenous gene is introduced, and further, the target product, such as secondary metabolite alkaloid, is expressed in the cynanchum wilfordii, and the target product can be produced in a large quantity and rapidly by taking the hairy root as a bioreactor.

To achieve the above object, one or more embodiments of the present invention provide the following technical solutions:

In a first aspect of the present invention, there is provided a method for genetic transformation of hairy roots of Cynanchum komarovii, comprising the steps of:

Placing the explant in an invaded dye solution containing agrobacterium rhizogenes, and performing ultrasonic and shake culture; placing the explant obtained in the previous step in a plant culture medium for co-culture and induction transformation; obtaining hairy roots of cynanchum glaucescens through screening culture and amplification culture;

Wherein, the ultrasonic conditions are: 30-50HZ and 20-40s.

The hairy roots of cynanchum glaucescens obtained in the invention can be cultivated in a solid or liquid culture medium in an enlarged way, so that complete plants are obtained, or the obtained hairy roots can be directly placed in nutrient soil for propagation.

In a specific embodiment of the invention, the explant is produced from a young stem segment, adventitious bud, leaf, petiole or hypocotyl;

Preferably tender stem segments;

More preferably, the method for obtaining the explant comprises the following steps: culturing young stem segments of rhizoma Cynanchi Stauntonii for 15-20d, placing on MS culture medium, dark culturing at 25-30deg.C until axillary buds of stem nodes sprout and extend, cutting off aseptic buds, and continuously culturing in MS culture medium for 20-28h to obtain explant.

In a specific embodiment of the present invention, the white front comprises willow leaf white front or lilac daphne flower bud white front.

In specific embodiments of the invention, the agrobacterium rhizogenes comprises K599, C58C1, MSU440, ar.a4, or ATCC 15834;

Preferably ar.a4 or ATCC 15834;

or, the agrobacterium rhizogenes comprises a target plasmid.

In a specific embodiment of the invention, the agrobacteria in the infesting solution are od600=0.4-0.8.

In a specific embodiment of the present invention, the plasmid of interest comprises a functional gene; the functional genes comprise genes with medicinal value, disease resistance genes, insect resistance genes or marker genes;

Further, the genes of medicinal value include carotene synthesis genes, anthocyanin synthesis genes and antibacterial peptide synthesis genes; the disease resistance genes comprise PcMYB, poMYB, moMYB1; the insect-resistant genes include NbMYB, nbMYB, 107, nbMYB, 163, and NbMYB, 423; the saponin related biosynthesis genes include CYP51G, CYP94D108, UGT73CR1, UGT80A40 and UGT80A41; the marker genes comprise RUBY system, chloramphenicol acetyl transferase gene cat, luciferase gene luc, beta-glucuronidase gene gus, secretory alkaline phosphatase gene seap and green fluorescent protein gene gfp.

In the present invention, the medicinal value refers to secondary metabolites such as secondary alkaloids and biological terpenes; wherein the alkaloid comprises indole alkaloid, benzyl isoquinoline alkaloid, and tropane alkaloid, such as antitumor vinblastine, camptothecine, and anti-heart dysfunction amarin, papaverine, anti-berberine, anisodamine, scopolamine, and scopolamine; ginsenoside, taxol, artemisinin, tanshinone, etc. as medicinal terpenoid.

In a specific embodiment of the invention, the plasmid of interest is 35 S:RUBY (He et al Hortic Res.2020Sep 19;7:152.Doi:10.1038/S41438-020-00390-1.eCollection 2020), i.e., the gene of interest is RUBY. The plasmid map is shown in FIG. 11, addgene number 160908.

Betalains are natural plant products which can be synthesized by using tyrosine as a substrate through catalysis of three enzymes (CYP 76AD1, DODA and GT). The bright red color seen in beets, dragon fruits and other plants is the result of betalain accumulation. RUBY is a novel reporter system utilizing the "2A" peptide to fuse three key genes CYP76AD1, DODA and glucosyltransferase for betalain biosynthesis. In a specific embodiment of the present invention, the conditions of the mixing are: the oscillating speed is 80-100 r/min, the oscillating time is 10-30 min, and the temperature is 25-30 ℃.

In a specific embodiment of the present invention, the co-cultivation method specifically comprises: removing surface moisture of the explant and excessive agrobacterium rhizogenes bacterial liquid by using sterile filter paper, clamping the explant onto a plant culture medium by using sterilized forceps, and placing the explant in a dark incubator at 22-27 ℃ for co-culture for 3-7d;

Preferably, the culture conditions are dark culture at 25℃for 3-5d.

In a specific embodiment of the invention, the plant culture medium is a 1/2MS solid culture or liquid culture medium.

In a specific embodiment of the invention, the conditions of the screening culture are: transferring the explants after co-culture to a screening medium, and carrying out photoperiod culture for 15-18d;

wherein the photoperiod is 16h of light culture and 8h of dark culture;

The screening culture medium is 1/2MS solid culture medium, 380-420 mug/mL cefotaxime sodium is added; preferably 400. Mu.g/mL.

In a second aspect of the invention there is provided the use of the method of the first aspect for large scale cultivation of cynanchum bungei or use of cynanchum bungei as a bioreactor for the production of a drug or secondary metabolite;

More specifically, the application includes any one or more of the following:

(1) Extracting and preserving medicinal components of hairy roots of rhizoma Cynanchi Stauntonii;

(2) Extracting material of rhizoma Cynanchi Stauntonii saponin component;

(3) A genetic transformation gene of cynanchum glaucescens;

(4) Improvement of the transgenic germplasm of the cynanchum glaucescens;

(5) And (5) performing enlarged culture of the cynanchum atratum.

The one or more of the above technical solutions have the following beneficial effects:

the invention establishes a genetic transformation method of hairy roots of cynanchum glaucescens by means of agrobacterium rhizogenes. The method provided by the invention makes up the blank of the genetic transformation of the cynanchum glaucescens, overcomes the defects of complex conditions and low genetic transformation rate compared with the traditional tissue culture mode, and simultaneously avoids transformation difference of tissue culture caused by the technical level of operators.

The method provided by the invention has the advantages of convenient material taking, simple operation, great shortening of the culture time of the cynanchum wilfordii, and sufficient hairy roots can be obtained after 8 weeks; and the transformation efficiency is high, when the explant is a tender stem segment, the generation rate of hairy roots of Ar.A4 agrobacterium is about 90%, and the positive rate is 56.33%.

According to the invention, the young stem section subjected to dark culture is found as an explant for the first time, so that the genetic transformation efficiency of the cynanchum glaucescens is remarkably improved.

The established genetic transformation system of the hairy root of the willow leaf and the white front has high positive expression after transformation, and the content of the betalains detected by the genetic transformation system is up to 12.67mg/g.

Additional aspects of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 shows hairy roots at various time points after Ar.A4 infection of young stem segment explants in example one of the present invention.

FIG. 2 shows hairy root morphology of the Cynanchum candidum after 20d infestation according to the present invention.

FIG. 3 shows genetic transformation efficiencies of different Agrobacterium rhizogenes in accordance with the first embodiment of the present invention.

FIG. 4 shows a liquid suspension culture of hairy roots of Cynanchum komarovii in a second embodiment of the present invention.

FIG. 5 shows the effect of light-protected co-cultivation time on induction after infection according to experimental example II of the present invention.

FIG. 6 is a diagram showing the phenotype of the hairy root of Cynanchum candidum transformed with Ar.A4-RUBY according to the application example of the present invention.

FIG. 7 shows a RUBY hairy root subculture phenotype according to an embodiment of the present invention.

FIG. 8 shows a hairy root PCR identification band according to an embodiment of the present invention.

FIG. 9 shows comparison of the extraction of different transformed hairy root betalains in application example two of the present invention.

FIG. 10 shows the comparison of the amounts of the different transformed hairy root betalains in application example two of the present invention.

FIG. 11 is a map of plasmid 35S:: RUBY of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention.

The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.

1. Preparing a culture medium:

MS medium: commercially available MS medium was adjusted to 4.74g/L with water and 30g sucrose per liter to adjust ph=5.8. 7.0g agar per liter of solid medium was added.

1/2MS medium: the commercially available 1/2MS medium was adjusted to 2.47g/L with water and 30g sucrose per liter to adjust pH=5.8. 7.0g agar per liter of solid medium was added.

MGL medium: 5g of commercially available tryptone, 5g of sodium chloride, 0.1g of magnesium sulfate heptahydrate, 0.25g of potassium dihydrogen phosphate, 5g of mannitol, 1.0g of glycine and pH=5.8 are added per liter of water.

TY medium: commercially available solutions of 5 g/liter tryptone, 3 g/liter yeast extract, and sterile 1M aqueous calcium chloride solution were added. If the solid culture medium is prepared, 15g of agar powder is added per liter.

(4) Major reagents and hormones:

Mother liquor: kanamycin (KM) is 100mg/mL, L cefotaxime sodium (solvent is dimethyl sulfoxide) 400 mug/mL, spectinomycin is 100mg/mL, hygromycin is 100mg/mL. Working solution: kanamycin (KM) is 50 mug/mL, L cefotaxime sodium (solvent is dimethyl sulfoxide) is 200-500mg/L, spectinomycin is 50 mug/mL, hygromycin is 50 mug/mL.

Note that: hormone without solvent is prepared by filtering with 0.22 μm sterile filter membrane, and packaging at-20deg.C. And after the solvent is hormone configuration of dimethyl sulfoxide, film coating is not needed.

2. Material

K599 is purchased from Shanghai Biotechnology Co., ltd. Cat#: AC1080ATCC15834 is purchased from Shanghai Biotechnology Co., ltd. Cat#: AE1100;

MSU440 is purchased from Shanghai Biotechnology Inc., cat#: AC1070;

Ar.A4 is available from Shanghai Biotechnology Inc., cat#: AC1050;

C58C1 was purchased from Shanghai Biotechnology Co., ltd., cat# AC1110

35S: : the RUBY plasmid was supplied by the company Zhang Xianlong, university of agriculture.

In the present invention, the objective plasmid means a plasmid capable of expressing a functional gene. If exogenous gene R2R3-MYB is inserted into the plant expression vector to realize anthocyanin production, the target plasmid is 35S:: RUBY in the embodiment of the invention.

Example 1

The embodiment discloses a method for genetic transformation of hairy roots of cynanchum glaucescens and application, and the method comprises the following specific steps:

1. Obtaining of Cynanchum Komarovii explants

Tissue cultured stem segments from 15 days of willow leaf are harvested. The stem segments of the willow leaves which are picked back are cleaned, and the leaves are removed. Then placing the materials into an ultra-clean workbench for operation, firstly soaking the stem with 75% alcohol, then soaking and sterilizing with 2% sodium hypochlorite for 15-20min for sterilization, and finally cleaning with sterile water to finish the sterilization stage. In an ultra-clean workbench, placing the sterilized material on sterilized sterile filter paper, absorbing water, and cutting the stem segments into 2cm stem segments by using a scalpel after high-temperature sterilization. Finally, inoculating on MS culture medium, culturing at 28deg.C, and culturing in 16h light/8 h dark until aseptic axillary bud grows. And cutting off the sterile buds, and continuously culturing in an MS culture medium for 20-28h to obtain the explants.

2. Preparation of infection liquid of agrobacterium rhizogenes

The Agrobacterium rhizogenes preserved at-80 ℃ is taken out and melted before transformation, bacterial liquid is dipped by a sterile inoculating loop, and the plane streaking is carried out on TY culture medium containing 50mg/L kanamycin. The inverted plate was then grown in a constant temperature incubator at 28℃for 48h until the strain recovered to form a single colony.

And (3) on an ultra-clean workbench, picking a single colony to 1mL of TY culture medium by using an inoculating loop, placing the single colony into a 1.5mL centrifuge tube, and culturing the single colony in a shaking table at 28 ℃ for 48 hours, wherein the OD600 of the bacterial liquid is about 0.6. Centrifuging at 8000rpm for 10min, removing supernatant, re-suspending the bacterial liquid with sterile water, centrifuging again, and repeating the washing of the residual culture medium twice. Re-suspending the bacterial block with MGL culture medium in the same volume to ensure the bacterial liquid OD600 of about 0.6.

The method is used for preparing the invader liquor of agrobacterium rhizogenes K599, C58C1, MSU440, ar.A4 and ATCC 15834. Note that 50mg/L kanamycin was required in the TY medium of Ar.A4.

3. Agrobacterium rhizogenes infected explant

Immersing the explant in the activated Agrobacterium rhizogenes leaching solution, carrying out ultrasonic treatment at 40HZ for 30s, and slowly shaking for 10-20 min at 100rpm on a shaking table at 28 ℃.

4. Co-cultivation

The surface moisture and excess agrobacterium rhizogenes bacteria solution of the explant after the dip-dyeing in the step 3 are removed by sterile filter paper, and the explant is clamped onto a co-culture medium (1/2 MS solid medium+filter paper) by using sterilized forceps. In order to simulate the environment in the soil and improve the infection efficiency of agrobacterium, the explants are placed in a dark incubator at 25 ℃ and are subjected to dark culture for 3d.

5. Screening culture

To inhibit Agrobacterium rhizogenes, induced hairy roots were obtained, after co-culturing the above steps for 3 days, the explants were transferred to a screening medium (1/2 MS solid medium +400. Mu.g/mL cefotaxime sodium), placed in a 28℃incubator for 15-18d according to a cycle of 16h light and 8h dark culture, and replaced with a new solid medium containing cefotaxime sodium every 15 days.

6. Expansion culture

After screening for 15-18 days we can see curled roots at the wound site of a few explants; screening medium (1/2 MS solid medium+400. Mu.g/mL cefotaxime sodium) was continued at 25℃for 16 hours light culture and 8 hours dark culture, after 5-6 weeks of culture, positive root screening was performed by PCR. Note that, every 2 weeks of proliferation culture, fig. 1 demonstrates that the method of the present invention can effectively induce hairy roots of cynanchum glaucescens, and that ar.a4 can effectively induce hairy root production after ar.a4 transformation at different time points, and that hairy roots are more and more with the increase of culture time, as shown in fig. 1.

7. Morphological identification

As shown in FIG. 2, axillary bud stem segments begin to form thicker roots about 20d after infection, gradually elongating into bifurcations. The branches are more and dense, and the root growth speed is high. The roots are mostly generated from the cut stem wound, young leaves are unfolded at the top ends of the axillary buds, a plurality of roots are generated near the vein wound after the leaves are infected, and no wound is generated at the wound.

8. Calculating the induction rate of hairy roots

Hairy root induction rate = (number of explants per total number of explants healed) ×100%

The hairy roots of the willow leaves are induced by shake co-culture, and the hairy roots are induced under the induction of five purchased agrobacterium rhizogenes, but the induction efficiency is completely different, so that data analysis is carried out on different agrobacterium rhizogenes. In general, the willow leaf white stem can be induced to form hairy roots by different strains (K599, C58C1, MSU440, ar.a4, ATCC 15834). The results in FIG. 3 show that different Agrobacterium rhizogenes induced in the stems at rates of 12.5.+ -. 1.5%, 26.7.+ -. 2.1%, 26.7.+ -. 2.8%, 88.5.+ -. 3.5% and 45.0.+ -. 3.5% for young stem segments of P.candidum, respectively, indicate that the above five Agrobacterium rhizogenes can induce hairy root production in P.candidum, ar.A4 is more suitable for inducing hairy root production in P.candidum than several other common Agrobacterium rhizogenes.

Example two liquid suspension culture expansion propagation

After subculturing the hairy roots of the plate culture in the first embodiment, selecting the monoclonal hairy roots for liquid suspension culture propagation. Firstly, cutting the robust hairy roots in an ultra-clean workbench, putting the cut hairy roots into a 1/2MS liquid culture medium, and performing degerming by shaking the liquid culture medium at 37 ℃ for 48 hours without adding antibiotics.

After the degerming is completed, the hairy roots are transferred into a new 1/2MS liquid culture medium and placed in a constant temperature shaking table, and the temperature is kept at 25 ℃, and the culture is carried out for 140r,16 hours and 8 hours in a dark way. A large number of proliferating hairy roots were obtained after 12 days as shown in FIG. 4 by inoculating 0.5g hairy roots in 50mL of the medium.

Experimental example one explant selection

The selection of the explant is critical to the induction rate of hairy roots and the transformation positive rate of exogenous genes, in some species, the root, the stem and the leaf cultivated under the same condition are used as the explant to show great difference in inducing callus, and the related research also shows that the three are used as the explant to show difference in hairy root induction, so that the invention adopts agrobacterium rhizogenes Ar.A4 to respectively infect the stem, the leaf and the root which germinate and the aseptic root, the stem and the leaf explant cultivated for about 60 days. The specific method is shown in the first embodiment, and the induction efficiency and the germination time are counted.

The results are shown in Table 1, table 1 shows that 15 days of culture the highest stem induction efficiency, up to 82.5%, and the leaf induction efficiency 79.4% was next to that of the stem without significant difference, but leaves were too little material at 20d culture stage. The stem has more early growth and is more suitable for being used as an external body induction material.

Table 1: induction efficiency of hairy root by induction of different explants and different culture time

Experimental example two Co-cultivation time determination

The agrobacteria resistance of each plant is different, so that the induction effect of the agrobacterium infection time on hairy roots of the medicinal plants is different. If the infection time is long, the concentration of the agrobacterium is too high, the propagation speed of the strain is increased, the later sterilization is difficult, and the death of the explant is easy to cause, so that the induction rate is reduced; the infection time is too short, the agrobacterium cannot be effectively attached to the wound part of the explant, the T-DNA of the agrobacterium Ri plasmid cannot be effectively transferred and integrated into the genome of the explant, and hairy roots cannot be effectively induced, so that the induction rate of the hairy roots is greatly reduced. In general, the genetic transformation rate is high for a co-cultivation time of 2-3d, and if the co-cultivation time is prolonged, the transformation efficiency is rather drastically reduced. The reason is that the growth rate of agrobacterium is far greater than that of plants, and agrobacterium encapsulates plant explants, resulting in death of the explants.

Removing surface moisture and excessive agrobacterium rhizogenes bacterial liquid of the impregnated explant by using sterile filter paper, clamping the explant to a co-culture medium (1/2 MS solid culture medium plus filter paper) by using sterilized forceps, placing the co-culture medium in a dark incubator at 25 ℃, setting the co-culture time to 1, 3, 5 and 7 days, and counting the induction efficiency and germination time.

As shown in FIG. 5, the results indicate that the induction efficiency of the cultures 3d and 5d is highest. The 7d bacterial liquid wraps the explant, so that the bacteria is easy to pollute and dye, the induction efficiency is reduced, and the induction effect is poor due to the too short 1d culture time.

Experimental example three suspension culture degerming condition investigation

Hairy roots are excised from various explants and cultured ex vivo, and the hairy roots have hormone autonomy, so that the ex vivo roots can grow rapidly in a culture medium without exogenous hormone. After the agrobacterium infects plants, part of the plasmid integrates hairy root DNA and plant nuclear DNA, but most of bacteria remain in plant cell gaps, and the growth speed of the bacteria is far greater than that of plant bodies. Thus, the isolated hairy roots are sterilized. The hairy eradication bacteria is a sterilization process and also a process for screening excellent strains. Agrobacterium rhizogenes is a gram-negative bacterium, and the most suitable growth temperature is 28 ℃ and the bacterial death can be caused by culturing for more than 12 hours at the temperature of more than 35 ℃. Hairy roots may be cultured at 19-32deg.C. Therefore, the temperature can be increased to rapidly degerming.

And (3) after subculturing the plate-cultured hairy roots, selecting the monoclonal hairy roots for liquid suspension culture propagation. Firstly, cutting the robust hairy roots in an ultra-clean workbench, and putting the cut hairy roots into a 1/2MS liquid culture medium, wherein the liquid culture medium is not added with antibiotics. Placing hairy roots into a constant temperature shaking table, adjusting the temperature to 35, 37 and 39 ℃ and waiting for about 24, 48 and 72 hours until degerming is completed. And (5) counting the growth rate and death rate of the explants.

The results in Table 2 show that the treatment at 37 ℃ for 48 hours is optimal, the sterilization effect is good, the sterilization effect at 35 ℃ is not strong, agrobacterium rhizogenes still exists in the later culture, the material is necrotic after the treatment at 39 ℃, and the subsequent culture cannot be carried out.

Table 2: comparison of degerming effects at different temperatures and treatment times

Application example 1

In order to verify whether the exogenous gene can be expressed in the white front or not by the method, a basis is provided for the subsequent expression of the gene related to the secondary metabolite in the white front and the acquisition of the secondary metabolite, the RUBY system is adopted as an example for transformation, whether the transformation is successful or not can be judged by whether the rooting is red or not, and further the PCR identification is adopted.

The plasmid of the gene of interest was selected for transformation into Agrobacterium rhizogenes Ar.A4. The gene in this application example is a plasmid of betalain synthesis related gene RUBY.

1. Strain preparation

Firstly, taking Ar.A4 Agrobacterium rhizogenes competence stored at the temperature of minus 80 ℃ and immediately inserting the Ar.A4 Agrobacterium rhizogenes competence into ice when part of the Agrobacterium rhizogenes competence is melted at room temperature or palm, and the Agrobacterium rhizogenes competence is in an ice-water mixture state. About 0.3-1 mug of plasmid DNA is added into each 100 mu L of competent cell, the competent cell bottom is rapidly and vigorously dialed by hand or repeatedly blown by a pipette, and the mixture is sequentially kept stand for 5 minutes, liquid nitrogen for 5 minutes, water bath at 37 ℃ for 5 minutes and ice bath for 5 minutes. After ice bath, the mixture was taken out and left at room temperature, 700. Mu.L of TY liquid medium was added, and the mixture was shake-cultured at 28℃and 150r for 2 hours on a shaker. The bacterial cells were collected by centrifugation at 6000r for 1 min, and about 100. Mu.L of the supernatant was gently blown against the resuspended pellet, which was spread evenly on a TY solid plate containing spectinomycin, and incubated at 28℃for 2-3 days.

Identification of the transformed bacterial liquid: in an ultra clean bench, individual colonies were picked from plates where monoclonal colonies had appeared, and the single colony picked up by gently aligning the viscous thalli with a sterilized sterile gun head was placed in a 1.5mL sterile centrifuge tube loaded with TY liquid medium containing spectinomycin. Each sterile centrifuge tube corresponds to each monoclonal colony and is labeled. And then placing the strain into a shaking table at 28 ℃ for shake culture for 1-2 days under 150r condition, and carrying out PCR identification after the strain is turbid. The primers are shown in Table 3, the PCR systems and the procedures are shown in tables 4 and 5, respectively.

PCR products were electrophoretically detected in 1% agarose gel, 140V,20 min. The colonies verified as positive preserve the bacterial liquid.

TABLE 3RUBY tandem Gene identification PCR

TABLE 4Taq enzyme PCR identification target gene system

TABLE 5Taq enzyme PCR amplification cycle reaction

TABLE 6 primers for hairy root PCR identification

2. Infection and cultivation

The obtained bacterial liquid is split-packed in a sterile 1.5mL centrifuge tube for centrifugation at 8000rpm for 10min in an ultra-clean workbench, the supernatant is removed, the bacterial liquid is re-suspended by sterile water for re-centrifugation, and the residual culture medium is repeatedly washed twice. Re-suspending the bacterial block by using an equal volume of MGL culture medium to ensure that the OD600 of bacterial liquid is about 0.6.

Taking sterile axillary buds, cutting new axillary bud stem segments into about 1cm, immersing the explants in activated different agrobacterium rhizogenes soaking solutions respectively, performing ultrasonic treatment for 30s, and slowly shaking for 10min at 100rpm on a shaking table at 28 ℃. Removing surface moisture and excessive agrobacterium rhizogenes bacterial liquid of the impregnated explant by using sterile filter paper, clamping the explant to a co-culture medium (1/2 MS solid culture medium) by using sterilized forceps, and placing the explant in a dark incubator at 25 ℃ for co-culture for 3-5 days.

Screening culture, namely transferring the explant to a screening culture medium (1/2 MS solid culture medium plus 400 mug/mL cefotaxime sodium) after co-culture for 3 days, and placing the explant in a 28 ℃ dark incubator for screening culture. The new solid medium containing cefotaxime sodium was changed every 15 days. After degerming, the strain was transferred to hygromycin-containing 1/2MS to screen hairy roots containing the target gene, and as shown in FIG. 6, the roots successfully transformed can be seen as red.

Subculture and identification statistics of the positive hairy roots, namely, subculturing the obtained positive hairy roots to about 2-3cm, and independently culturing each as a single clone and marking. Meanwhile, the transformation efficiency of the positive hairy roots is counted. Several positive hairy roots were randomly selected for PCR detection, and target genes (Table 3) and hairy root genes (Table 6) were identified. FIG. 7 shows hairy root phenotype after subculture.

The primers to be detected are the genes listed in Table 3 and Table 6, the PCR system and the reaction procedure are those listed in Table 4 and Table 5, and the PCR products were electrophoretically detected in 1% agarose gel at 140V for 20 min.

The statistical result PCR detection result is shown in figure 8, and the rolB and rolC genes can be detected in the root system and the agrobacterium RUBY positive strain; and no band is detected by the root system of the white-front seedling (negative control), which indicates that the root system is hairy root induced by agrobacterium rhizogenes Ar.A4. 3 hairy root systems are detected, and the target gene RUBY can be detected in 2 hairy root lines, which shows that the exogenous target gene RUBY is successfully integrated into the hairy roots of the willow leaves through the mediation of agrobacterium rhizogenes Ar.A4, and the conversion rate is 56.33 percent by (the total number of the induced positive hairy roots/the total number of the induced hairy roots after infection) multiplied by 100.

Application example determination of the content of betalains

0.5G of the positive hairy root material obtained in application example I was taken and put into liquid nitrogen to be ground into powder. Then adding 5mL of 80% methanol aqueous solution, carrying out ultrasonic treatment at room temperature for 30 minutes, putting into a light-shielding shaking table at 25-28 ℃ for shaking for 1 hour, centrifuging to extract supernatant, adding 5mL of deionized water again into the residual plant solid, and repeating the previous operation to extract. After centrifugation, the two solutions were combined. The betalain content was measured in an ultraviolet spectrophotometer at a wavelength of 538 nm. The absolute content was calculated from the molar extinction coefficient of betalains 5.66 x 10 ⁴.

Measurement results: according to different chromatic aberration, positive hairy roots are divided into three strains of R0, R1, R2 and content measurement is carried out. Fig. 9 shows comparison of extraction of different transformed hairy root betalains, fig. 10 shows comparison of different transformed hairy root betalains, fig. 9 and 10 show that the R1-R2 lines all contain betalains, and the betalains content is obviously increased in sequence, which proves that RUBY tandem gene transformation is successful, and the method disclosed by the invention is applicable to genetic transformation of willow leaf rhizoma bletillae.

To sum up: the invention provides a method for establishing an efficient system for genetic transformation of the willow leaf and the willow leaf can be used for efficiently and quickly obtaining the willow leaf and the willow leaf positive hairy root. The method comprises the steps of activating the willow leaf cynanchum wilfordii explant, constructing a plant expression vector of a target gene, culturing agrobacterium rhizogenes engineering bacteria liquid of a recombinant vector, impregnating the explant, co-culturing, rooting induction and culturing, and detecting by PCR. For solving the key method of the problem of supplying the active ingredients of the willow leaf cynanchum wilfordii by utilizing the transgenic hairy root technology, a simple and efficient construction foundation is sought for constructing a genetic transformation system of the willow leaf cynanchum wilfordii transgenic hairy root.

While the foregoing description of the embodiments of the present invention has been presented in conjunction with the drawings, it should be understood that it is not intended to limit the scope of the invention, but rather, it is intended to cover all modifications or variations within the scope of the invention as defined by the claims of the present invention.

Claims (10)

1. A method for genetic transformation of hairy roots of cynanchum glaucescens, which is characterized by comprising the following steps:

Placing the explant in an invaded dye solution containing agrobacterium rhizogenes, and carrying out ultrasonic and shaking; placing the explant obtained in the previous step in a plant culture medium for co-culture and induction transformation; obtaining hairy roots of cynanchum glaucescens through screening culture and amplification culture;

Wherein, the ultrasonic conditions are: 30-50HZ and 20-40s.

2. A method of genetic transformation of hairy roots of cynanchum glaucescens according to claim 1, wherein the explants are produced from young stem segments, adventitious buds, leaves, petioles or hypocotyls;

Preferably tender stem segments;

More preferably, the method for obtaining the explant comprises the following steps: culturing young stem segments of rhizoma Cynanchi Stauntonii for 15-20d, placing on MS culture medium, dark culturing at 25-30deg.C until axillary buds of stem nodes sprout and extend, cutting off aseptic buds, and continuously culturing in MS culture medium for 20-28h to obtain explant.

3. A method of genetic transformation of hairy roots of cynanchum glaucescens according to claim 1, wherein the cynanchum glaucescens comprises cynanchum salicifolium or cynanchum genkwa.

4. A method of genetic transformation of hairy roots of cynanchum glaucescens according to claim 1, wherein the agrobacterium rhizogenes comprises K599, C58C1, MSU440, ar.a4 or ATCC 15834;

Preferably ar.a4 or ATCC 15834;

or, the agrobacterium rhizogenes comprises a target plasmid.

5. A method of genetic transformation of hairy roots of cynanchum glaucescens as claimed in claim 4, wherein the agrobacteria in the infested solution are od600=0.4-0.8.

6. The method for genetic transformation of hairy roots of Cynanchum komarovii of claim 4, wherein the objective plasmid comprises functional genes, disease-resistant genes, insect-resistant genes or marker genes;

Further, the genes of medicinal value include carotene synthesis genes, anthocyanin synthesis genes and antibacterial peptide synthesis genes; the disease resistance genes comprise PcMYB, poMYB, moMYB1; the insect-resistant genes include NbMYB, nbMYB, 107, nbMYB, 163, and NbMYB, 423; the saponin related biosynthesis genes include CYP51G, CYP94D108, UGT73CR1, UGT80A40 and UGT80A41; the marker genes comprise RUBY system, chloramphenicol acetyl transferase gene cat, luciferase gene luc, beta-glucuronidase gene gus, secretory alkaline phosphatase gene seap and green fluorescent protein gene gfp.

7. The genetic transformation method of hairy roots of cynanchum glaucescens according to claim 1, wherein the condition of mixing is as follows: the oscillating speed is 80-100 r/min, the oscillating time is 10-30 min, and the temperature is 25-30 ℃.

8. The genetic transformation method of hairy roots of cynanchum glaucescens according to claim 1, wherein the co-culture method comprises the following steps: removing surface moisture of the explant and excessive agrobacterium rhizogenes bacterial liquid by using sterile filter paper, clamping the explant onto a plant culture medium by using sterilized forceps, and placing the explant in a dark incubator at 22-27 ℃ for co-culture for 3-7d;

Preferably, the co-cultivation condition is dark cultivation at 25 ℃ for 3-5d; the culture medium is 1/2MS solid culture medium.

9. A method for genetic transformation of hairy roots of Cynanchum komarovii according to claim 8, wherein,

The conditions of the screening culture are as follows: transferring the explants after co-culture to a screening medium, and carrying out photoperiod culture for 15-18d;

wherein the photoperiod is 16h of light culture and 8h of dark culture;

The screening culture medium is 1/2MS solid culture medium, 380-420 mug/mL cefotaxime sodium is added; preferably 400. Mu.g/mL.

10. Use of the method according to any one of claims 1-9 for large scale cultivation of cynanchum bungei or use of cynanchum bungei as a bioreactor for the production of a drug or a secondary metabolite;

More specifically, the application includes any one or more of the following:

(1) Extracting and preserving medicinal components of hairy roots of rhizoma Cynanchi Stauntonii;

(2) Extracting material of rhizoma Cynanchi Stauntonii saponin component;

(3) A genetic transformation gene of cynanchum glaucescens;

(4) Improvement of the transgenic germplasm of the cynanchum glaucescens;

(5) And (5) performing enlarged culture of the cynanchum atratum.