CN107119070B - Method for improving induction efficiency and genetic transformation efficiency of hairy roots of bupleurum chinense and application of method - Google Patents
Method for improving induction efficiency and genetic transformation efficiency of hairy roots of bupleurum chinense and application of method Download PDFInfo
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Abstract
The invention provides a method for improving induction efficiency and genetic transformation efficiency of hairy roots of bupleurum chinense and application thereof. The optimal combination condition for improving the infection efficiency of the agrobacterium rhizogenes is confirmed based on the exploration of the agrobacterium rhizogenes strain, the OD value of the infection bacterial liquid, the co-culture time and other conditions, and compared with the traditional hairy root induction rate of about 10%, the hairy root induction rate obtained by the technical scheme provided by the invention reaches 70% -85%. The method for improving the genetic transformation efficiency of the hairy roots of bupleurum chinense can obtain the transformation rate of more than 80 percent of exogenous genes, and provides better, more efficient and simpler selection for researching the functional genes of bupleurum chinense and/or industrialization of metabolites of the hairy roots of bupleurum chinense.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for improving induction efficiency and genetic transformation efficiency of hairy roots of bupleurum chinense and application thereof.
Background
Hairy roots, also called hairy roots, are a large number of by-products produced at the infected site by inserting the T-DNA on Ri plasmid contained in Agrobacterium rhizogenes into the nuclear genome of plants or organs, tissues (including callus), single cells, protoplasts. The induction culture of the hairy roots can be used as a bioreactor to produce plant metabolites with application value. In addition, exogenous genes can be introduced through agrobacterium rhizogenes to regulate and control the synthesis of target metabolites.
Radix bupleuri is a typical traditional bulk medicinal material in China, and has an application history of more than 2000 years. Is the essential herb for dispelling and allaying fever, soothing liver and invigorating yang. Bupleurum chinense DC is one of Bupleurum chinense based plants regulated by national formulary of China, and is a perennial herb plant of Bupleurum genus in Umbelliferae. China, northeast, northwest, east, Hubei and Sichuan are distributed and planted, and are the main sources of bupleurum medicinal materials.
At present, a plurality of plants including medicinal plants have hairy root induction culture systems, but the induction culture systems of each plant are different, and the success of changing one plant into another plant is difficult when an effective method is used for one plant. In the process of exploring a specific plant hairy root induction culture system, the optimal conditions are obtained through tests from the conditions of the type of agrobacterium rhizogenes strain, the type of a plant explant, the infection mode, the infection time and the like.
In the article of establishing the hairy root induction and plant regeneration system of bupleurum chinense (sun crystal, etc., pharmaceutical science, 2013), we have researched and established a stable hairy root induction system of bupleurum chinense from designing various induction conditions. The agrobacterium rhizogenes A4 infects the leaf base of the aseptic seedling of bupleurum chinense, after 3 days of culture, the aseptic seedling is transferred to bacteriostatic culture, hairy roots appear after 10 days, after 4-5 weeks of culture, the liquid shaking culture is transferred to obtain a large amount of hairy roots, and the hairy roots generated by induction can be regenerated into plants. The hairy root can be regenerated into plant by two ways, one is continuous culture that the hairy root which is cultured by liquid shaking falls off spontaneously to form seedling, and the other is that the callus which is produced by the hairy root and falls off is placed on a regeneration solid culture medium which is screened to regenerate into plant. The establishment of the hairy root and the plant regeneration system of the bupleurum lays a foundation for the development of research on the secondary metabolism of the bupleurum, in particular to the research and the utilization of agrobacterium-mediated genetic transformation. However, the system is used for research and application of multi-gene transformation of the hairy root, the efficiency is low, and the induction rate of the hairy root is only about 10%.
In the article of Agrobacterium induced northern bupleurum hairy root system establishment and UGT gene function preliminary study (Sun Jing et al, microbiology 2014), referring to the induction method provided in the foregoing, by designing various induction conditions including different strain species, explant type, infection mode and infection time, the stable northern bupleurum hairy root is explored and establishedAnd (4) an induction system. The following are found: infecting the leaf base of aseptic seedling of bupleuri radix with Agrobacterium rhizogenes A4, soaking the bacterial solution for 20min, and placing in solid culture medium (B)5+ acetosyringone 200 μ tmol/L + sucrose 20g/L or MS + NAA 6mg/L + acetosyringone 200mol/L + sucrose 20 g/L) for 3 days, transferring to bacteriostatic culture, and allowing hairy roots to grow after 10 days. Therefore, the hairy root induction system of bupleurum chinense is established. The inductivity is still about 10%. And the article states that: whether the induction rate of the hairy roots of the bupleurum can be improved or not is to be continuously researched from the aspects of strains, induction conditions and the like.
In addition, in "preliminary study on hairy root system establishment and UGT gene function of northern bupleurum chinense Miller induced by Agrobacterium" (Sunjing et al, microbiology 2014), "preliminary study on genetic transformation of northern bupleurum chinense Miller BcUGT10 gene mediated by Agrobacterium rhizogenes" was conducted, but the results were found: "3 resistant hairy roots obtained by screening induced hairy roots with 25mgm hygromycin and no positive transformed hairy roots obtained after PCR detection"
The invention aims to provide a method for improving the induction rate of hairy roots of bupleurum chinense and realizing genetic transformation of exogenous genes.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for improving the induction efficiency and the genetic transformation efficiency of hairy roots of bupleurum chinense and application thereof.
The technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a method for improving the induction efficiency of hairy roots of bupleurum chinense, which comprises the following steps:
(1) obtaining an explant for infection;
(2) obtaining agrobacterium rhizogenes bacterial liquid for infection;
(3) co-culturing transformed explants: placing the explant obtained in the step 1) into the agrobacterium rhizogenes bacterial liquid for infection obtained in the step 2), and shaking and dip-dyeing; then transferring into MS culture medium, and treating according to a) or b):
a) co-culturing for 3-5 days under a dark condition;
b) alternately culturing for 4-7 days according to illumination/darkness (preferably 16 hours of illumination/8 hours of darkness);
(4) induction of hairy roots: placing the explant treated in the step 3) in an antibacterial culture medium, culturing in the dark, and inducing hairy roots to obtain induced hairy roots.
In an embodiment of the present invention, in the step (1), the step of obtaining an explant for infection specifically includes: after the disinfected seeds are germinated to obtain seedlings, the roots of the seedlings are cut off and cultured in a subculture medium to obtain sterile and rootless differentiated seedlings, namely explants for infection by the first strain.
In one embodiment of the invention, the sterilized seeds are inoculated into MS solid culture medium to be cultured to obtain sterile seedlings.
It will be understood by those skilled in the art that, in addition to the examples shown in the examples of the present invention, other methods may be used to sterilize and disinfect the seeds, and the seeds may be cultured until they germinate to obtain sterile seedlings.
Further, placing the obtained sterile rootless differentiated seedling on a hormone-free MS culture medium for pre-culturing for 0-3 days to obtain an explant for infection by a first strain; preferably, the pre-culture is for 0, 1, 2 or 3 days.
Preferably, the first bacterial species is one or more of agrobacterium rhizogenes a4, ACCC10060, ACCC15834, SW 101.
In a specific embodiment of the present invention, the subculture medium is an MS medium containing the following components: 2.5mg/L of 6-BA, 2.5mg/L of IAA2, 30g/L of cane sugar and 5g/L of plant gel.
In one embodiment of the present invention, in the step (1), the leaves and the petioles of the seedling of bupleurum chinense (preferably cultivated in soil) are sterilized to obtain the explant for the infection of the second bacterial species.
Further, the leaves and the petioles of the obtained sterilized bupleurum seedlings are placed on a hormone-free MS culture medium for pre-culture for 0-3 days, and explants for infection by a second strain are obtained; preferably, the pre-culture is for 0, 1, 2 or 3 days.
Still further, the method of sterilization comprises: soaking the materials in 75% alcohol for 30-50S, sealing with 20% sodium hypochlorite (NaClO) solution for 5-10min, and washing with sterile water for 3-5 times.
Preferably, the second species is agrobacterium rhizogenes SW 101.
In an embodiment of the invention, in the step (2), the OD value of the bacterial liquid of the strain is 0.8-1.2.
In a preferred embodiment of the present invention, in the step (2), the OD value of the bacterial liquid of the strain used is 0.8, 1 or 1.2.
In a preferred embodiment of the present invention, in the step (3), the shake-culture dip dyeing time is 20-25 min.
In one embodiment of the present invention, when the step (3) is a), the strains used are one or more of agrobacterium rhizogenes a4, ACCC10060, ACCC15834 and SW 101.
Further, when the step (3) is the step a), the co-culture time is 3, 4 or 5 days.
In one embodiment of the present invention, when the step (3) is the step b), the strain used is Agrobacterium rhizogenes SW 101.
Further, when the step (3) is the step b), culturing is carried out for 4, 5, 6 or 7 days alternately.
In one embodiment of the present invention, in the step (4), the bacteriostatic medium is MS medium containing 500mg/L cefotaxime sodium (or car).
It will be understood by those skilled in the art that, in addition to the examples shown in the examples of the present invention, other bacteriostatic media known to those skilled in the art may be used, such as MS medium using 400 or 300mg/L cefotaxime sodium (or car).
In one embodiment of the invention, in the step (4), the induced hairy roots are obtained after dark culture for 20-30 days.
In an embodiment of the present invention, in the step (4), the obtained induced hairy roots are subcultured once every 30 days for 90-100 days of bacteriostatic treatment, and then transferred into a common culture medium (such as MS liquid culture medium without cefotaxime sodium (or kana)), and continuously shake-cultured (specifically, dark culture at 150 rpm) to obtain a large amount of induced hairy roots.
The second aspect of the invention provides a method for improving the genetic transformation efficiency of hairy roots of bupleurum chinense, which comprises the following steps:
(1) obtaining an explant for infection;
(2) obtaining a rhizogenes agrobacterium liquid for infection, wherein the rhizogenes agrobacterium carries exogenous genes;
(3) co-culturing transformed explants: placing the explant obtained in the step 1) into the agrobacterium rhizogenes bacterial liquid for infection obtained in the step 2), and shaking and dip-dyeing; then transferring into MS culture medium, and treating according to a) or b):
a) co-culturing for 3-5 days under a dark condition;
b) alternately culturing for 4-7 days according to illumination/darkness (preferably 16 hours of illumination/8 hours of darkness);
(4) induction of hairy roots: placing the explant treated in the step 3) in an antibacterial culture medium, culturing in the dark, and inducing hairy roots to obtain induced hairy roots.
In an embodiment of the present invention, in the step (1), the step of obtaining an explant for infection specifically includes: after the disinfected seeds are germinated to obtain seedlings, the roots of the seedlings are cut off and cultured in a subculture medium to obtain sterile and rootless differentiated seedlings, namely explants for infection by the first strain.
In one embodiment of the invention, the sterilized seeds are inoculated into MS solid culture medium to be cultured to obtain sterile seedlings.
It will be understood by those skilled in the art that, in addition to the examples shown in the examples of the present invention, other methods may be used to sterilize and disinfect the seeds, and the seeds may be cultured until they germinate to obtain sterile seedlings.
Further, placing the obtained sterile rootless differentiated seedling on a hormone-free MS culture medium for pre-culturing for 0-3 days to obtain an explant for infection by a first strain; preferably, the pre-culture is for 0, 1, 2 or 3 days.
Preferably, the first bacterial species is one or more of agrobacterium rhizogenes a4, ACCC10060, ACCC15834, SW 101.
In a specific embodiment of the present invention, the subculture medium is an MS medium containing the following components: 2.5mg/L of 6-BA, 2.5mg/L of IAA2, 30g/L of cane sugar and 5g/L of plant gel.
In one embodiment of the present invention, in the step (1), the leaves and the petioles of the seedling of bupleurum chinense (preferably cultivated in soil) are sterilized to obtain the explant for the infection of the second bacterial species.
Further, the leaves and the petioles of the obtained sterilized bupleurum seedlings are placed on a hormone-free MS culture medium for pre-culture for 0-3 days, and explants for infection by a second strain are obtained; preferably, the pre-culture is for 0, 1, 2 or 3 days.
Still further, the method of sterilization comprises: soaking the materials in 75% alcohol for 30-50S, sealing with 20% sodium hypochlorite (NaClO) solution for 5-10min, and washing with sterile water for 3-5 times.
Preferably, the second species is agrobacterium rhizogenes SW 101.
In one embodiment of the present invention, in the step (2), the agrobacterium rhizogenes comprises a foreign gene or a plasmid into which a foreign gene is inserted. Preferably, the plasmid is PK2GW 7.
In an embodiment of the invention, in the step (2), the OD value of the bacterial liquid of the strain is 0.8-1.2.
In a preferred embodiment of the present invention, in the step (2), the OD value of the bacterial liquid of the strain used is 0.8, 1 or 1.2.
In a preferred embodiment of the present invention, in the step (3), the shake-culture dip dyeing time is 20-25 min.
In one embodiment of the present invention, when the step (3) is a), the strains used are one or more of agrobacterium rhizogenes a4, ACCC10060, ACCC15834 and SW 101.
Further, when the step (3) is the step a), the co-culture time is 3, 4 or 5 days.
In one embodiment of the present invention, when the step (3) is the step b), the strain used is Agrobacterium rhizogenes SW 101.
Further, when the step (3) is the step b), culturing is carried out for 4, 5, 6 or 7 days alternately.
In one embodiment of the present invention, in the step (4), the bacteriostatic medium is MS medium containing 500mg/L cefotaxime sodium (or car).
It will be understood by those skilled in the art that, in addition to the examples shown in the examples of the present invention, other bacteriostatic media known to those skilled in the art may be used, such as MS medium using 400 or 300mg/L cefotaxime sodium (or car).
In one embodiment of the invention, in the step (4), the induced hairy roots are obtained after dark culture for 20-30 days.
In an embodiment of the present invention, in the step (4), the obtained induced hairy roots are subcultured once every 30 days for 90-100 days of bacteriostatic treatment, and then transferred into a common culture medium (such as MS liquid culture medium without cefotaxime sodium (or kana)), and continuously shake-cultured (specifically, dark culture at 150 rpm) to obtain a large amount of induced hairy roots.
In a third aspect, the invention provides a method for improving the induction efficiency of the hairy root of bupleurum chinense for application in induction, genetic transformation, gene function research and/or production of metabolites of bupleurum chinense (preferably, secondary metabolites, and more preferably, secondary metabolites with medicinal value).
In a fourth aspect, the invention provides a method for improving the genetic transformation efficiency of the hairy root of bupleurum chinense in the induction, genetic transformation, functional gene research of the hairy root of bupleurum chinense and/or the production of metabolites (preferably secondary metabolites, more preferably secondary metabolites with medicinal value) of the hairy root of bupleurum chinense.
Compared with the prior art, the invention has the beneficial effects that:
the optimal combination condition for improving the infection efficiency of the agrobacterium rhizogenes is determined based on the exploration of the agrobacterium rhizogenes strain, the OD value of the infection bacterial liquid, the co-culture time and other conditions. And the optimal induction conditions of SW101 for inducing the hairy roots of bupleurum chinense and A4, ACCC10060 and ACCC15834 for inducing the hairy roots of bupleurum chinense are given. Compared with the traditional hairy root induction rate of about 10 percent, the hairy root induction rate obtained by the technical scheme provided by the invention reaches 70 to 85 percent.
Furthermore, the invention also provides a technical scheme for improving the genetic transformation efficiency of the hairy roots of bupleurum chinense, and experiments prove that the technical scheme provided by the invention can obtain the exogenous gene transformation rate of more than 80 percent, thereby providing better, more efficient and simpler selection for researching the functional genes of bupleurum chinense and/or industrialization of metabolites of the hairy roots of bupleurum chinense.
Drawings
FIG. 1 is a photograph of Agrobacterium rhizogenes infecting a bupleuri radix explant to produce hairy roots, provided by an embodiment of the present invention;
FIG. 2 shows the growth of the hairy roots of Bupleurum scorzonerifolium provided by the embodiment of the present invention after 60 days of culture;
FIG. 3 shows the electrophoresis result of the Carna resistance gene fragment of the PCR amplification vector provided in the embodiment of the present invention.
Detailed Description
The invention is described in further detail below with reference to the figures and examples.
It is to be understood that commercially available reagents are used in the present invention unless otherwise specified.
The Chinese explanation of the English abbreviation of the invention is as follows:
cef: cephamycin, cefotaxime sodium;
6-BA: 6-benzylamino adenine;
AS: acetosyringone;
IAA: indole acetic acid.
Example 1
The embodiment of the invention provides a method for improving the induction efficiency of hairy roots of bupleurum chinense, which comprises the following steps:
first, obtaining of explant
1. Washing dry seed of bupleuri radix with tap water, and washing with sterilized distilled water for several times;
2. the following steps are performed in the clean bench: soaking the raw materials in 75% alcohol for 30-50S;
3. sealing with 20% sodium hypochlorite (NaClO) solution for 15min, and washing with sterile water for 3-5 times;
4. absorbing water of the disinfected seeds by sterile filter paper, carrying out dark culture at 25 ℃ on the basis of common MS culture, transferring out contaminated seeds every 2-3 days, basically causing no contamination phenomenon after 8-9 days, and culturing until the seeds start to germinate after about 14 days;
5. cutting off the roots of the seedlings, and adding the seedlings into a subculture medium (MS +6-BA2.5mg/L + IAA2.5mg/L +30g/L sucrose +5g/L plant gel) to obtain sterile and rootless differentiated seedlings, namely explants for infection subsequently.
Second, Agrobacterium rhizogenes activation
1. Inoculating Agrobacterium rhizogenes in LB culture medium containing 50mg/L rifampicin (Rif), dark culturing at 28 deg.C for 2 days;
2. picking single colony, and performing shaking dark culture at 28 ℃ and 180r/min for 24h in an LB liquid culture medium containing 50mg/L rifampicin;
3. diluting the bacterial liquid according to the ratio of 1: 100, and continuously shaking and culturing until OD600 is 0.8-1.2 and 5000 r.min-1Centrifuging for 10min, and collecting thallus;
4. the pellet was resuspended in an equal volume of MS + 200. mu.M AS broth for infection.
Third, root-growing agrobacterium infection (hairy root induction)
1. Placing the explant obtained in the step one into a heavy suspension, and shaking at 25 ℃ at 100rpm for 20-25 min;
2. transferring the mixture into an MS culture medium, and carrying out dark co-culture for 3-5 days;
3. culturing the co-cultured explant in a medium containing 500mg/L cef (or car) MS at 25 ℃ in the dark, and selecting a root system with good growth vigor after 20-30 days, wherein the root system is different induced hairy root systems;
4. subculturing every 30d for 90-100d, transferring into MS liquid culture medium without cef (or car), and continuously culturing under shaking (150rpm dark culture).
The picture of the rhizogenes generated by infecting bupleurum chinense explants with agrobacterium rhizogenes provided by the embodiment of the invention is shown in figure 1; the growth status of the hairy root of Bupleurum falcatum after 60 days of culture is shown in FIG. 2. Through analysis, compared with the prior art (10% of hairy root induction rate), the method for improving the induction efficiency of the hairy root of bupleurum chinense provided by the embodiment of the invention has the unexpected effects: 70-85% of explants can induce hairy roots (that is, the induction efficiency of the hairy roots is more than 70%), and the number of the hairy roots on each explant is 5-10.
To further illustrate the beneficial effects of the present invention, the present invention provides the following alternative example 1, where alternative example 1 replaces some of the conditions in steps one to three of example 1, and others are unchanged, and the corresponding test results are shown in Table 1 (d: day; min: min; h: h):
table 1.
In table 1, the pre-culture specifically comprises the steps of step one in example 1, obtaining the "explant" at point 5 to obtain the sterile and rootless differentiated plantlet, i.e. the explant used for infection later "is replaced by: placing the obtained sterile rootless differentiated plantlets on a hormone-free MS culture medium for pre-culturing for 0-3 days to obtain explants for subsequent strain infection; specifically, preculture was for 0, 1, 2 or 3 days ". If the explant used is "leaf and petiole", instead of the "rootless differentiated plantlet" described in example 1, the "leaf and petiole" is obtained by the following method: taking leaves and petioles of seedlings of bupleurum chinense cultivated in soil, and disinfecting and sterilizing for infection, wherein the disinfecting and sterilizing method comprises the following steps: soaking the materials in 75% alcohol for 30-50S, sealing with 20% sodium hypochlorite (NaClO) solution for 5-10min, and washing with sterile water for 3-5 times. Note: the injection method is to inject the bacterial liquid into each part of the seedling and then directly culture the seedling on a culture medium for inducing hairy roots.
As can be seen from Table 1: based on various conditions in the alternative example 1, it was preliminarily confirmed that the infection efficiency of Agrobacterium rhizogenes is mainly related to the strains, OD value of the infected bacterial liquid, co-culture time and other factors.
To further illustrate the beneficial effects of the present invention, the present invention provides the following alternative example 2, wherein the alternative example 2 replaces some of the conditions in the first to third steps of example 1, and others are not changed, and the corresponding test results are shown in tables 2 and 3 (d: day; min: min; h: h):
table 2.
Table 3.
As can be seen from tables 2 and 3, the too high or too low OD value affects the rooting rate; too long or too short co-cultivation time affects the rooting rate.
Comparative examples
To further illustrate the beneficial effects of the present invention, the present invention provides the following comparative examples, which replace some of the conditions in step one of example 1, and do not change others, and the corresponding test results are shown in table 4:
table 4.
| Formulation of | Induced differentiation results |
| MS + KT0.5mg/L +30g/L sucrose +5g/L plant gel | The increment rate is about 1:1 |
| MS +6-BA2.5mg/L + IAA2.5mg/L + sucrose +5g/L plant gel | The increment rate is about 1:5 |
| MS +6-BA 0.5mg/L + IAA 0.5mg/L + sucrose +5g/L plant gel | The increment rate is about 1:2 |
| MS +6-BA 5mg/L + IAA.5mg/L +30g/L sucrose +5g/L plant gel | The increment rate is about 1:10 but the seedlings are vitrified |
The experimental conditions for step one of example 1 of the present invention, i.e., set 2 of conditions in table 4, are identified in table 4.
Example 2
The embodiment of the invention provides a method for improving the genetic transformation efficiency of hairy roots of bupleurum chinense, which comprises the following steps:
first, genetic transformation of hairy root
Firstly, preparing competent cells:
taking a strain, streaking and inoculating the strain on an LB culture medium containing 50mg/L rifampicin, and carrying out dark culture at 28 ℃ for 2 days; picking single colony, and performing shaking dark culture at 28 ℃ and 180r/min for 24h in an LB liquid culture medium containing 50mg/L rifampicin; diluting the bacterial liquid according to the ratio of 1: 50, continuously shaking and culturing until OD600 is 0.6, carrying out ice bath on the bacterial liquid for 15min, subpackaging the bacterial liquid in a 1.5mL sterile centrifuge tube, and centrifuging at 4 ℃ at 5000rpm for 5 min; discarding the supernatant, resuspending the thallus in 600 μ L of ice-precooled 0.05mol/L sterile CaCl2, ice-cooling for 30min, and centrifuging at 5000rpm at 4 ℃ for 10 min; discarding the supernatant, and suspending the thallus in 100 μ L of sterile CaCl2 precooled with ice at 0.05 mol/L; storage at-80 ℃ for transformation;
secondly, transformation by a freeze-thaw method:
1. adding plasmid (PK 2GW7, Invitrogen in the example) with exogenous gene into 100 μ L of competent cells, gently mixing, and standing on ice for 30 min; quickly freezing in liquid nitrogen for 5min, warm bathing at 37 deg.C for 5min, and ice-cooling for 2 min; adding 700 μ L LB liquid culture medium, shaking at 28 deg.C for 12 hr, and centrifuging at 6000rpm for 3 min; discarding the supernatant, reserving 100 μ L of bacterial liquid, uniformly mixing the liquid with the gun head, uniformly coating the mixture on an LB plate containing plasmid corresponding resistance antibiotics (kanamycin in the example) and 50mg/L rifampicin by using an applicator, and performing inverted culture at 28 ℃ for 2d until a single colony appears;
2. selecting a single colony, carrying out dark shaking culture at 28 ℃ and 180r/min for 24h, and confirming the successful transformation by bacterial liquid PCR;
3. selecting 1 successfully transformed single colony, and enlarging the culture amount of the bacteria for hairy root induction; see step three of example 1 and alternative examples 1-2 for the induction step; the experimental procedures of example 2 of the present invention were obtained separately;
thirdly, verifying the transformation effect by PCR:
1. rapidly extracting genome DNA of hairy roots: taking a hairy root sample with the length of about 2cm into a centrifuge tube, adding 20 mu L of 0.2M NaOH solution, working for 10s at 4000rpm of a high-throughput tissue grinder, intermittently operating for 10s, totally operating for 3 times, after crushing, centrifuging at 3500rpm for 1min, adding 490 mu L of 0.1M Tris-HCl, carrying out vortex oscillation, centrifuging at 3500rpm for 1min, and taking 1.5 mu L of supernatant for PCR amplification.
2. The genomic DNA of hairy roots is taken as a template, the peculiar Kanna resistance gene fragment of a plasmid vector (PK 2GW7 in the example) is amplified, hairy roots induced by a non-transformed plasmid strain are taken as a Control (CK), and a primer sequence (Baozuo biosynthesis) K-2F: ACTGGGCACAACAGACAATC (SEQ ID NO.1) and K-2R: CTTCAGCAATATCACGGGTAG (SEQ ID NO.2) are adopted.
PCR System (10. mu.l) and procedure:
prim STAR HS (Bio/industry): 5. mu.L, template: 1.5. mu.L, primers each 0.5. mu.L, ddH20: 2.5. mu.L.
The procedure is as follows: at 95 ℃ for 5min, (98 ℃ for 10s, 58 ℃ for 5s, 72 ℃ for 20s) for 35 cycles, at 72 ℃ for 5 min.
In the embodiment 2 of the invention, the agrobacterium rhizogenes is transformed by the vector PK2GW7(Invitrogen), the agrobacterium rhizogenes is infected by the bupleurum chinense explant according to the method in the embodiment 1 and the step three of the alternative embodiment 1-2, and the generation of hairy roots is identified by adopting the Carna resistance gene on the PCR amplification vector, so that the hairy root induction transformation efficiency of the method can reach 70% -85%.
FIG. 3 shows the electrophoresis results of the PCR amplification vector Carna resistance gene fragment of example 2 when Agrobacterium rhizogenes infects Bupleurum falcatum explants according to the method of step three of example 1, M: DNA molecular weight standard (Marker), lanes 1-7: transgenic hairy root system, CK: hairy root system without PK2GW7 vector.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (4)
1. A method for improving the induction efficiency of hairy roots of bupleurum chinense is characterized by comprising the following steps:
(1) obtaining an explant for infection;
after the seeds are disinfected and germinated to obtain seedlings, cutting off the roots of the seedlings, culturing the seedlings in a subculture medium to obtain sterile and rootless differentiated seedlings and obtain explants for infection;
the subculture medium is an MS culture medium containing the following components: 2.5mg/L, IAA 2.5.5 mg/L of 6-BA, 30g/L of sucrose and 5g/L of plant gel;
(2) obtaining agrobacterium rhizogenes bacterial liquid for infection;
(3) co-culturing transformed explants: placing the explant obtained in the step 1) into the agrobacterium rhizogenes liquid for infection obtained in the step 2), and shaking and staining, wherein the used bacterial strain is agrobacterium rhizogenes ACCC10060, the OD value of the agrobacterium rhizogenes liquid is 0.8, and the shaking and staining time is 20-25 minutes; then transferring the mixture into an MS culture medium, and co-culturing for 3-4 days under a dark condition;
(4) induction of hairy roots: placing the explant treated in the step 3) in an antibacterial culture medium, culturing in the dark, and inducing hairy roots to obtain induced hairy roots.
2. A method for improving the genetic transformation efficiency of hairy roots of bupleurum chinense is characterized by comprising the following steps:
(1) obtaining an explant for infection;
after the seeds are disinfected and germinated to obtain seedlings, cutting off the roots of the seedlings, culturing the seedlings in a subculture medium to obtain sterile and rootless differentiated seedlings and obtain explants for infection;
the subculture medium is an MS culture medium containing the following components: 2.5mg/L, IAA 2.5.5 mg/L of 6-BA, 30g/L of sucrose and 5g/L of plant gel;
(2) obtaining a liquid of agrobacterium rhizogenes for infection, wherein the agrobacterium rhizogenes comprises exogenous genes or plasmids inserted with the exogenous genes;
(3) co-culturing transformed explants: placing the explant obtained in the step 1) into the agrobacterium rhizogenes liquid for infection obtained in the step 2), and shaking and staining, wherein the used bacterial strain is agrobacterium rhizogenes ACCC10060, the OD value of the agrobacterium rhizogenes liquid is 0.8, and the shaking and staining time is 20-25 minutes; then transferring the mixture into an MS culture medium, and co-culturing for 3-4 days under a dark condition;
(4) induction of hairy roots: placing the explant treated in the step 3) in an antibacterial culture medium, culturing in the dark, and inducing hairy roots to obtain induced hairy roots.
3. Use of the method of claim 1 for enhancing the induction efficiency of hairy root of Bupleurum falcatum Linn in the induction, genetic transformation, gene function studies and/or the production of metabolites of Bupleurum falcatum Linn.
4. Use of the method of claim 2 for increasing the efficiency of genetic transformation of hairy root of Bupleurum falcatum Linn in the induction, genetic transformation, gene function studies and/or production of metabolites of Bupleurum falcatum Linn.
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