CN107119070A - It is a kind of to improve Bupleurum Chinese hairy root induction efficiency and the method and its application of genetic transformation efficiency - Google Patents
It is a kind of to improve Bupleurum Chinese hairy root induction efficiency and the method and its application of genetic transformation efficiency Download PDFInfo
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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Abstract
Bupleurum Chinese hairy root induction efficiency and the method and its application of genetic transformation efficiency are improved the invention provides a kind of.The present invention based on to agrobacterium rhizogene strain, infect the conditions such as bacterium solution OD values, co-cultivation time and grope, confirm the optimum combination condition of the infect efficiency of raising agrobacterium rhizogenes, compared to 10% or so traditional hairy root induction rate, the hairy root induction rate that the technical scheme that the present invention is provided is obtained is up to 70% 85%.The invention provides raising Bupleurum Chinese hairy root genetic transformation efficiency method, more than 80% gene transformation rate can be obtained, is studied for Bupleurum Chinese functional gene and/or the industrialization of Bupleurum Chinese hairy root metabolite provides more preferable, more efficient, easier selection.
Description
Technical field
Biological technical field of the present invention, and in particular to one kind improves Bupleurum Chinese hairy root induction efficiency and genetic transformation efficiency
Method and its application.
Background technology
Hairy root, also known as Hairy root, root of hair is that the T-DNA on the Ri plasmids contained by agrobacterium rhizogenes is inserted into plant
Or organ, tissue (including callus), individual cells, the nuclear genome of protoplast, and produced in infection site
A large amount of accessory substances.The Fiber differentiation of hairy root can produce the plant metabolites for having application value as bioreactor.Separately
Outside, foreign gene can also be imported by agrobacterium rhizogenes, with the synthesis of goal of regulation and control metabolite.
Radix bupleuri is the typical traditional bulk medicinal materials of China, the applicating history for having more than 2 000 years.Be evacuation bring down a fever, relax liver, rise
The key medicine of sun.Bupleurum Chinese (Bupleurum chinense DC.) is one of radix bupleuri Original plant as defined in China's NF,
For Umbelliferae Bupleurum herbaceos perennial.The ground such as China northeast, North China, northwest, East China, Hubei, Sichuan be distributed and
Plantation, is the main source of Radix Bupleuri.
Have various plants at present has hairy root induction cultivating system, but the induction training of every kind of plant including medicinal plant
Foster system all has differences, and effective method, changes a kind of plant and be often difficult to success on a kind of plant.Groping specified plant
It is typically the classification, plant outside shade type, the side of infecting from agrobacterium rhizogenes strain during hairy root induction cultivating system
The conditions such as formula, time of infection are set out, and optimum condition is obtained by testing.
《The foundation of Bupleurum Chinese hairy root induction and its plant regeneration system》(Sun Jing etc., Acta Pharmaceutica Sinica, 2013) one text
In, we have studied from a variety of inductive conditions are designed, explore and set up stable radix bupleuri hairy root induction system.Root of hair agriculture bar
Bacterium A4 infects Bupleurum Chinese aseptic seedling leaf base, after co-culturing 3 days, is transferred to micro-organisms, starts within 10 days hairy root occur, through 4~5
Week culture, switchs to liquid and shakes training, obtain a large amount of hairy roots, induce the hairy root produced renewable into plant.Hairy root regeneration into
Plant has two ways:A kind of is that liquid shakes the hairy root of training from the continuation culture of falling out formation seedling, and another is by hair
The class callus that the generation of shape root comes off, which is placed on the regenerated solids culture medium through screening, regenerates plant.Radix bupleuri hairy root and its
The foundation of plant regeneration system, to carry out the research of radix bupleuri secondary metabolism, is studied especially with Agrobacterium-mediated genetic transformation
Laid a good foundation with utilizing.However, this system is used for the research and application of polygenes transformed hairy root, efficiency comparison is low, hairy
Root induction rate is only 10% or so.
《Agrobacterium induces Bupleurum Chinese hairy root Establishing and UGT gene function Primary Studies》(Sun Jing etc., microorganism
Learn, 2014) in a text, with reference to the abductive approach provided above, by designing a variety of inductive conditions, including different strain species,
Explant type, mode of infection and time of infection etc., explore and set up stable Bupleurum Chinese hairy root induction system.It was found that：Adopting
Bupleurum Chinese aseptic seedling leaf base is infected with Agrobacterium rhizogenesA4, bacterium solution contaminates 20min, is placed in solid medium (B5+ acetyl cloves
The μ tmol/L+ sucrose 20g/L or MS+NAA 6mg/L+ acetosyringone 200mol/L+ sucrose 20g/L of ketone 200) co-culture after 3d,
Micro-organisms are transferred to, 10d or so sends hairy root.Thus, Bupleurum Chinese hairy root induction system is established.Inductivity still exists
10% or so.And article is pointed out：Whether radix bupleuri hairy root induction rate, which can also improve, needs to be continued from the side such as bacterial strain and inductive condition
Face is groped.
In addition,《Agrobacterium induces Bupleurum Chinese hairy root Establishing and UGT gene function Primary Studies》(Sun Jing etc.,
Microbiology, 2014) in a text, " genetic transformation for carrying out the Bupleurum Chinese BcUGT10 genes of agrobacterium rhizogenes mediation is tentatively ground
Study carefully ", but result discovery：" the hairy root that induction is produced obtains 3 resistance hairy roots through 25mgm hygromycin selections, after PCR detections,
Positive transformants hairy root is not obtained "
Bupleurum Chinese hairy root induction rate can be improved it is an object of the invention to provide one kind, and so as to realizing foreign gene
Genetic transformation method.
The content of the invention
In order to solve problems of the prior art, the present invention provide it is a kind of improve Bupleurum Chinese hairy root induction efficiency and
The method and its application of genetic transformation efficiency.
The technical solution adopted by the present invention is as follows：
First aspect present invention provides a kind of method for improving Bupleurum Chinese hairy root induction efficiency, comprises the following steps：
(1) explant for infecting is obtained；
(2) the agrobacterium rhizogenes bacterium solution for infecting is obtained；
(3) conversion explant is co-cultured：By step 1) obtained by explant be placed in step 2) hair that infects of gained
In root Agrobacterium bacterium solution, shake training and contaminate；Then MS culture mediums are transferred to, according to a) or b) handling：
A) co-cultured 3~5 days under dark condition；
B) according to illumination/dark (preferably 16 hours illumination/8 hour dark) alternate culture 4~7 days；
(4) induction of hairy root：Take step 3) processing explant be placed in micro-organisms base, dark culturing, induction hair
Root, obtains the hairy root of induction.
In an embodiment of the present invention, in the step (1), the step of described acquisition is used for the explant infected is specific
Including：Disinfection seed rudiment is obtained after seedling, cuts seedling root, is cultivated into subculture medium, obtains point of sterile unrooted
Change seedling, that is, obtain the explant infected for the first strain.
In a specific embodiment of the invention, culture in MS solid mediums is inoculated in after seed disinfection and obtains sterile children
Seedling.
It will be appreciated by persons skilled in the art that in addition to example shown of the embodiment of the present invention, other can also be used
Method seed is carried out disinfection, it is degerming, and cultivate to seed germination, obtain aseptic seedlings.
Further, the differentiation seedling of the sterile unrooted of the acquisition is placed in preculture 0~3 day on no hormone MS culture mediums
Afterwards, the explant infected for the first strain is obtained；Preferably, preculture 0,1,2 or 3 days.
Preferably, first strain be Agrobacterium rhizogenesA4, ACCC10060, ACCC15834, SW101 in one kind or
It is a variety of.
In a specific embodiment of the invention, the subculture medium is the MS culture mediums containing following component：6-
BA2.5mg/L, IAA2.5mg/L, 30g/L sucrose, 5g/L plant gels.
In an embodiment of the present invention, in the step (1), take and (preferably cultivated in soil) blade of radix bupleuri seedling
And petiole, the sterilized degerming rear acquisition explant infected for the second strain.
Further, the blade and petiole of the radix bupleuri seedling of the sterilized sterilizing of the acquisition are placed in no hormone MS culture mediums
Upper preculture obtains the explant infected for the second strain after 0~3 day；Preferably, preculture 0,1,2 or 3 days.
Further, the degerming method of the sterilization includes：Using 75% alcohol-pickled 30~50S, 20% time
Sodium chlorate (NaClO) solution Seal treatment 5-10min, aseptic water washing 3~5 times.
Preferably, second strain is agrobacterium rhizogenes SW101.
In an embodiment of the present invention, in the step (2), the bacterium solution OD values of bacterial strain uses therefor are 0.8~1.2.
In a preferred embodiment, in the step (2), the bacterium solution OD values of bacterial strain uses therefor are 0.8,1 or 1.2.
In a preferred embodiment, in the step (3), the time for shaking training dip-dye is 20~25min.
In an embodiment of the present invention, when the step (3) takes a) step when, bacterial strain uses therefor be Agrobacterium rhizogenesA4,
One or more in ACCC10060, ACCC15834, SW101.
Further, when the step (3) takes a) step when, the time that co-cultures is 3,4 or 5 days.
In an embodiment of the present invention, when the step (3) takes b) step when, bacterial strain uses therefor be agrobacterium rhizogenes SW101.
Further, when the step (3) takes b) step when, alternate culture 4,5,6 or 7 days.
In an embodiment of the present invention, in the step (4), micro-organisms base for containing 500mg/L Cefotaxime Sodium (or
Car MS culture mediums).
It will be appreciated by persons skilled in the art that in addition to example shown of the embodiment of the present invention, other can also be used
Micro-organisms base well-known to those skilled in the art, such as using 400 or 300mg/L Cefotaxime Sodium (or car) MS
Culture medium.
In an embodiment of the present invention, in the step (4), dark culturing obtains the hairy root of induction after 20~30 days.
In an embodiment of the present invention, in the step (4), the hairy root of the induction of acquisition, every 30 days subcultures are once, common
Antibacterial processing 90~100 days, is transferred in ordinary culture medium (the MS fluid nutrient mediums such as without Cefotaxime Sodium (or Ka Na)), continues
Shaken cultivation (specifically, 150rpm dark culturings), obtains the hairy root largely induced.
Second aspect of the present invention provides a kind of method for improving Bupleurum Chinese hairy root genetic transformation efficiency, including following step
Suddenly：
(1) explant for infecting is obtained；
(2) the agrobacterium rhizogenes bacterium solution for infecting, the agrobacterium rhizogenes foreign gene-carrying are obtained；
(3) conversion explant is co-cultured：By step 1) obtained by explant be placed in step 2) hair that infects of gained
In root Agrobacterium bacterium solution, shake training and contaminate；Then MS culture mediums are transferred to, according to a) or b) handling：
A) co-cultured 3~5 days under dark condition；
B) according to illumination/dark (preferably 16 hours illumination/8 hour dark) alternate culture 4~7 days；
(4) induction of hairy root：Take step 3) processing explant be placed in micro-organisms base, dark culturing, induction hair
Root, obtains the hairy root of induction.
In an embodiment of the present invention, in the step (1), the step of described acquisition is used for the explant infected is specific
Including：Disinfection seed rudiment is obtained after seedling, cuts seedling root, is cultivated into subculture medium, obtains point of sterile unrooted
Change seedling, that is, obtain the explant infected for the first strain.
In a specific embodiment of the invention, culture in MS solid mediums is inoculated in after seed disinfection and obtains sterile children
Seedling.
It will be appreciated by persons skilled in the art that in addition to example shown of the embodiment of the present invention, other can also be used
Method seed is carried out disinfection, it is degerming, and cultivate to seed germination, obtain aseptic seedlings.
Further, the differentiation seedling of the sterile unrooted of the acquisition is placed in preculture 0~3 day on no hormone MS culture mediums
Afterwards, the explant infected for the first strain is obtained；Preferably, preculture 0,1,2 or 3 days.
Preferably, first strain be Agrobacterium rhizogenesA4, ACCC10060, ACCC15834, SW101 in one kind or
It is a variety of.
In a specific embodiment of the invention, the subculture medium is the MS culture mediums containing following component：6-
BA2.5mg/L, IAA2.5mg/L, 30g/L sucrose, 5g/L plant gels.
In an embodiment of the present invention, in the step (1), take and (preferably cultivated in soil) blade of radix bupleuri seedling
And petiole, the sterilized degerming rear acquisition explant infected for the second strain.
Further, the blade and petiole of the radix bupleuri seedling of the sterilized sterilizing of the acquisition are placed in no hormone MS culture mediums
Upper preculture obtains the explant infected for the second strain after 0~3 day；Preferably, preculture 0,1,2 or 3 days.
Further, the degerming method of the sterilization includes：Using 75% alcohol-pickled 30~50S, 20% time
Sodium chlorate (NaClO) solution Seal treatment 5-10min, aseptic water washing 3~5 times.
Preferably, second strain is agrobacterium rhizogenes SW101.
In an embodiment of the present invention, in the step (2), the agrobacterium rhizogenes is comprising foreign gene or inserted with outer
The plasmid of source gene.Preferably, the plasmid is PK2GW7.
In an embodiment of the present invention, in the step (2), the bacterium solution OD values of bacterial strain uses therefor are 0.8~1.2.
In a preferred embodiment, in the step (2), the bacterium solution OD values of bacterial strain uses therefor are 0.8,1 or 1.2.
In a preferred embodiment, in the step (3), the time for shaking training dip-dye is 20~25min.
In an embodiment of the present invention, when the step (3) takes a) step when, bacterial strain uses therefor be Agrobacterium rhizogenesA4,
One or more in ACCC10060, ACCC15834, SW101.
Further, when the step (3) takes a) step when, the time that co-cultures is 3,4 or 5 days.
In an embodiment of the present invention, when the step (3) takes b) step when, bacterial strain uses therefor be agrobacterium rhizogenes SW101.
Further, when the step (3) takes b) step when, alternate culture 4,5,6 or 7 days.
In an embodiment of the present invention, in the step (4), micro-organisms base for containing 500mg/L Cefotaxime Sodium (or
Car MS culture mediums).
It will be appreciated by persons skilled in the art that in addition to example shown of the embodiment of the present invention, other can also be used
Micro-organisms base well-known to those skilled in the art, such as using 400 or 300mg/L Cefotaxime Sodium (or car) MS
Culture medium.
In an embodiment of the present invention, in the step (4), dark culturing obtains the hairy root of induction after 20~30 days.
In an embodiment of the present invention, in the step (4), the hairy root of the induction of acquisition, every 30 days subcultures are once, common
Antibacterial processing 90~100 days, is transferred in ordinary culture medium (the MS fluid nutrient mediums such as without Cefotaxime Sodium (or Ka Na)), continues
Shaken cultivation (specifically, 150rpm dark culturings), obtains the hairy root largely induced.
Third aspect present invention provide it is a kind of improve Bupleurum Chinese hairy root induction efficiency method Bupleurum Chinese hairy root induction,
Genetic transformation, gene functional research and/or Bupleurum Chinese metabolite (preferably, are secondary metabolite, it is highly preferred that serving as reasons
The secondary metabolite of medical value) production in application.
Fourth aspect present invention provides a kind of method for improving Bupleurum Chinese hairy root genetic transformation efficiency in Bupleurum Chinese hairy root
Induction, genetic transformation, functional gene research and/or Bupleurum Chinese hairy root metabolite (are preferably, secondary metabolite, more
Preferably, it is secondary metabolite by medical value) application in production.
Compared with prior art, the beneficial effects of the present invention are：
The present invention based on to agrobacterium rhizogene strain, infect the conditions such as bacterium solution OD values, co-cultivation time and grope, it is thus identified that
Improve the optimum combination condition of the infect efficiency of agrobacterium rhizogenes.And give SW101 induction Bupleurum Chinese hairy root and A4,
ACCC10060, ACCC15834 induce the optimal inductive condition of Bupleurum Chinese hairy root.Lured compared to 10% or so traditional hairy root
Conductance, the hairy root induction rate that the technical scheme that the present invention is provided is obtained reaches 70%-85%.
Further, present invention also offers the technical scheme for improving Bupleurum Chinese hairy root genetic transformation efficiency, by reality
Checking, the technical scheme that the present invention is provided can obtain more than 80% gene transformation rate, be Bupleurum Chinese functional gene
Research and/or the industrialization of Bupleurum Chinese hairy root metabolite provide more preferable, more efficient, easier selection.
Brief description of the drawings
Fig. 1 is that agrobacterium rhizogenes provided in an embodiment of the present invention infects Bupleurum Chinese explant generation root of hair photo；
Fig. 2 is upgrowth situation after Bupleurum Chinese hairy root culture provided in an embodiment of the present invention 60 days；
Fig. 3 is that PCR amplification vectors card provided in an embodiment of the present invention receives the electrophoresis result of resistance gene fragment.
Embodiment
The present invention is described in further details with example below in conjunction with the accompanying drawings.
If it is understood that without specified otherwise, the present invention uses commercial reagents using reagent.
The Chinese lexical or textual analysis of english abbreviation of the present invention is as follows：
cef：Cephalosporin, Cefotaxime Sodium；
6-BA：6- benzyl aminoadenines；
AS：Acetosyringone；
IAA：Heteroauxin.
Embodiment 1
The embodiments of the invention provide a kind of method for improving Bupleurum Chinese hairy root induction efficiency, comprise the following steps：
First, the acquisition of explant
1st, Bupleurum Chinese dry seedses, rinse floating dust, then rinsed several times with sterile purified water with running water；
2nd, step is operated in super-clean bench afterwards：With 75% alcohol-pickled 30~50S；
3rd, 20% sodium hypochlorite (NaClO) solution Seal treatment 15min, clean 3~5 times of aseptic water washing；
4th, by the aseptic filter paper suck dry moisture of the seed after sterilization, common MS cultures are based on 25 DEG C of dark culturings, per 2-3
My god, microbiological contamination seed is produced, substantially without microbiological contamination phenomenon after 8-9 days, culture was to 14 days or so, and seed starts to sprout；
5th, seedling root is cut, (MS+6-BA2.5mg/L+IAA2.5mg/L+30g/L sucrose+the 5g/ into subculture medium
L plant gels), the differentiation seedling of sterile unrooted is obtained, the explant infected is as subsequently used for.
2nd, agrobacterium rhizogenes activates
1st, agrobacterium rhizogenes is inoculated in the LB culture mediums of rifampin containing 50mg/L (Rif), 28 DEG C of light cultures 2 days；
2nd, picking single bacterium colony, in the LB fluid nutrient mediums of the rifampin containing 50mg/L, 28 DEG C, 180r/min vibration light cultures
24h；
3rd, by 1: 100 dilution bacterium solution, continue to shake training to OD600=0.8~1.2,5 000rmin-110min is centrifuged, is received
Collect thalline；
4th, it is resuspended and is precipitated with MS+200 μM of isometric AS fluid nutrient medium, for infects.
3rd, agrobacterium rhizogenes is infected (hairy root induction)
1st, the explant obtained by step one is placed in re-suspension liquid and 20~25min of training is shaken at 100rpm25 DEG C；
2nd, MS culture mediums are transferred to, dark co-cultures 3~5d；
3rd, the explant after co-culturing is in cef containing 500mg/L (or car) MS culture mediums, 25 DEG C of dark culturings, 20-
The root system grown fine is selected after 30d, the different hairy root root systems as induced；
4th, per 30d subcultures once, in common antibacterial processing 90-100d, the MS fluid nutrient mediums for being transferred to no cef (or car), after
Persistent oscillation culture (150rpm dark culturings).
It is as shown in Figure 1 that agrobacterium rhizogenes provided in an embodiment of the present invention infects Bupleurum Chinese explant generation root of hair photo；North
Upgrowth situation is as shown in Figure 2 after radix bupleuri hairy root culture 60 days.By analysis, the embodiments of the invention provide raising Bupleurum Chinese
Hairy root induction efficiency method, induction result obtains unexpected effect compared to existing method (10% hairy root induction rate)
Really：70%~85% explant can induce hairy root (i.e. hairy root induction efficiency reaches more than 70%), and each outer
Hairy radical mesh is at 5~10 in implant.
Alternative embodiment 1
In order to further illustrate beneficial effects of the present invention, the invention provides following alternative embodiment 1, alternative embodiment
1 replaces some conditions in the step of embodiment 1 one to three, and other are constant, and corresponding test result is referring to (the d of table 1：My god；min：
Minute；h：Hour)：
Table 1.
In table 1, the preculture is specially by embodiment 1 Step 1: " acquisition of explant " the 5th point of " sterile nothing of acquisition
The differentiation seedling of root, is as subsequently used for the explant infected " replace with：" the differentiation seedling of the sterile unrooted of acquisition, is placed in nothing
Preculture is after 0~3 day on hormone MS culture mediums, and acquisition is subsequently used for the explant that strain infects；Specifically, preculture 0,1,2
Or 3 days ".If the explant used is " blade and petiole ", rather than described in embodiment 1 when " no Furcation defects seedling ", " blade
And petiole " acquisition methods be：Fetch earth the blade and petiole of the radix bupleuri seedling cultivated in earth, it is sterilized it is degerming after be used to infect,
Sterilize degerming method：With 75% alcohol-pickled 30~50S, 20% sodium hypochlorite (NaClO) solution Seal treatment 5-
10min, aseptic water washing 3~5 times.Note：Injection is that bacterium solution is expelled into each position of seedling, then directly in induction root of hair
Cultivated on culture medium.
As shown in Table 1：Groped based on each condition in alternative embodiment 1, the preliminary infect efficiency master for confirming agrobacterium rhizogenes
Will to bacterial strain, to infect the factors such as bacterium solution OD values, co-cultivation time related.
Alternative embodiment 2
In order to illustrate further beneficial effects of the present invention, the invention provides following alternative embodiment 2, replace and implement
Example 2 replaces some conditions in the step of embodiment 1 one to three, and other are constant, and corresponding test result is referring to table 2,3 (d：My god；
min：Minute；h：Hour)：
Table 2.
Table 3.
From table 2,3, OD values are too high or too low influences Rooting percent；Co-culture overlong time or too short influence root of hair
Rate.
Comparative example
In order to illustrate further beneficial effects of the present invention, the invention provides following comparative example, contrast is implemented
Example replaces some conditions in the step of embodiment 1 one, and other are constant, and corresponding test result is referring to table 4：
Table 4.
| Formula | Induce differentiated result |
| MS+KT0.5mg/L+30g/L sucrose+5g/L plant gels | Appreciation rate is about 1:1 |
| MS+6-BA 2.5mg/L+IAA 2.5mg/L+30g/L sucrose+5g/L plant gels | Appreciation rate is about 1:5 |
| MS+6-BA 0.5mg/L+IAA 0.5mg/L+30g/L sucrose+5g/L plant gels | Appreciation rate is about 1:2 |
| MS+6-BA 5mg/L+IAA.5mg/L+30g/L sucrose+5g/L plant gels | Appreciation rate is about 1:10 but vitrifying occurs in seedling |
The 2nd group of condition in the experiment condition of the step one of the embodiment of the present invention 1, i.e. form 4 is determined by table 4.
Embodiment 2
The embodiments of the invention provide a kind of method for improving Bupleurum Chinese hairy root genetic transformation efficiency, including following step
Suddenly：
First, the genetic transformation of hairy root
First, the preparation of competent cell：
Take strain, streak inoculation is on the LB culture mediums of the rifampin containing 50mg/L, 28 DEG C of light cultures 2 days；Picking single bacterium colony,
In the LB fluid nutrient mediums of the rifampin containing 50mg/L, 28 DEG C, 180r/min vibration light cultures 24h；Bacterium solution is diluted by 1: 50, after
It is continuous to shake training to OD600=0.6, bacterium solution ice bath 15min is sub-packed in 1.5mL sterile centrifugation tubes, 4 DEG C of centrifugations of 5000rpm
5min；Supernatant is abandoned, in the sterile CaCl2 of 0.05mol/L that thalline is resuspended in 600 μ L ice precoolings, ice bath 30min, 5000rpm 4
DEG C centrifugation 10min；Supernatant is abandoned, in the sterile CaCl2 of 0.05mol/L that thalline is resuspended in 100 μ L ice precoolings；- 80 DEG C of preservations, are supplied
Conversion is used；
2nd, freeze-thaw method is converted：
1st, that the plasmid (this example be PK2GW7, Invitrogen) with foreign gene is added into 100 μ L competence is thin
In born of the same parents, mixing is flicked, 30min is placed on ice；Quick-frozen 5min in liquid nitrogen, 37 DEG C of warm bath 5min, then ice bath 2min；Add 700 μ L
LB fluid nutrient mediums, 28 DEG C are shaken training 12h, 6000rpm centrifugation 3min；Supernatant is abandoned, retains 100 μ L bacterium solutions, pipette tips are mixed, with coating
Device is spread evenly across on the antibiotic of the corresponding resistant containing plasmid (this example is Ka Na) and the LB flat boards of 50mg/L rifampins, 28 DEG C
Culture 2d is inverted to there is single bacterium colony；
2nd, picking single bacterium colony, 28 DEG C, 180r/min vibration light culture 24h, bacterium solution PCR confirm convert successfully；
3rd, 1 successful single bacterium colony of conversion is selected, expands the culture amount of bacterium, for hairy root induction；Induction step referring to
Embodiment 1 and three the step of alternative embodiment 1-2；The experimental procedure of the embodiment of the present invention 2 is obtained respectively；
3rd, PCR verifies changing effect：
1st, hairy kan gene group DNA rapid extractions：Take and be about 2cm hairy root samples in centrifuge tube, add 20 μ L0.2M
NaOH solution, using high-flux tissue mill 4000rpm work 10s, interval 10s, totally 3 times, after crushing, 3500rpm is centrifuged
1min, plus 490 μ L 0.1M Tris-HCl, vortex oscillation, 3500rpm centrifugation 1min take 1.5 μ L of supernatant liquid to be expanded for PCR.
2nd, using hairy kan gene group DNA as template, amplification plasmid vector (this example be PK2GW7) distinctive card receives resistance
Genetic fragment, control (CK), primer sequence (precious biosynthesis) K-2F are used as using the hairy root that unconverted plasmid-bearing strains are induced:
ACTGGGCACAACAGACAATC (SEQ ID NO.1), K-2R:CTTCAGCAATATCACGGGTAG(SEQ ID NO.2).
PCR system (10 μ l) and program：
Prim STAR HS (raw work):5 μ L, template:1.5 μ l, primer each 0.5 μ l, ddH20:2.5μl.
Program：95 DEG C, 5min, (98 DEG C of 10s, 58 DEG C of 5s, 72 DEG C of 20s) 35 are circulated, 72 DEG C of 5min.
The embodiment of the present invention 2 is by carrier PK2GW7 (Invitrogen) transforming agrobacterium rhizogenes, according to embodiment 1 and replacement
The method of embodiment 1-2 step 3 carries out agrobacterium rhizogenes and infects Bupleurum Chinese explant, is resisted using the Ka Na on PCR amplification vectors
Property gene, differentiate hairy root generation, can draw this method hairy root induction transformation efficiency reach 70%-85%.
Fig. 3 is embodiment when carrying out agrobacterium rhizogenes according to the method for the step 3 of embodiment 1 to infect Bupleurum Chinese explant
2PCR amplification vector cards receive the electrophoresis result of resistance gene fragment, M:DNA molecular amount standard (Marker), swimming lane 1-7：Transgenosis
Hairy root root system, CK：The hairy root root system of PK2GW7 carriers is not carried.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (10)
1. a kind of method for improving Bupleurum Chinese hairy root induction efficiency, it is characterised in that comprise the following steps：
(1) explant for infecting is obtained；
(2) the agrobacterium rhizogenes bacterium solution for infecting is obtained；
(3) conversion explant is co-cultured：By step 1) obtained by explant be placed in step 2) the root of hair agriculture infected of gained
In bacillus bacterium solution, shake training and contaminate；Then MS culture mediums are transferred to, according to a) or b) handling：
A) co-cultured 3~5 days under dark condition；
B) according to illumination/dark alternate culture 4~7 days；
(4) induction of hairy root：Take step 3) processing explant be placed in micro-organisms base, dark culturing, induce root of hair, obtain
The hairy root that must be induced.
2. the method for Bupleurum Chinese hairy root induction efficiency is improved as claimed in claim 1, it is characterised in that the step (1)
In specifically include a) or b)：
A) disinfection seed rudiment is obtained after seedling, cuts seedling root, is cultivated into subculture medium, obtains point of sterile unrooted
Change seedling, obtain the explant for infecting；Or
B) take the blade and petiole of radix bupleuri seedling, it is sterilized it is degerming after obtain explant for infecting.
3. the method for Bupleurum Chinese hairy root induction efficiency is improved as claimed in claim 2, it is characterised in that the step (a)
In, the subculture medium is the MS culture mediums containing following component：6-BA 2.5mg/L, IAA 2.5mg/L, 30g/L sucrose,
5g/L plant gels.
4. the method for Bupleurum Chinese hairy root induction efficiency is improved as claimed in claim 1, it is characterised in that the step (2)
In, the bacterium solution OD values of bacterial strain uses therefor are 0.8~1.2.
5. the method for Bupleurum Chinese hairy root induction efficiency is improved as claimed in claim 1, it is characterised in that the step (3)
In, the time for shaking training dip-dye is 20~25min.
6. the method for Bupleurum Chinese hairy root induction efficiency is improved as claimed in claim 1, it is characterised in that when the step
(3) take a) step when, bacterial strain uses therefor be Agrobacterium rhizogenesA4, ACCC10060, ACCC15834, SW101 in one or more.
7. the method for Bupleurum Chinese hairy root induction efficiency is improved as claimed in claim 1, it is characterised in that when the step
(3) take b) step when, bacterial strain uses therefor be agrobacterium rhizogenes SW101.
8. a kind of method for improving Bupleurum Chinese hairy root genetic transformation efficiency, it is characterised in that comprise the following steps：
(1) explant for infecting is obtained；
(2) the agrobacterium rhizogenes bacterium solution for infecting is obtained, the agrobacterium rhizogenes is comprising foreign gene or inserted with external source base
The plasmid of cause；
(3) conversion explant is co-cultured：By step 1) obtained by explant be placed in step 2) the root of hair agriculture infected of gained
In bacillus bacterium solution, shake training and contaminate；Then MS culture mediums are transferred to, according to a) or b) handling：
A) co-cultured 3~5 days under dark condition；
B) according to illumination/dark alternate culture 4~7 days；
(4) induction of hairy root：Take step 3) processing explant be placed in micro-organisms base, dark culturing, induce root of hair, obtain
The hairy root that must be induced.
9. it is a kind of it is as claimed in claim 1 improve Bupleurum Chinese hairy root induction efficiency method Bupleurum Chinese hairy root induction,
Application in genetic transformation, gene functional research and/or the production of Bupleurum Chinese metabolite.
10. a kind of method as claimed in claim 8 for improving Bupleurum Chinese hairy root genetic transformation efficiency is in Bupleurum Chinese hairy root
Application in induction, genetic transformation, gene functional research and/or the production of Bupleurum Chinese metabolite.
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| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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| CN107760712A (en) * | 2017-12-14 | 2018-03-06 | 湖南科技大学 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
| CN108913716A (en) * | 2018-08-01 | 2018-11-30 | 成都大学 | A kind of rapid induction quinoa hairy method |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
| CN112795590A (en) * | 2021-03-29 | 2021-05-14 | 兰州大学 | Method for transforming hairy root of sweet clover |
| CN113980885A (en) * | 2021-12-07 | 2022-01-28 | 西南科技大学 | Preparation and instantaneous transformation method of bupleurum protoplast |
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