CN113652446B - Agrobacterium rhizogenes mediated one-step transformation method for hairy roots of Caragana carryover - Google Patents
Agrobacterium rhizogenes mediated one-step transformation method for hairy roots of Caragana carryover Download PDFInfo
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Abstract
The invention discloses an agrobacterium rhizogenes mediated one-step transformation method of hairy roots of middle caragana, which comprises the following steps: s1, electrically transforming a plasmid containing an e-GFP expression vector into Agrobacterium rhizogenes K599 competent cells; s2, preparing OD 600nm 0.8 to 0.95 of agrobacterium rhizogenes suspension, and acetosyringone is added; s3, culturing bacterial paste used for infection; s4, selecting middle caragana seedlings, chamfering the 1cm position below cotyledonary nodes of the seedlings, discarding the lower part, infecting the incision, transplanting the seedlings back into soil, pouring 1/4B5 culture medium into the seedlings, and culturing in an illumination incubator. Compared with the method for transforming the hairy roots of the middle caragana by using a tissue culture method, the method for transforming the hairy roots of the middle caragana by using the one-step method enables the gene to be excessively expressed in the hairy roots, and has the advantages of simpler operation, no need of preparing aseptic seedlings, shorter detection time, high gene expression level and convenient detection.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to an agrobacterium rhizogenes mediated one-step transformation method of hairy roots of middle caragana.
Background
The concept of hairy roots was proposed by STEWART for the earliest 1900 years. In 1907, smith and Townsend found that agrobacterium rhizogenes could infect plants to produce hairy roots. The agrobacterium plasmid mediated method is a high-efficiency transformation method in plant genetic engineering. The majority of transgenic plants are obtained by mediation of the Ti plasmid of Agrobacterium tumefaciens. Chilton in 1982 reported that hairy roots were caused by the Agrobacterium rhizogenes Ri plasmid. So far, the hairy root technology has entered a brand new era. Related studies of Ri plasmid-mediated genetic transformation of plant hairy roots have been carried out among a number of plant species. The hairy root culture technology is mainly used for synthesizing plant secondary metabolites and promoting gene expression, and is also used as an important technical means for enhancing plant adaptability and improving plant stress tolerance. Research on plant hairy root culture was initiated in Zhou Ligang et al 1998. 2015, pan Yan et al discuss pseudo-ginseng hairy root induction technology and application. 2018 roqueen and the like establish a cotton hairy root transformation system. Research progress of the hairy root of transgenic fleece-flower root was published in 2020, luo Qiaoni et al. At present, hairy root induction has been widely used in herbaceous plants, woody plants, shrubs, etc., mainly in economic plants and medicinal plants. In recent years, plant hairy root transformation systems are established by corn, spinach, liquorice, broccoli, black nightshade, stevia rebaudiana, cucumber, red sage root and the like.
The middle caragana (Caragana intermedia) is caragana shrub belonging to the family Leguminosae, and is a main perennial drought deciduous shrub species in loess plateau areas. The caragana plants are widely distributed in inner Mongolia areas, and are used as dominant species for wind prevention, sand fixation and water and soil conservation in Ningxia, gansu and Shanxi arid areas. The adaptability and stress resistance of the middle caragana can be improved through hairy root induction, and the variety is improved, so that the method has important significance for production, living and environmental construction. At present, the genetic transformation system of the hairy roots of the middle caragana is not established. Methods for transforming hairy roots of Caragana intermedia using a one-step method have also been rarely reported. Therefore, it is necessary to provide a method for one-step transformation of hairy roots of Caragana microphylla mediated by Agrobacterium rhizogenes, which provides a convenient way for researching the gene function of Caragana microphylla and lays a foundation for the deep research of genetic transformation of Caragana microphylla.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention aims to provide an agrobacterium rhizogenes mediated one-step transformation method of the hairy roots of the middle caragana.
An agrobacterium rhizogenes mediated one-step transformation method of hairy roots of Caragana intermedia, comprising the following steps:
s1, electrically transforming a plasmid containing an e-GFP expression vector into Agrobacterium rhizogenes K599 competent cells;
s2, inoculating agrobacterium rhizogenes K599 obtained in the step S1 into an LB liquid medium for culture to prepare OD 600nm Agrobacterium rhizogenes bacterial suspension with the concentration of 0.8-0.95 and preparing the obtained rhizogenesAdding acetosyringone with the final concentration of 25-100 mu mol/mL into the bacillus suspension;
s3, taking part of agrobacterium rhizogenes bacterial suspension added with acetosyringone in the step S2, centrifuging, removing supernatant, uniformly mixing the rest precipitate, then sucking the uniformly mixed precipitate, adding the uniformly mixed precipitate into an LB solid culture medium for coating, and placing the coated precipitate into an incubator to culture bacterial paste used for infection, wherein the operations are carried out under the condition of an ultra-clean workbench at room temperature;
s4, selecting middle caragana seedlings, chamfering the 1cm position below the cotyledonary node of the middle caragana seedlings, discarding the lower part, immersing the cotyledonary node incision of the middle caragana seedlings in agrobacterium rhizogenes bacterial suspension after acetosyringone is added in the step S2 in dark environment, scraping the cotyledonary node incision of the middle caragana seedlings to obtain bacterial paste obtained in the step S3, directly transplanting the middle caragana seedlings back to soil after scraping the bacterial paste, pouring 1/4B5 culture medium to the middle caragana seedlings, culturing the middle caragana seedlings in an illumination incubator, and opening a vent of a seedling culturing tray where the middle caragana seedlings are positioned after 12-20 d.
Preferably, the e-GFP-containing reporter gene expression vector is pEGAD.
Preferably, the OD of the agrobacterium rhizogenes suspension described in step S2 600nm The value is 0.9, and the final concentration of acetosyringone is 50 mu mol/mL.
Preferably, the bacterial paste used for the infection in step S3 is cultivated: the temperature of the incubator was set at 37℃and the incubation time was 10 to 16 hours.
Preferably, the soaking time of the middle caragana seedling cotyledonary node incision in the step S4 is 1min, and the thickness of the bacterial paste at the incision section is 0.1-0.5 mm.
Preferably, the amount of 1/4B5 medium used in step S4 is 5mL.
Preferably, the specific steps of the step S2 are as follows: inoculating Agrobacterium rhizogenes K599 obtained in step S1 into LB liquid medium, shake culturing at 28deg.C to OD 600nm Obtaining a primary bacterial suspension of 0.9, and then transferring the primary bacterial suspension to fresh LB liquid medium againAt 28 ℃, the primary bacterial suspension is cultured to OD in a shaking way 600nm After 0.8 to 0.95, obtaining agrobacterium rhizogenes bacterial suspension, adding acetosyringone into the obtained agrobacterium rhizogenes bacterial suspension, and standing; the volume ratio of the primary bacterial suspension to the fresh LB liquid medium is 1:100.
Preferably, step S4 selects middle caragana seedlings which grow for 4-5 d and have good vigour.
Preferably, the culture conditions in step S4 are: the humidity was 90% and the temperature was 28 ℃.
The one-step conversion method further comprises the following steps:
s5, observing the growth condition of the hairy roots of the caragana intermedia, and detecting the expression condition of the e-GFP reporter gene at 14-28 d.
The LB liquid culture medium is prepared according to the following method: taking 1g of Tryptone (Tryptone), 0.5g of Yeast extract (Yeast extract) and 1g of sodium chloride (NaCl), adjusting the pH value to 7.0 by using NaOH or HCl, adding deionized water to a volume of 100mL, and carrying out high-pressure sterilization at 121 ℃ for 20min.
The LB liquid medium contains 50. Mu.g/mL kanamycin and 50. Mu.g/mL streptomycin.
The 1/4B5 culture medium refers to a 1/4 concentration culture medium of a B5 standard culture medium.
The OD is 600nm The value is an optical density value measured when the wavelength is set to 600nm, and is a standard index for tracking the microbial density in a liquid culture, and is generally used to indicate the cell density of a cell.
Compared with the prior art, the invention has the beneficial effects that:
1. compared with the method for transforming the hairy roots of the middle caragana by using a tissue culture method, the method has the advantages of simpler operation, no need of preparing aseptic seedlings, shorter detection time, high gene expression level and convenient detection, and the generated hairy roots of the plants can be directly used for subsequent experiments after being re-detected, thereby providing a new way for carrying out gene function verification and screening functional genes.
2. Compared with the injection method for stabbing plant hypocotyls to perform hairy root transformation, the one-step transformation method has higher transformation efficiency, the transformation efficiency of the transformation of the hairy roots of the middle caragana can reach more than 40%, and the generated hairy roots are more.
3. In the process of converting the hairy roots of the middle caragana by a one-step method, the invention optimizes the bacterial liquid concentration of agrobacterium rhizogenes and the concentration of added acetosyringone, and finally shows that the bacterial liquid concentration is OD 600nm The transformation efficiency is optimal when the transformation efficiency is 0.8-0.95, and the transformation efficiency of hairy roots can be further improved when acetosyringone with the working concentration of 25-100 mu mol/mL is added, so that a foundation is laid for the deep research of hairy roots of middle caragana.
4. In the method, besides the step of infecting the hypocotyl of the middle caragana by using the agrobacterium rhizogenes bacterial suspension, more importantly, the beveled plant hypocotyl is scraped to obtain the agrobacterium rhizogenes bacterial paste, and the step of scraping the bacterial paste is added, so that the success rate of transformation of hairy roots of the middle caragana is greatly improved.
5. According to the invention, the research shows that pouring the 1/4B5 culture medium into the middle caragana seedlings planted back into the soil after infection is more beneficial to the growth of the hairy roots of the middle caragana, and is also beneficial to the growth of the middle caragana seedlings, and the middle caragana seedlings are not required to be watered within 2 weeks after pouring the 1/4B5 culture medium.
Drawings
FIG. 1 is a schematic diagram of the structure of a plant expression vector pEGAD;
FIG. 2 shows the infection and cultivation process of middle caragana seedlings in the example of the present invention: wherein, figure a is middle caragana seedling growing for 5 days, figure B is the middle caragana seedling cotyledon node is subjected to beveling treatment, figure c is bacterial paste scraping, figure d is the middle caragana seedling is poured with 1/4B5 culture medium, figure e is the middle caragana seedling is placed in an illumination incubator for culture, and figure f is a hairy root phenotype figure after the middle caragana seedling is transformed for 14 d;
FIG. 3 is a hairy root chart (B) of middle Caragana seedlings after 14d infestation with Agrobacterium rhizogenes and a middle Caragana seedling chart (A) without Agrobacterium rhizogenes infestation in an example of the present invention;
FIG. 4 is a diagram showing the detection of the intermediate Caragana hairy root eGFP after transformation in the example of the present invention.
Detailed Description
The present invention is described more fully below in order to enable those skilled in the art to better understand the aspects of the invention.
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. In the present specification, "part" and "%" respectively mean "part by mass" and "% by mass" unless otherwise specified.
The expression vector pEGAD in the invention is derived from the gift of agricultural university in China, and Agrobacterium rhizogenes K599 is purchased from Shanghai Weidi Biotechnology Co.
Example 1
An agrobacterium rhizogenes mediated one-step transformation method of hairy roots of Caragana intermedia, comprising the following steps:
s1, electrically transforming a plasmid containing an e-GFP expression vector into competent cells of Agrobacterium rhizogenes K599
S11, taking out the pEGAD plasmid, thawing, and placing on ice;
s12, taking out agrobacterium rhizogenes K599 competent cells from a refrigerator at the temperature of minus 80 ℃ to be thawed and placed on ice;
s13, cleaning an electrode cup: soaking the electrode cup in 75% ethanol for 10min, soaking in 100% ethanol for 10min, cleaning with clear water, drying in a fume hood, and precooling after drying;
s14, adding 1 mu L pEGAD plasmid into competent cells of agrobacterium rhizogenes K599, flicking and uniformly mixing, and then sucking all agrobacterium rhizogenes K599 out by using a sheared and sterilized gun head, and adding the all agrobacterium rhizogenes K599 into a cleaned precooled electrode cup (U=1400V, T=5.4-5.8 ms) to obtain a conversion solution;
s15, sucking out all the conversion liquid in the step S14, adding the conversion liquid into a 1.5mL EP tube containing 800 mu L of LB liquid medium, and carrying out shaking culture for 2 hours at 28 ℃ at the shaking speed of 160rpm to obtain bacterial liquid;
s16, sucking 50 mu L of the bacterial liquid in the step S15, coating the bacterial liquid in a double-resistance plate containing 50 mu g/mL kanamycin and 50 mu g/mL streptomycin, and inversely culturing at 28 ℃ for 36-48 h;
s17, picking single colonies on the double-resistance plate in the step S16, and identifying whether the expression vector pEGAD is successfully transferred into agrobacterium rhizogenes K599 by colony PCR.
S2, preparing agrobacterium rhizogenes bacterial suspension
S21 Agrobacterium rhizogenes K599 identified as containing the plasmid vector pEGAD-e-GFP was inoculated into 4mL LB liquid medium (containing 50. Mu.g/mL kanamycin and 50. Mu.g/mL streptomycin), shaken overnight at 28℃at a shaking speed of 180rpm, and shake-cultured overnight to OD 600nm The value is about 0.9, and the primary bacterial suspension is obtained;
s22, the next day, a part of the primary fungus suspension is preserved, and the step of preserving is as follows: 1mL of the primary bacterial suspension is put into a 1.5mL EP tube, the primary bacterial suspension is centrifuged for 2min at 4000rpm, the supernatant is discarded, 800 mu L of fresh LB liquid medium and 200 mu L of 75% glycerol are added, the mixture is uniformly mixed, quick-frozen by liquid nitrogen and stored in a refrigerator at-80 ℃ for a long time, the other part of the primary bacterial suspension is subjected to expansion culture, the part of the primary bacterial suspension is transferred into fresh LB liquid medium (containing 50 mu g/mL kanamycin and 50 mu g/mL streptomycin) again, and bacterial shaking is carried out at 28 ℃ at 180rpm until the OD 600nm Obtaining agrobacterium rhizogenes bacterial suspension with the concentration of 0.9; the volume ratio of the primary bacterial suspension to the fresh LB liquid culture medium is 1:100, the primary bacterial suspension is preferably 1mL, and the fresh LB liquid culture medium is preferably 100mL.
S23, adding acetosyringone with the final concentration of 50 mu mol/mL into agrobacterium rhizogenes obtained in the step S22 as an inducer, fully and uniformly mixing, and standing for 12 hours in darkness at room temperature, wherein the preparation method of the acetosyringone with the final concentration of 50 mu mol/mL comprises the following steps: 0.3924g acetosyringone is weighed and dissolved in 10mL DMSO, after the acetosyringone is completely dissolved, ultrapure water is used for fixing the volume to 20mL, and the acetosyringone is packaged into a 1mL centrifuge tube and is preserved at the temperature of minus 20 ℃ for standby.
S3, preparing bacterial paste for infection
Taking 1ml of agrobacterium rhizogenes suspension added with acetosyringone, centrifuging, removing part of supernatant, uniformly mixing and precipitating, absorbing 50 mu l of uniformly mixed precipitate, adding the precipitate into LB solid medium for coating, placing the solid medium into a 37 ℃ incubator for culturing for 10-16 hours to prepare bacterial paste used for infection, and carrying out experiments under the condition of an ultra-clean workbench at room temperature.
S4, one-step conversion procedure
S41, planting and selecting the middle caragana: weighing laboratory special nutrient soil and vermiculite, sterilizing in a sterilizing pot, picking full and insect-free seeds of a wild middle caragana, sowing the seeds in a pot filled with the nutrient soil and the vermiculite in a ratio of 1:3, putting the pot into a seedling tray, covering a cover, placing the seedling tray into a temperature-controllable illumination incubator for culturing at 28 ℃, and after growing for 4-5 days, selecting seedlings with good growth vigor for experiments.
S42, subtracting the primary roots of the seedlings by using sterilized scissors, chamfering the position about 1cm below cotyledonary node of the seedlings by using a sterilized blade, chamfering, increasing the contact area of an explant, discarding the lower part, immersing the incision in the agrobacterium rhizogenes bacterial suspension added with acetosyringone in the step S2 for 1min under a darker environment, scraping the incision to obtain bacterial paste prepared in the step S3 for infection, scraping the incision to a thickness of 0.1-0.5 mm, transplanting the seedlings back into soil, pouring 5ml of 1/4B5 culture medium into each seedling, culturing in an illumination incubator at a humidity of 90% and a temperature of 28 ℃, and opening a vent of a seedling tray in which the middle caragana seedlings are positioned after 12-20 d so that the grown hairy roots gradually adapt to the environmental humidity.
S5, observing the growth condition of the hairy roots of the middle caragana, pulling out the hairy root plants for detection when 14-28 d, directly irradiating positive hairy roots by using a LUYOR-3415 fluorescent protein excitation light source, and detecting the expression condition of the e-GFP reporter gene.
Example 2
The difference from example 1 is that: the method for preparing the agrobacterium rhizogenes bacterial suspension in the step S2 comprises the following steps: s21, inoculating agrobacterium rhizogenes K599 identified as a plasmid vector pEGAD-e-GFP into 4mL of LB liquid culture medium (containing 50 mug/mL kanamycin and 50 mug/mL streptomycin), shaking overnight at 28 ℃ at a shaking speed of 180rpm, and culturing overnight under shaking until the OD600nm value is about 0.9 to obtain a primary bacterial suspension;
s22, the next day, a part of the primary fungus suspension is preserved, and the step of preserving is as follows: 1mL of the primary bacterial suspension is filled into a 1.5mL EP tube, the primary bacterial suspension is centrifuged at 4000rpm for 2min, the supernatant is discarded, 800 mu L of fresh LB liquid medium and 200 mu L of 75% glycerol are added, the primary bacterial suspension is uniformly mixed, quick-frozen by liquid nitrogen and placed in a refrigerator at-80 ℃ for long-term storage, the other part of the primary bacterial suspension is subjected to expansion culture, 1mL of primary bacterial suspension is transferred into 100mL of fresh LB liquid medium (containing 50 mu g/mL kanamycin and 50 mu g/mL streptomycin), and bacteria are shaken at 28 ℃ at an oscillating speed of 180rpm until OD600nm is 0.8, so that the agrobacterium rhizogenes bacterial suspension is obtained.
S23, adding acetosyringone with the final concentration of 25 mu mol/mL into the agrobacterium rhizogenes bacterial suspension obtained in the step S22 as an inducer, fully and uniformly mixing, and standing in the dark at room temperature for 12 hours.
Example 3
The difference from example 1 is that: the method for preparing the agrobacterium rhizogenes bacterial suspension in the step S2 comprises the following steps:
s21 Agrobacterium rhizogenes K599 identified as containing the plasmid vector pEGAD-e-GFP was inoculated into 4mL LB liquid medium (containing 50. Mu.g/mL kanamycin and 50. Mu.g/mL streptomycin), shaken overnight at 28℃at a shaking speed of 180rpm, and shake-cultured overnight to OD 600nm The value is about 0.9, and the primary bacterial suspension is obtained;
s22, the next day, a part of the primary fungus suspension is preserved, and the step of preserving is as follows: 1mL of the initial bacterial suspension is filled into a 1.5mL EP tube, the mixture is centrifuged at 4000rpm for 2min, the supernatant is discarded, 800 mu L of fresh LB liquid medium and 200 mu L of 75% glycerol are added, the mixture is uniformly mixed, quick-frozen by liquid nitrogen and stored in a refrigerator at-80 ℃ for a long time, the other part of the initial bacterial suspension is subjected to expansion culture, 1mL of the initial bacterial suspension is transferred into 100mL of fresh LB liquid medium (containing 50 mu g/mL kanamycin and 50 mu g/mL streptomycin) again, and bacterial shaking is carried out at 28 ℃ at 180rpm until the OD 600nm 0.9, the Agrobacterium rhizogenes suspension is obtained.
S23, adding acetosyringone with the final concentration of 100 mu mol/mL into the agrobacterium rhizogenes suspension obtained in the step S22 as an inducer, fully and uniformly mixing, and standing in the dark at room temperature for 12 hours.
Example 4
The difference from example 1 is that: the method for preparing the agrobacterium rhizogenes bacterial suspension in the step S2 comprises the following steps: s21 Agrobacterium rhizogenes K599 identified as containing the plasmid vector pEGAD-e-GFP was inoculated into 4mL LB liquid medium (containing 50. Mu.g/mL kanamycin and 50. Mu.g/mL streptomycin), shaken overnight at 28℃at a shaking speed of 180rpm, and shake-cultured overnight to OD 600nm The value is about 0.9, and the primary bacterial suspension is obtained;
s22, the next day, a part of the primary fungus suspension is preserved, and the step of preserving is as follows: 1mL of the initial bacterial suspension is filled into a 1.5mL EP tube, the mixture is centrifuged at 4000rpm for 2min, the supernatant is discarded, 800 mu L of fresh LB liquid medium and 200 mu L of 75% glycerol are added, the mixture is uniformly mixed, quick-frozen by liquid nitrogen and stored in a refrigerator at-80 ℃ for a long time, the other part of the initial bacterial suspension is subjected to expansion culture, 1mL of the initial bacterial suspension is transferred into 100mL of fresh LB liquid medium (containing 50 mu g/mL kanamycin and 50 mu g/mL streptomycin) again, and bacterial shaking is carried out at 28 ℃ at 180rpm until the OD 600nm 0.95, and obtaining agrobacterium rhizogenes bacterial suspension.
S23, adding acetosyringone with the final concentration of 50 mu mol/mL into the agrobacterium rhizogenes bacterial suspension obtained in the step S22 as an inducer, fully and uniformly mixing, and standing in the dark at room temperature for 12 hours.
Comparative example 1
The difference from example 1 is that: the one-step conversion procedure in step S4 is as follows:
s41, planting and selecting the middle caragana: weighing laboratory special nutrient soil and vermiculite, sterilizing in a sterilizing pot, picking full and insect-free seeds of a wild middle caragana, sowing the seeds in a pot filled with the nutrient soil and the vermiculite in a ratio of 1:3, putting the pot into a seedling tray, covering a cover, placing the seedling tray into a temperature-controllable illumination incubator for culturing at 28 ℃, and after growing for 4-5 days, selecting seedlings with good growth vigor for experiments.
S42, subtracting the primary roots of the seedlings by using sterilized scissors, chamfering the position about 1cm below cotyledonary node of the seedlings by using a sterilized blade, discarding the lower part, soaking the incision in the agrobacterium rhizogenes suspension prepared in the step S2 for 1min, transplanting the seedlings back into soil, pouring 5ml of 1/4B5 culture medium into each seedling, and culturing in an illumination incubator under the environment with the humidity of 90% and the temperature of 28 ℃.
Comparative example 2
The difference from example 1 is that: the one-step conversion procedure in step S4 is as follows:
s41, planting and selecting the middle caragana: weighing laboratory special nutrient soil and vermiculite, sterilizing in a sterilizing pot, picking full and insect-free seeds of a wild middle caragana, sowing the seeds in a pot filled with the nutrient soil and the vermiculite in a ratio of 1:3, putting the pot into a seedling tray, covering a cover, placing the seedling tray into a temperature-controllable illumination incubator for culturing at 28 ℃, and after growing for 4-5 days, selecting seedlings with good growth vigor for experiments.
S42, injecting the agrobacterium rhizogenes suspension prepared in the step S2 into cotyledonary nodes of seedlings, transplanting the seedlings back into soil after infection, pouring 5ml of 1/4B5 culture medium into each seedling, and culturing in an illumination incubator under the environment with the humidity of 90% and the temperature of 28 ℃.
According to research, the pouring of the 1/4B5 culture medium into the middle caragana seedlings planted back into the soil after infection is more beneficial to the growth of the hairy roots of the middle caragana, and meanwhile, the growth of the middle caragana seedlings is also beneficial, and watering of the middle caragana seedlings is not needed within 2 weeks after the pouring of the 1/4B5 culture medium.
After examination of the long hairy root intermediate caragana plants of example 1-example 4 and comparative example 2 after 14 days of dip-dyeing, it was found that when OD 600nm When the final concentration of the acetosyringone added is 50 mu mol/L, the hairy root induction rate is the highest and can reach more than 40%. In contrast, the transformation method in comparative example 1, in which the cotyledonary node of the seedling was immersed in the Agrobacterium rhizogenes suspension alone without scraping the bacterial paste infection, had a lower transformation rate of hairy roots of Caragana intermedia than the transformation method in example 1. The conversion rate of the intermediate caragana hairy roots by injection in comparative example 2 is much lower than that in example 1, and the conversion rate of the intermediate caragana hairy roots by injection is only about 30%.
As can be seen from FIGS. 3 and 4, the intermediate Caragana plant obtained by the one-step transformation method of the intermediate Caragana hairy root of the present invention can grow hairy roots after being impregnated for 14 days, and the positive hairy roots can be seen to have stronger fluorescence by directly irradiating the positive hairy roots with LUYOR-3415 fluorescent protein excitation light source. The transgenic-GFP hairy roots were found to be significantly more fluorescent than normal roots when observed under a fluorescence microscope.
The method for transforming the hairy roots of the middle caragana by using the one-step transformation method of the hairy roots of the middle caragana has the induction rate reaching more than 40 percent, and has shorter time and higher efficiency compared with the method for transforming the hairy roots of the middle caragana by using an injection method.
The above embodiments are preferred embodiments of the present invention, and besides, the present invention may be implemented in other ways, and any obvious substitution is within the scope of the present invention without departing from the concept of the present invention.
Claims (6)
1. An agrobacterium rhizogenes mediated one-step transformation method of hairy roots of Caragana intermedia, which is characterized by comprising the following steps:
s1, electrically transforming a plasmid containing an e-GFP expression vector into Agrobacterium rhizogenes K599 competent cells;
s2, inoculating agrobacterium rhizogenes K599 obtained in the step S1 into an LB liquid medium for culture to prepare OD 600nm 0.9. Mu. Mol/mL acetosyringone is added into the prepared agrobacterium rhizogenes suspension;
s3, taking part of agrobacterium rhizogenes bacterial suspension added with acetosyringone in the step S2, centrifuging, removing supernatant, uniformly mixing the rest precipitate, then sucking 50 μl of the uniformly mixed precipitate, adding the precipitate into an LB solid culture medium for coating, and placing the coated precipitate into an incubator to culture bacterial paste used for infection, wherein the operations are carried out under the condition of an ultra-clean workbench at room temperature;
s4, selecting middle caragana seedlings, chamfering the 1cm lower part of the cotyledon node of the middle caragana seedlings, discarding the lower part, soaking the cotyledon node incision of the middle caragana seedlings in agrobacterium rhizogenes bacterial suspension after acetosyringone is added in the step S2 in dark environment, scraping the cotyledon node incision of the middle caragana seedlings from bacterial paste obtained by culturing in the step S3, directly transplanting the middle caragana seedlings back to soil after scraping the bacterial paste, pouring 1/4B5 culture medium on the middle caragana seedlings, placing the middle caragana seedlings in an illumination incubator for culturing, opening a vent of a seedling tray where the middle caragana seedlings are positioned after 12-20 d, wherein the soaking time of the cotyledon node incision of the middle caragana seedlings in the step S4 is 1min, and the thickness of the bacterial paste at the incision section is 0.1-0.5 mm; wherein the dosage of the 1/4B5 culture medium is 5mL;
the specific steps of the step S2 are as follows: inoculating Agrobacterium rhizogenes K599 obtained in step S1 into LB liquid medium, shake culturing at 28deg.C to OD 600nm Obtaining a primary bacterial suspension of 0.9, transferring the primary bacterial suspension into fresh LB liquid medium again, and shaking culturing the primary bacterial suspension to OD at 28 DEG C 600nm After 0.8 to 0.95, obtaining agrobacterium rhizogenes bacterial suspension, adding acetosyringone into the obtained agrobacterium rhizogenes bacterial suspension, and standing; the volume ratio of the primary bacterial suspension to the fresh LB liquid medium is 1:100.
2. The one-step transformation method according to claim 1, wherein the e-GFP-containing reporter gene expression vector is pEGAD.
3. The one-step transformation method according to claim 1, wherein the bacterial paste used for the infection in step S3 is cultured under conditions of: the temperature of the incubator was set at 37℃and the incubation time was 10 to 16 hours.
4. The one-step transformation method according to claim 1, wherein step S4 is to select young middle caragana plants which grow for 4-5 d and are well grown.
5. The one-step transformation method according to claim 1, wherein the culture conditions in step S4 are: the humidity was 90% and the temperature was 28 ℃.
6. The one-step conversion method according to claim 1, further comprising the steps of: s5, observing the growth condition of the hairy roots of the caragana intermedia, and detecting the expression condition of the e-GFP reporter gene at 14-28 d.
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