CN115058447A - Method for realizing exogenous target gene transient expression in azalea petals by utilizing agrobacterium-mediated method - Google Patents
Method for realizing exogenous target gene transient expression in azalea petals by utilizing agrobacterium-mediated method Download PDFInfo
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Abstract
The invention provides a method for realizing exogenous target gene transient expression in rhododendron petals by utilizing an agrobacterium-mediated method, which comprises the following steps of: vector construction, agrobacterium transformation, agrobacterium liquid culture, infection liquid preparation, sample treatment and agrobacterium infection. The method of the invention greatly improves the transformation efficiency of the target gene on the azalea petals by performing syringe injection treatment on the lower epidermis of the azalea petals, and lays a foundation for the research of an instantaneous expression system of the azalea petals and molecular breeding of the azalea.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a method for realizing exogenous target gene transient expression in azalea petals by utilizing an agrobacterium-mediated method.
Background
Transient expression technology is a technology for obtaining high-level expression of a target gene in a short time, and transient expression exogenous genes cannot be integrated into a host genome and cannot be stably inherited, compared with the technology for integrating exogenous target genes into a host genome through stable expression; however, transient expression has the advantages of short time, simple operation, low cost, good transformation effect and the like, and has the defect that the application is limited by the affinity of species and agrobacterium, and most of the current researches are leaf tissues; the agrobacterium itself has potential influence on plant disease resistance, and has been applied to most plants by numerous scholars at home and abroad, and common transient expression methods include a gene gun mediated method, an agrobacterium mediated method, a PEG mediated protoplast transformation method, a plant virus vector mediated method and the like, and among the numerous methods, the agrobacterium mediated method is the most common.
In the research process at home and abroad, transient expression has been applied to various plant researches such as crops of tobacco (Nicotiana tabacum), pea (Pisum sativum), wheat (Triticum monococcum), corn (Zea mays), and the like; fruits such as peach (Prunus persica), strawberry (Fragaria × ananassa), orange (Citrus reticulata), etc.; ornamental plants such as Petunia hybrida, Catharanthus roseus and Typha orientalis show that transient expression can provide a rapid and reliable basis for genetic transformation of plants.
Rhododendron Planch (Rhododendron simsii Planch) belongs to ornamental flowers and has important ecological influence, ornamental value and medicinal value. At present, the physiological and biochemical research on rhododendron is very wide, but the transient expression of rhododendron is not researched at home and abroad, so that the transient expression technology of the rhododendron petals is valuable for researching the physiological and biochemical functions of the rhododendron and molecular breeding of the rhododendron.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for realizing the transient expression of a foreign target gene in azalea petals by utilizing an agrobacterium-mediated method so as to make up for the vacancy of transient expression in the azalea petals in the prior art.
In order to solve the problems, the invention provides a method for realizing the transient expression of exogenous target genes in rhododendron petals by utilizing an agrobacterium-mediated method, which comprises the following steps:
s1, vector construction: carrying out single enzyme digestion on pCAMBIA1302 plasmid by using Ncol-HF enzyme, detecting the enzyme digestion product by 1.5% agarose gel electrophoresis, carrying out gel cutting, recovering and purifying the enzyme digestion product, connecting the purified product according to a vector fragment and an insertion fragment by using ligase, gently and uniformly mixing the connected pCAMBIA1302 vector with a target gene, adding the mixed vector into an escherichia coli competent cell, and coating the thallus on an LB (lysogeny broth) resistant solid culture medium containing kanamycin for culture to obtain the pCAMBIA1302 recombinant plasmid with the target gene;
s2 Agrobacterium transformation: transforming the pCAMBIA1302 recombinant plasmid with the target gene obtained in the step S1 into Agrobacterium GV3101 by a freeze-thaw method, and culturing the transformed bacterial cells by coating the bacterial cells on LB resistant solid medium containing kanamycin to obtain GV3101-pCAMBIA1302 containing the target gene plasmid;
s3, culturing agrobacterium liquid: inoculating the GV3101-pCAMBIA1302 containing the target gene plasmid obtained in the step S2 on an agrobacterium tumefaciens plate, selecting a single colony of the plate, mixing the single colony with an LB resistance culture medium for shake cultivation, and inoculating a bacterium solution after the cultivation into an LB liquid culture medium containing antibiotics by suction; simultaneously inoculating the helper plasmid P19 bacterial liquid into another LB liquid culture medium for shake culture;
s4, preparing an invasion liquid: re-suspending the GV3101-pCAMBIA1302 containing the target gene plasmid and the auxiliary plasmid P19 bacterial liquid treated in the step S3 by buffer solution, mixing, and standing to obtain an infection liquid;
s5: infection with agrobacterium: and (4) taking azalea petals in the full-bloom stage as transient expression injection samples, injecting the infection liquid prepared in the step S4 into the petals, and shading and lighting for culture to finish the transient gene expression of the azalea petals in the exogenous purpose.
Preferably, in step S1, the ligase is
plus One step PCR Cloning Kit ligase.
Preferably, in step S1, the molar ratio of the vector fragment to the insert fragment is 1: 2.
Preferably, in the step S1, the step S2 and the step S3, the formulation of the LB-resistant solid medium is: LB medium with 50mg/mL Kan,50mg/mL Rif,50mg/mL Gent was added.
Preferably, in the step S1 and the step S2, the culture conditions are: the culture was inverted overnight at 28 ℃.
Preferably, in step S3, the conditions for shaking culture in combination with LB resistant medium are: culturing in a shaker at 28 deg.C and 200rpm for 12h to obtain orange-yellow bacterial liquid without white floccule; the culture conditions of the shaking culture inoculated in the other LB liquid culture medium are as follows: the cells were cultured for 8 hours at 28 ℃ on a shaker at 200rpm to obtain an orange-colored bacterial suspension having white flocs.
Preferably, in step S4, the GV3101-pCAMBIA1302 containing the desired gene plasmid is resuspended at OD600 ═ 0.8, and the helper plasmid P19 is resuspended at OD600 ═ 1.
Preferably, in step S4, the formulation of the resuspension is: 0.5M MES,0.1M AS,1M MgCl 2 。
Preferably, the conditions for the light-shielding and light-irradiation culture are as follows: the light was kept away from the sun for 1 day and then continued for 1 day.
Compared with the prior art, the invention has the following technical advantages:
the method for realizing the transient expression of the exogenous target gene in the azalea petals by utilizing the agrobacterium-mediated method firstly applies the transient expression to the azalea petals, and realizes the agrobacterium transient expression on the azalea petals. Meanwhile, the flower petal is suitable for petals of most ornamental plants; the method for carrying out agrobacterium-mediated injection on azalea petals is easier to inject than a leaf agrobacterium-mediated injection method, so that the infection efficiency and area of agrobacterium are improved, and the content of a target gene is conveniently measured by carrying out gene expression quantity (qRT-PCR) on petal samples after transient expression; the auxiliary plasmid P19 bacterial liquid is utilized to protect the degradation of target gene coding protein, and the infection rate is greatly improved; after the infection liquid is prepared, standing at room temperature for 3h, so that the GV3101-pCAMBIA1302 bacterial liquid with target genes can be better activated, and compared with the existing agrobacterium-mediated method, the method has the advantages of improving the activity and infection efficiency of the bacteria. The method greatly improves the transformation efficiency of target genes on the azalea petals by performing syringe injection treatment on the lower epidermis of the azalea petals, and lays a foundation for the research of an instantaneous expression system of the azalea petals and molecular breeding of the azalea.
Drawings
FIG. 1 is a diagram of fluorescence imaging results of petals of azalea after infection by Agrobacterium;
sequentially preparing a green fluorescent protein, a bright field and an overlying field result graph from left to right;
FIG. 2 is a graph showing the comparison of the expression levels of RhHI genes in a normally growing Rhododendron, petals infected with the objective gene plasmid GV3101-pCAMBIA1302 and petals infected with the unloaded GV3101-pCAMBIA1302 Agrobacterium by qRT-PCR;
the expression levels of the RhHI genes of the normal growing rhododendron, the petals infected by the target gene plasmid GV3101-pCAMBIA1302 and the petals infected by the unloaded GV3101-pCAMBIA1302 agrobacterium are sequentially shown from left to right.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention discloses a method for realizing exogenous target gene transient expression in rhododendron petals by utilizing an agrobacterium-mediated method, which comprises the following steps of:
s1, vector construction: carrying out single enzyme digestion on pCAMBIA1302 plasmid by using Ncol-HF enzyme, detecting the enzyme digestion product by 1.5% agarose gel electrophoresis, carrying out gel cutting, recovering and purifying the enzyme digestion product, connecting the purified product according to a vector fragment and an insertion fragment by using ligase, gently and uniformly mixing the connected pCAMBIA1302 vector with a target gene, adding the mixed vector into an escherichia coli competent cell, and coating the thallus on an LB (lysogeny broth) resistant solid culture medium containing kanamycin for culture to obtain the pCAMBIA1302 recombinant plasmid with the target gene;
s2 Agrobacterium transformation: transforming the pCAMBIA1302 recombinant plasmid with the target gene obtained in the step S1 into agrobacterium GV3101 by a freeze-thaw method, and coating transformed bacteria on an LB resistant solid culture medium containing kanamycin to culture so as to obtain GV3101-pCAMBIA1302 containing the target gene plasmid;
s3, culturing agrobacterium liquid: inoculating the GV3101-pCAMBIA1302 containing the target gene plasmid obtained in the step S2 to an agrobacterium plate, selecting a single colony of the plate, mixing the colony with an LB resistance culture medium for shake cultivation, and sucking the cultured bacterial liquid to inoculate the LB liquid culture medium containing antibiotics; simultaneously inoculating the helper plasmid P19 bacterial liquid into another LB liquid culture medium for shake culture;
s4, preparing an invasion liquid: re-suspending the GV3101-pCAMBIA1302 containing the target gene plasmid and the auxiliary plasmid P19 bacterial liquid treated in the step S3 by buffer solution, mixing, and standing to obtain an infection liquid;
s5: infection with agrobacterium: and (4) taking azalea petals in the full-bloom stage as transient expression injection samples, injecting the infection liquid prepared in the step S4 into the petals, and shading and lighting for culture to finish the transient gene expression of the azalea petals in the exogenous purpose.
Preferably, in the step S1, the ligase is
plus One step PCR Cloning Kit ligase.
Preferably, the following components: in step S1, the molar ratio of the vector fragment to the insert fragment is 1: 2.
Preferably, in the step S1, the step S2 and the step S3, the formulation of the LB-resistant solid medium is: LB medium supplemented with 50mg/mL Kan,50mg/mL Rif,50mg/mL Gent.
Preferably, in the step S1 and the step S2, the culture conditions are: the culture was inverted overnight at 28 ℃.
Preferably, in the step S3, the conditions for shaking culture in combination with the LB resistant medium are: culturing in a shaker at 28 deg.C and 200rpm for 12h to obtain orange-yellow bacterial liquid without white floccule; the culture conditions of the shaking culture inoculated in the other LB liquid culture medium are as follows: the cells were cultured for 8 hours at 28 ℃ on a shaker at 200rpm to obtain an orange-colored bacterial suspension having white flocs.
Preferably, in step S4, the GV3101-pCAMBIA1302 containing the target gene plasmid is resuspended at OD600 ═ 0.8, and the helper plasmid P19 is resuspended at OD600 ═ 1.
Preferably, in step S4, the formulation of the resuspension is: 0.5M MES,0.1M AS,1M MgCl 2 。
Preferably, the conditions for the light-shielding and light-irradiation culture are as follows: the light was kept away from the sun for 1 day and then continued for 1 day.
The above-described method of the present invention is illustrated and explained below with reference to specific data:
preparing raw materials:
the plant material is the experimental material for the good growing azalea plants in the improved variety garden of Ningbo North Lun firewood bridge Wanjing azalea (121 deg. 27 ' 40-122 deg. 10 ' 22 ' from east longitude, 29 deg. 41 ' 44-29 deg. 58 ' 48 from north latitude) of China. Well-grown Rhododendron plants in full-bloom stage were selected for Agrobacterium infection transformation experiments and grown in a climate chamber at 26 ℃ with 12h/12h (light/dark) cycle. The method realizes the transient expression of the exogenous gene in the petals of the rhododendron in the full-bloom stage, and specifically comprises the following steps:
s1: vector construction
The pCAMBIA1302 plasmid is subjected to single enzyme digestion by Ncol-HF enzyme, an enzyme digestion product is detected by 1.5% agarose gel electrophoresis, and the enzyme digestion product is subjected to gel cutting, recovery and purification. Use of
The purified product was ligated with the molar ratio of vector fragment to insert fragment 1:2 using plus One step PCR Cloning Kit ligase. The pCAMBIA1302 vector with the target gene after ligation was gently mixed and added to E.coli (DH 5. alpha.) competent cells. Uniformly coating a certain amount of thallus on LB resistant solid culture medium containing kanamycinThe cells were cultured overnight at 28 ℃ in an inverted state. Single colonies on the plate were picked, identified by bacterial liquid PCR, and the plasmids were sequenced.
S2: agrobacterium transformation
The pCAMBIA1302 recombinant plasmid with the target gene and the idle plasmid are transformed into agrobacterium GV3101 by a freeze-thaw method, a certain amount of thallus is evenly coated on LB resistant solid culture medium containing kanamycin, and the thallus is inverted at 28 ℃ for overnight culture.
S3: culture of Agrobacterium liquid
Single colonies of resistant plates were picked from GV3101-pCAMBIA1302 containing the desired gene plasmid and unloaded GV3101-pCAMBIA1302 Agrobacterium plates and mixed with 5mL LB resistant medium (50mg/mL Kan,50mg/mL Rif,50mg/mL Gent). The cells were incubated at 28 ℃ on a shaker at 200rpm for 12h, and the cells were orange-yellow and free of white flocs. Measuring bacterial liquid OD600 to be 0.8-1.2, sucking 2mL of orange bacterial liquid, and inoculating the orange bacterial liquid into 150mL of LB liquid culture medium containing three antibiotics; the helper plasmid P19 was inoculated into 150mL of LB liquid medium (50mg/mL Kan). Culturing in a shaker at 28 deg.C and 200rpm for 8h, wherein the strain containing target gene plasmid GV3101-pCAMBIA1302 and the unloaded strain containing GV3101-pCAMBIA1302 is orange yellow and has white floccule; the helper plasmid P19 was white in color.
S4: preparation of the invaded dye liquor
The bacterial solution containing the target gene plasmid GV3101-pCAMBIA1302, the unloaded GV3101-pCAMBIA1302 and the helper plasmid P19 was centrifuged at 4000rpm at 4 ℃ for 10min, the GV3101-pCAMBIA1302 with the target gene and the unloaded GV3101-pCAMBIA1302 were resuspended to OD600 of about 0.8 with buffer (0.5M MES,0.1M AS,1M MgCl2), and the helper plasmid P19 was resuspended to OD600 of about 1 with buffer. The suspension of the re-suspended GV3101-pCAMBIA1302 with the target gene and the unloaded GV3101-pCAMBIA1302 and the re-suspended solution 1 of the helper plasmid P19: 1, mixing evenly. At room temperature, the mixture was allowed to stand for 3 hours.
Preparing a buffer solution: 1.5mL of 1M MgCl2 sterilized, 3mL of 0.5M MES sterilized, 225uL of 0.1M AS filtered from the bacteria plus sterile water to 150 mL.
S5: infection with Agrobacterium
Sample processing
The petals of the rhododendron in the full-bloom stage with good growth and development conditions are taken as transient expression injection samples.
Injecting into petals of rhododendron in full bloom stage with a sterile syringe, keeping out of the sun for 1d, illuminating for 1d, and observing the expression part of GFP (Green Fluorescent protein) Fluorescent protein signals in the cells of the petals of the rhododendron by using a laser confocal microscope.
The invention discloses a method for realizing exogenous target gene transient expression in azalea petals by utilizing an agrobacterium-mediated method, which greatly improves the transformation efficiency of a target gene on the azalea petals by performing syringe injection treatment on the lower epidermis of the azalea petals, and lays a foundation for the research of an azalea petal transient expression system and the molecular breeding of azalea. The invention applies transient expression to the petals of rhododendron for the first time and is simultaneously applicable to the petals of most ornamental plants. Compared with a leaf agrobacterium-mediated injection method, the method for carrying out agrobacterium-mediated injection on azalea petals is easier to inject, improves the infection efficiency and area of agrobacterium, is convenient for measuring the content of a target gene by carrying out gene expression quantity (qRT-PCR) on petal samples after transient expression, protects the degradation of target gene coding protein by using the auxiliary plasmid P19 bacterial liquid, and greatly improves the infection rate;
after the preparation of the infection solution, the method is kept still for 3 hours at room temperature, so that the GV3101-pCAMBIA1302 bacterial solution with the target gene can be better activated, and compared with the existing agrobacterium-mediated method, the method has the advantages of improving the activity of the bacteria and the infection efficiency. The experimental results are as follows: the fluorescence imaging results of FIG. 1 show that GFP-labeled RhHI is localized mainly in the nucleus and cell membrane. FIG. 2 uses qRT-PCR to identify that the expression level of the petal RhHI gene infected by the target gene plasmid GV3101-pCAMBIA1302 is larger than that of the petal and normal-growing rhododendron RhHI gene infected by the unloaded GV3101-pCAMBIA1302 agrobacterium.
Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and such changes and modifications will fall within the scope of the present invention.
Claims (9)
1. A method for realizing the transient expression of exogenous target genes in rhododendron petals by utilizing an agrobacterium-mediated method is characterized by comprising the following steps: the method comprises the following steps:
s1, vector construction: carrying out single enzyme digestion on pCAMBIA1302 plasmid by using Ncol-HF enzyme, detecting the enzyme digestion product by 1.5% agarose gel electrophoresis, carrying out gel cutting, recovering and purifying the enzyme digestion product, connecting the purified product according to a vector fragment and an insertion fragment by using ligase, gently and uniformly mixing the connected pCAMBIA1302 vector with a target gene, adding the mixed vector into an escherichia coli competent cell, and coating the thallus on an LB (lysogeny broth) resistant solid culture medium containing kanamycin for culture to obtain the pCAMBIA1302 recombinant plasmid with the target gene;
s2 Agrobacterium transformation: transforming the pCAMBIA1302 recombinant plasmid with the target gene obtained in the step S1 into Agrobacterium GV3101 by a freeze-thaw method, and culturing the transformed bacterial cells by coating the bacterial cells on LB resistant solid medium containing kanamycin to obtain GV3101-pCAMBIA1302 containing the target gene plasmid;
s3, culturing agrobacterium liquid: inoculating the GV3101-pCAMBIA1302 containing the target gene plasmid obtained in the step S2 to an agrobacterium plate, selecting a single colony of the plate, mixing the colony with an LB resistance culture medium for shake cultivation, and sucking the cultured bacterial liquid to inoculate the LB liquid culture medium containing antibiotics; simultaneously inoculating the helper plasmid P19 bacterial liquid into another LB liquid culture medium for shake culture;
s4, preparing an invasion liquid: re-suspending the GV3101-pCAMBIA1302 containing the target gene plasmid and the auxiliary plasmid P19 bacterial liquid treated in the step S3 by buffer solution, mixing, and standing to obtain an infection liquid;
s5: infection with agrobacterium: and (4) taking azalea petals in the full-bloom stage as transient expression injection samples, injecting the infection liquid prepared in the step S4 into the petals, and shading and lighting for culture to finish the transient gene expression of the azalea petals in the exogenous purpose.
2. The method of claim 1, wherein the Agrobacterium-mediated transformation is performed on Rhododendron simsiiThe method for realizing the transient expression of the exogenous target gene in the flower petals is characterized in that: in the step S1, the ligase is
plus One step PCR Cloning Kit ligase.
3. The method for realizing the transient expression of the exogenous target gene in the azalea petals by utilizing the agrobacterium-mediated method according to claim 1, which is characterized in that: in step S1, the molar ratio of the vector fragment to the insert fragment is 1: 2.
4. The method for realizing the transient expression of the exogenous target gene in the azalea petals by utilizing the agrobacterium-mediated method according to claim 1, which is characterized in that: in the step S1, the step S2, and the step S3, the formulation of the LB-resistant solid medium is: LB medium with 50mg/mL Kan,50mg/mL Rif,50mg/mL Gent was added.
5. The method for realizing the transient expression of the exogenous target gene in the azalea petals by utilizing the agrobacterium-mediated method according to claim 1, which is characterized in that: in step S1 and step S2, the culture conditions are: the culture was inverted overnight at 28 ℃.
6. The method for realizing the transient expression of the exogenous target gene in the azalea petals by utilizing the agrobacterium-mediated method according to claim 1, which is characterized in that: in step S3, the conditions for shaking culture in combination with LB-resistant medium are: culturing in a shaker at 28 deg.C and 200rpm for 12h to obtain orange-yellow bacterial liquid without white floccule; the culture conditions of the shaking culture inoculated in the other LB liquid culture medium are as follows: the cells were cultured for 8 hours at 28 ℃ on a shaker at 200rpm to obtain an orange-colored bacterial suspension having white flocs.
7. The method for realizing the transient expression of the exogenous target gene in the azalea petals by utilizing the agrobacterium-mediated method according to claim 1, which is characterized in that: in step S4, GV3101-pCAMBIA1302 containing the desired gene plasmid was resuspended at OD600 ═ 0.8, and the helper plasmid P19 was resuspended at OD600 ═ 1.
8. The method for realizing the transient expression of the exogenous target gene in the azalea petals by utilizing the agrobacterium-mediated method according to claim 7, which is characterized in that: in step S4, the formula of the resuspension is: 0.5M MES,0.1M AS,1M MgCl 2 。
9. The method for realizing the transient expression of the exogenous target gene in the azalea petals by utilizing the agrobacterium-mediated method according to claim 1, which is characterized in that: the conditions of shading and illumination culture are as follows: the light was kept away from the sun for 1 day and then continued for 1 day.
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