Genetic transformation method based on agrobacterium rhizogenes mediated eggplant hairy root transgenosis

Abstract

The invention discloses a genetic transformation method for eggplant hairy root transgenosis based on agrobacterium rhizogenes mediation, and particularly relates to the field of biological transgenosis breeding, by using an agrobacterium rhizogenes mediation method, exogenous gene plasmids are transformed into agrobacterium rhizogenes competent cells, positive bacteria containing target genes are identified through PCR, and the transgenic eggplant hairy root transgenosis is obtained. According to the eggplant hairy root transgenic genetic transformation method based on agrobacterium rhizogenes mediation, the number of needed seeds is small, the detection method is simple, the detection time is short, the detection efficiency is high, the detection cost is low, and the method is suitable for large-scale popularization and application of the eggplant hairy root transgenic genetic transformation method based on agrobacterium rhizogenes mediation. The experiment period is greatly shortened compared with that of a traditional explant genetic transformation method.

Description

Technical Field

[0001] The invention relates to the technical field of genetic transformation, and more specifically to a genetic transformation method of eggplant hairy roots mediated by Agrobacterium rhizogenes. Background Art

[0002] Eggplant is an important vegetable crop widely planted worldwide. However, due to the narrow genetic basis of eggplant cultivars and the difficulty in constructing interspecific populations, it is difficult to achieve rapid and efficient aggregation of excellent traits using conventional breeding. The genetic transformation system of eggplant is still immature and has strong genotype dependence, which limits the progress of eggplant molecular breeding and basic theoretical research to a certain extent. At present, the development of eggplant biological breeding technology is relatively weak, and it is particularly difficult to quickly and accurately obtain excellent varieties with stable genetic traits. The present invention aims to quickly, accurately and stably transform and integrate exogenous target genes into the eggplant genome through Agrobacterium rhizogenes as a mediating pathway. The hairy root transformation system can be used to evaluate the efficiency of the eggplant gene editing system, provide a new approach for eggplant gene function research and biological breeding, and is an important supplement to the stable genetic transformation of eggplant. Summary of the invention

[0003] In order to overcome the above defects of the prior art, an embodiment of the present invention provides a method for genetic transformation of eggplant hairy roots mediated by Agrobacterium rhizogenes to solve the problems raised in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solutions: Step A1: Transform Agrobacterium rhizogenes competent cells with exogenous plasmid; Step A2: Verification by Agrobacterium colony PCR; Step A3: germination and sowing of eggplant; Step A4: Preparation of infection solution; Step A5: Infecting eggplant seedlings with Agrobacterium rhizogenes; Step A6: Induction of hairy roots; Step A7: Identification of positive plants.

[0005] Step A1: The step of transforming Agrobacterium rhizogenes competent cells is specifically to use the heat shock method to transfer the expression vector plasmid into Agrobacterium competent cells K599. Preferably, the specific steps of transferring the expression vector plasmid into Agrobacterium competent cells in step A1 are as follows: Step A11: Take out the competent Agrobacterium stored in the -80°C ultra-low temperature freezer, wait for it to partially melt at room temperature or in your hands for a while, and insert it into ice when it is in an ice-water mixture state; Step A12: Add 0.5 μg of plasmid DNA for every 50 μg of competent cells, and gently stir the bottom of the tube to mix it; place it on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min; Step A13: Add 700 μL of LB liquid medium without antibiotics and culture at 28°C at 200 rpm for 2-3 h; Step A14: Centrifuge the turbid bacterial solution at 6000r / min to collect the bacteria, take about 100 μL of the supernatant, gently blow to resuspend the bacteria, spread it on LB solid culture medium containing corresponding antibiotics, and place it upside down in a 28°C incubator for 2-3 days.

[0006] Step A2: The specific method for verifying the target gene by Agrobacterium colony PCR is as follows: Step A21: Pick a single colony of the transformed Agrobacterium, streak it in LB medium containing corresponding antibiotics, and place it in a 28°C incubator for 48 hours; Step A22: The total volume of reagents used for the Agrobacterium colony PCR amplification reaction is 20 μL, and the reaction system includes 2× Rapid Taq Master Mix 10 μL, Primer F 0.8 μL, Primer R 0.8 μL, ddH2O 7.4 μL, and DNA 1 μL; Step A23: Containing 35S-RUBY vector verification primer sequence including upstream primer Primer F: TGTGAACATGGTGGAGCA; downstream primer Primer R: TGAATCTTGGCGAGGTTG; Step A24: Containing DsRED (RFP) vector verification primer sequence includes upstream primer Primer F: TAAGGGATGACGCACAAT; downstream primer Primer R: CCTACAGGAACAGGTGGTG Step A25: PCR reaction steps are: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 sec, 60°C annealing for 15 sec, 72°C extension for 15 sec, return to 95°C denaturation for 15 sec for 35 cycles, 72°C final extension for 5 min, and storage at 4°C; Step A26: The specific steps of agarose gel electrophoresis are as follows: measure 100mL 1×TAE electrophoresis buffer, add 1g agarose powder into it, heat and boil until completely dissolved, add 10µL nucleic acid dye (10000×), mix well and pour into a mold, and set aside after solidification; put the solidified gel into an electrophoresis tank filled with 1×TAE electrophoresis buffer, add the marker matching the target band to the spotting well as a control, and add the PCR products to the subsequent spotting wells in sequence; electrophoresis at 150V, 400mA for 20min; detect the results with a gel imaging analyzer.

[0007] Step A27: Dip the single colony streaked out in step A21 as a template, use the vector plasmid as a template for the positive control, add 1 μL of sterile water to the negative control, add the primers shown in step A24, prepare the 20 μL reaction system shown in step A22, and go through the PCR amplification cycle shown in step A25. After the end, take 10 μL of PCR product for 1% agarose gel electrophoresis detection.

[0008] Step A28: Pick the positive monoclonal strain, put it into YEP liquid culture medium containing corresponding antibiotics, and culture it in a shaker at 28°C and 200rpm. After the culture is completed, aspirate 1ml of the bacterial solution, add about 150ml of sterilized glycerol, and store the glycerol bacteria in a -80°C ultra-low temperature refrigerator.

[0009] Step A3: Germination and sowing of eggplant. The specific method is as follows: place the test eggplant seeds between two layers of filter paper, place them in a culture dish, moisten the filter paper with 400 ppm gibberellin, place the culture dish in a 30°C incubator for germination for 12-48 hours, and when the seeds turn white, directly sow them in a plug tray filled with a substrate and place them in a plant light culture room for cultivation. When the seedlings grow to two cotyledons, they are used for subsequent infection tests.

[0010] Step A4: Preparation of infection solution.

[0011] Preferably, the specific steps of infection preparation are as follows: Step A41: strain activation: glycerol bacteria stored in a -80°C refrigerator were evenly spread on YEP solid medium containing the corresponding antibiotics and inverted for activation culture for 2 days. A single colony was picked up with a sterile pipette tip and inoculated into 500 μL YEP liquid medium at 28°C and 200 r min. -1 Shake and incubate for 12 h; Step A42: Preparation of infection solution: aspirate 100 μL of activated bacterial solution and inoculate it into 100 mL of YEP liquid culture medium and continue shaking culture overnight. When the OD600 value of the bacterial solution reaches 0.8, collect the bacteria and resuspend them in buffer solution. Let it stand for 3 hours before use.

[0012] Step A5: Infect eggplant seedlings with Agrobacterium rhizogenes containing the target gene.

[0013] The preferred Agrobacterium rhizogenes infection steps are specifically: Step A51: prepare MES infection solution, MES buffer includes 10mM MgCl2, 10mM MES, 100μM acetosyringone, pH is adjusted to 5.7, filter sterilize, collect bacteria and resuspend; Step A52: Bacteria solution immersion method: Cut off the main root system at the rhizome of the seedling, then immerse the seedling incision in the activated bacteria solution, ultrasonically treat for 60 seconds, immerse to a depth of about 3 cm, and immerse for 30 minutes.

[0014] Step A6: Induction of hairy roots.

[0015] The preferred hairy root induction method is specifically: Step A61: transplanting the seedlings into a substrate after infection, and using the substrate to cover the infected site; Step A62: After covering with soil, pour 1 ml of bacterial solution into the substrate; Step A63: Cover the seedling tray with a transparent moisture-retaining cover and co-cultivate in the dark for 2 days; Step A64: After the co-cultivation, transfer the culture to normal light conditions and spray water regularly to keep it moist (do not water in large quantities in the tray).

[0016] Step A7: The specific method for identifying positive plants is as follows: after culturing for 30 days, the number of plants with successfully induced hairy roots is counted and the transformation rate is calculated.

[0017] Transformation rate = (number of plants with positive roots / total number of infected plants) × 100% Technical effects and advantages of the present invention: 1. Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) are a class of Gram-negative bacteria with a broad host range. Their Ri plasmids can be modified with target genes to integrate genetic material into the plant genome.

[0018] 2. The Agrobacterium rhizogenes-mediated hairy root transformation system has the advantages of simple operation, short cycle and high transformation efficiency.

[0019] 3. The present invention makes full use of the characteristics of eggplant seedlings, such as strong differentiation ability, simple operation and short experimental cycle, which is of great significance for the establishment of eggplant gene editing system and gene function research. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the process of the present invention.

[0021] Figure 2 This is the colony PCR identification of Agrobacterium strain K599 transformed with 35S:RUBY and pW501 of the present invention.

[0022] Figure 3 The present invention shows the rooting conditions of three eggplant seedlings with different genotypes after transformation of 35S:RUBY gene and induction.

[0023] Figure 4 The figure shows the rooting conditions of three eggplant seedlings with different genotypes after transformation with pW501 and induction. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] See also Figure 1 - Figure 4 As shown, the present invention provides a genetic transformation method of eggplant hairy root transgene mediated by Agrobacterium rhizogenes, comprising the following steps: Step A1: The step of transforming Agrobacterium rhizogenes competent cells is specifically to transfer the expression vector plasmid into Agrobacterium competent cells K599 by heat shock method.

[0026] In step A1, the expression vector plasmid is transferred into Agrobacterium competent cells, and the specific steps are as follows: Step A11: Take out the competent Agrobacterium stored in the -80°C ultra-low temperature freezer, wait for it to partially melt at room temperature or in your hands for a while, and insert it into ice when it is in an ice-water mixture state; Step A12: Add 0.5 μg of plasmid DNA for every 50 μg of competent cells, and gently stir the bottom of the tube to mix it; place it on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min; Step A13: Add 700 μL of LB liquid medium without antibiotics and culture at 28°C at 200 rpm for 2-3 h; Step A14: Centrifuge the turbid bacterial solution at 6000r / min to collect the bacteria, take about 100 μL of the supernatant, gently blow to resuspend the bacteria, spread it on LB solid culture medium containing corresponding antibiotics, and place it upside down in a 28°C incubator for 2-3 days.

[0027] Step A2: PCR verification of target genes by Agrobacterium colony.

[0028] In step A2, the method for verifying the target gene is specifically as follows: Step A21: Pick a single colony of the transformed Agrobacterium, streak it in LB medium containing corresponding antibiotics, and place it in a 28°C incubator for 48 hours; Step A22: The total volume of reagents used for the Agrobacterium colony PCR amplification reaction is 20 μL, and the reaction system includes 2× Rapid Taq Master Mix 10 μL, Primer F 0.8 μL, Primer R 0.8 μL, ddH2O 7.4 μL, and DNA 1 μL; Step A23: Containing 35S-RUBY vector verification primer sequence including upstream primer Primer F: TGTGAACATGGTGGAGCA; downstream primer Primer R: TGAATCTTGGCGAGGTTG; Step A24: Containing DsRED (RFP) vector verification primer sequence includes upstream primer Primer F: TAAGGGATGACGCACAAT; downstream primer Primer R: CCTACAGGAACAGGTGGTG Step A25: PCR reaction steps are: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 sec, 60°C annealing for 15 sec, 72°C extension for 15 sec, return to 95°C denaturation for 15 sec for 35 cycles, 72°C final extension for 5 min, and storage at 4°C; Step A26: The specific steps of agarose gel electrophoresis are as follows: measure 100mL 1×TAE electrophoresis buffer, add 1g agarose powder into it, heat and boil until completely dissolved, add 10µL nucleic acid dye (10000×), mix well and pour into a mold, and set aside after solidification; put the solidified gel into an electrophoresis tank filled with 1×TAE electrophoresis buffer, add the marker matching the target band to the spotting well as a control, and add the PCR products to the subsequent spotting wells in sequence; electrophoresis at 150V, 400mA for 20min; detect the results with a gel imaging analyzer.

[0029] Step A27: Dip the single colony streaked out in step A21 as a template, use the vector plasmid as a template for the positive control, add 1 μL of sterile water to the negative control, add the primers shown in step A24, prepare the 20 μL reaction system shown in step A22, and go through the PCR amplification cycle shown in step A25. After the end, take 10 μL of PCR product for 1% agarose gel electrophoresis detection.

[0030] Step A28: Pick the positive monoclonal strain, put it into YEP liquid culture medium containing corresponding antibiotics, and culture it in a shaker at 28°C and 200rpm. After the culture is completed, aspirate 1ml of the bacterial solution, add about 150ml of sterilized glycerol, and store the glycerol bacteria in a -80°C ultra-low temperature refrigerator.

[0031] Step A3: Germination and sowing of eggplant. The specific method is as follows: place the test eggplant seeds between two layers of filter paper, place them in a culture dish, moisten the filter paper with 400 ppm gibberellin, place the culture dish in a 30°C incubator for germination for 12-48 hours, and when the seeds turn white, directly sow them in a plug tray filled with a substrate and place them in a plant light culture room for cultivation. When the seedlings grow to two cotyledons, they are used for subsequent infection tests.

[0032] Step A4: Preparation of infection solution.

[0033] The specific steps of step A4, preparing the infection solution are as follows: Step A41: strain activation: glycerol bacteria stored in a -80°C refrigerator were evenly spread on YEP solid medium containing the corresponding antibiotics and inverted for activation culture for 2 days. A single colony was picked up with a sterile pipette tip and inoculated into 500 μL YEP liquid medium at 28°C and 200 r min. -1 Shake and incubate for 12 h; Step A42: Preparation of infection solution: aspirate 100 μL of activated bacterial solution and inoculate it into 100 mL of YEP liquid culture medium and continue shaking culture overnight. When the OD600 value of the bacterial solution reaches 0.8, collect the bacteria and resuspend them in buffer solution. Let it stand for 3 hours before use.

[0034] Step A5: Infect eggplant seedlings with Agrobacterium rhizogenes containing the target gene.

[0035] In step A5, the specific steps of Agrobacterium infecting eggplant seedlings are: Step A51: prepare MES infection solution, MES buffer includes 10mM MgCl2, 10mM MES, 100μM acetosyringone, pH is adjusted to 5.7, filter sterilize, collect bacteria and resuspend; Step A52: Bacteria solution immersion method: Cut off the main root system at the rhizome of the seedling, then immerse the seedling incision in the activated bacteria solution, ultrasonically treat for 60 seconds, immerse to a depth of about 3 cm, and immerse for 30 minutes.

[0036] Step A6: Induction of hairy roots In step A6, the method of inducing hairy roots is specifically as follows: Step A61: transplanting the seedlings into a substrate after infection, and using the substrate to cover the infected site; Step A62: After covering with soil, pour 1 ml of bacterial solution into the substrate; Step A63: Cover the seedling tray with a transparent moisture-retaining cover and co-cultivate in the dark for 2 days; Step A64: After the co-cultivation, transfer the culture to normal light conditions and spray water regularly to keep it moist (do not water in large quantities in the tray).

[0037] Step A7: The specific method for identifying positive plants is as follows: after culturing for 30 days, the number of plants with successfully induced hairy roots is counted and the transformation rate is calculated.

[0038] Transformation rate = (number of plants with positive roots / total number of infected plants) × 100% It should be noted that the preset values ​​in the present invention are determined according to specific circumstances, that is, this embodiment does not specifically limit the specific values.

[0039] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A genetic transformation method for eggplant hairy roots mediated by Agrobacterium rhizogenes, characterized in that: The steps include: A1: Exogenous plasmid transforms Agrobacterium rhizogenes competent cells; A2: PCR verification of Agrobacterium colonies; A3: Germination and sowing of eggplant; A4: Preparation of infection solution; A5: Agrobacterium rhizogenes infects eggplant seedlings; A6: Induction of hairy roots; Step A7: Identification of positive plants; The exogenous target gene can be quickly, accurately and stably transformed and integrated into the eggplant genome. The hairy root transformation system can be used to evaluate the efficiency of the eggplant gene editing system, providing a new approach for eggplant gene function research and biological breeding.

2. The method for genetic transformation of eggplant hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In the step A1, the step of transforming the Agrobacterium rhizogenes competent cells is specifically to transfer the expression vector plasmid into the Agrobacterium competent cells (K599) by the heat shock method, and the specific steps are as follows: Step A11: Take out the competent Agrobacterium stored in the -80°C ultra-low temperature freezer, wait for it to partially melt at room temperature or in your hand for a while, and insert it into ice when it is in the state of ice-water mixture; Step A12: Add 0.5 μg of plasmid DNA for every 50 μg of competent cells, and gently stir the bottom of the tube to mix it; place it on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min; Step A13: Add 700 μL of LB liquid medium without antibiotics and culture at 28°C at 200 rpm for 2-3 h; Step A14: Centrifuge the turbid bacterial solution at 6000r / min to collect the bacteria, take about 100 μL of the supernatant, gently blow to resuspend the bacteria, spread it on LB solid culture medium containing corresponding antibiotics, and place it upside down in a 28°C incubator for 2-3 days.

3. The method for genetic transformation of eggplant hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step A2, the Agrobacterium colony PCR target gene verification method is specifically as follows: Step A21: Pick a single colony of the transformed Agrobacterium, streak it in LB medium containing corresponding antibiotics, and place it in a 28°C incubator for 48 hours; Step A22: The total volume of reagents used for the Agrobacterium colony PCR amplification reaction is 20 μL, and the reaction system includes 2×RapidTaq Master Mix 10 μL, Primer F 0.8 μL, Primer R 0.8 μL, ddH2O 7.4 μL, and DNA 1 μL; Step A23: Containing 35S-RUBY vector verification primer sequence including upstream primer Primer F: TGTGAACATGGTGGAGCA; Downstream primer Primer R: TGAATCTTGGCGAGGTTG; Step A24: Containing DsRED (RFP) vector to verify that the primer sequence includes the upstream primer Primer F: TAAGGGATGACGCACAA; Downstream primer Primer R: CCTACAGGAACAGGTGGTG; Step A25: PCR reaction steps are: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 sec, 60°C annealing for 15 sec, 72°C extension for 15 sec, return to 95°C denaturation for 15 sec for 35 cycles, 72°C final extension for 5 min, and storage at 4°C; Step A26: Agarose gel electrophoresis. The specific steps are as follows: measure 100mL 1×TAE electrophoresis buffer, add 1g agarose powder, heat and boil until completely dissolved, add 10µL nucleic acid dye (10000×), mix well and pour into a mold, set aside after solidification; put the solidified gel into an electrophoresis tank filled with 1×TAE electrophoresis buffer, add a marker matching the target band to the spot well as a control, and add the PCR products to the subsequent spot wells in sequence; perform electrophoresis at 150V, 400mA for 20min; and detect the results using a gel imaging analyzer; Step A27: Dip the single colony streaked out in step A21 as a template, use the vector plasmid as a template for the positive control, add 1 μL of sterile water to the negative control, add the primers shown in step A24, prepare the 20 μL reaction system shown in step A22, and perform the PCR amplification cycle shown in step A25. After the end, take 10 μL of the PCR product for 1% agarose gel electrophoresis detection; Step A28: Pick the positive monoclonal strain, put it into YEP liquid culture medium containing corresponding antibiotics, and culture it in a shaker at 28°C and 200rpm. After the culture is completed, aspirate 1ml of the bacterial solution, add about 150ml of sterilized glycerol, and store the glycerol bacteria in a -80°C ultra-low temperature refrigerator.

4. The method for genetic transformation of eggplant hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The eggplant germination and sowing method in step A3 is specifically as follows: the test eggplant seeds are placed between two layers of filter paper, placed in a culture dish, the filter paper is moistened with 400 ppm gibberellin, the culture dish is placed in a 30° C. incubator for germination for 12-48 hours, and after the seeds turn white, they are directly sown in a plug tray filled with a substrate, placed in a plant light culture room for cultivation, and used for subsequent infection tests when the seedlings grow to two cotyledons.

5. The method for genetic transformation of eggplant hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The preparation of the infection solution in step A4 is specifically carried out as follows: Step A41: strain activation: glycerol bacteria stored in a -80°C refrigerator were evenly spread on YEP solid medium containing the corresponding antibiotics and inverted for activation culture for 2 days. A single colony was picked up with a sterile pipette tip and inoculated into 500 μL YEP liquid medium at 28°C and 200 r min. -1 Shake and incubate for 12 h; Step A42: Preparation of infection solution: aspirate 100 μL of activated bacterial solution and inoculate it into 100 mL of YEP liquid culture medium and continue shaking culture overnight. When the OD600 value of the bacterial solution reaches 0.8, collect the bacteria and resuspend them in buffer solution. Let it stand for 3 hours before use.

6. The method for genetic transformation of eggplant hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step A5, the step of infecting eggplant seedlings with Agrobacterium rhizogenes containing the target gene is specifically as follows: Step A51: prepare MES infection solution, MES buffer includes 10mM MgCl2, 10mM MES, 100μM acetosyringone, pH is adjusted to 5.7, filter sterilize, collect bacteria and resuspend; Step A52: Bacteria solution immersion method: Cut off the main root system at the rhizome of the seedling, then immerse the seedling incision in the activated bacteria solution, ultrasonically treat for 60 seconds, immerse to a depth of about 3 cm, and immerse for 30 minutes.

7. The method for genetic transformation of eggplant hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step A6, the method for inducing hairy roots is specifically as follows: Step A61: transplanting the seedlings into a substrate after infection, and using the substrate to cover the infected site; Step A62: After covering with soil, pour 1 ml of bacterial solution into the substrate; Step A63: Cover the seedling tray with a transparent moisture-retaining cover and co-cultivate in the dark for 2 days; Step A64: After the co-cultivation, transfer the cells to normal light conditions for cultivation and spray water regularly to keep them moist.

8. The method for genetic transformation of eggplant hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step A7, the identification method of positive plants is specifically as follows: after culturing for 30 days, the number of plants with successfully induced hairy roots is counted, and the transformation rate is calculated: Transformation rate = (number of plants with positive roots / total number of infected plants) × 100%.

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