CN117384946A - Agrobacterium tumefaciens-mediated radish genetic transformation method and application thereof - Google Patents
Agrobacterium tumefaciens-mediated radish genetic transformation method and application thereof Download PDFInfo
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Abstract
The invention discloses a genetic transformation method of radishes mediated by agrobacterium tumefaciens and application thereof, which comprises the steps of selecting radishes NAU-LHZ' with shanks of 4-5 d seedling ages as explants, pre-culturing for 3d, using 1/2MS liquid culture medium to re-suspend the agrobacterium tumefaciens as an invasion solution, shaking and culturing the explants in a shaking table at 28 ℃ and 200rpm for 20min, airing on filter paper, co-culturing for 3d in darkness, transferring to a decarboxylation culture medium for differentiation culture until rooting culture to obtain radishes regenerated plants. The DNA of radish leaf is extracted and the general primer is used to identify whether the target gene is introduced. And meanwhile, RT-qPCR is adopted to verify whether the target gene is over-expressed. In addition, the electrophoretically identified radish transgenic positive seedlings are selfed to obtain T 2 Extracting DNA after the generation of strain, and adopting universal primer amplification to identify radish transgenic positive seedlings, T 2 Transformation efficiency of substitution linesUp to 46.15%. Compared with the flower dipping method reported by the former, the invention improves the transformation and screening efficiency, provides a new method for efficient transformation and gene function verification of radishes, and also provides technical support for transgenic research of the regenerated recalcitrant crops such as radishes.
Description
Technical Field
The invention belongs to the field of vegetable genetic breeding and biotechnology. According to the invention, proper genotypes are selected to infect radish cotyledons with handles, so that the problems of low screening efficiency, time consumption and labor consumption in the traditional flower dipping method transformation are solved, the operation is simple, convenient and efficient, and the transgenic radish plants with stable target genes can be obtained rapidly.
Background
Radish (Raphanus sativus L.) is a vegetable crop of the genus Raphanus of the family Brassicaceae, native to China, the first and second year root vegetables. The edible parts are mainly expanded fleshy roots, are rich in nutrition and various in application, are ranked in the vegetable planting area of China, and play an important role in vegetable production and supply.
The establishment of a plant high-frequency in-vitro regeneration system is a prerequisite for carrying out the functional verification of gardening trait genes. However, radish is a recalcitrant vegetable crop that has been reported to transform radish by the floral dip method, but the frequency of obtaining positive transgenic seedlings is very low. Although there is a genetic transformation method using the hypocotyl of radish as an explant, it is greatly affected by genotype and has low transformation efficiency; moreover, the transformation of the radishes by the needling method is greatly influenced by the development time of the apical meristem, and the number of obtained transgenic plants is small. In addition, the radish blooming and seed setting time period is longer, the radish has self-incompatibility, and the harvested radish seeds are fewer, so that the wide application of the technology is limited. At present, the key gene function verification of the radish can only be carried out in the heterologous mode crop Arabidopsis thaliana and tobacco, and the gene function verification in the species is difficult to realize except for a virus-induced gene silencing (VIGS) system. In addition, because of the lack of a high-efficiency stable radish genetic transformation system, the target character can not be accurately improved and target gene function analysis can not be performed in the radish by utilizing a gene editing technology and the like. Therefore, a stable and efficient radish target gene genetic transformation system needs to be established, and the problems of low transformation rate, difficult acquisition of transgenic plants and the like in radish molecular breeding are solved.
Disclosure of Invention
Aiming at the problems that a radish efficient genetic transformation system is not established and the like, the invention aims to provide an agrobacterium tumefaciens-mediated radish genetic transformation method and application thereof, solve the problems of low radish genetic transformation efficiency and long time consumption, and provide technical support for efficient transformation and gene function verification of radishes.
The invention aims at realizing the following technical scheme:
an agrobacterium tumefaciens-mediated radish genetic transformation method and application thereof, comprising the following steps:
(1) Taking the cotyledon with the stem of the radish early bolting high-generation inbred line NAU-LHZ' as an explant, and placing the explant on an MS solid culture medium for pre-culture.
(2) The agrobacterium solution transformed with the recombinant expression vector is streaked, activated and cultured, then the bacteria are enriched, and resuspended with 1/2MS liquid medium as an invader solution. The recombinant expression vector is a plant over-expression vector into which a target gene is inserted. Shaking culture of the agrobacterium liquid to OD 600 About 2.0, the bacterial cells collected by suspending and centrifuging the 1/2MS suspension are immersed in the dye solution to adjust the OD 600 From 0.5 to 0.8, preferably OD 600 0.6 to 0.7.
(3) And (3) infecting the cotyledons with handles after the preculture in the step (1). The precultured cotyledons with stalks and the infection solution are placed in a 250mL conical flask and the explant is completely immersed in the infection solution, preferably 100-150 mL of the infection solution, and the container is oscillated at 28 ℃ at 200rpm for about 15-30 min, preferably 20min.
(4) Placing the infected explant on an MS solid culture medium, performing co-culture under a dark condition, then performing induced differentiation culture on a decarboxylation culture medium, performing subculture until differentiation and bud formation, and transferring to a rooting culture medium to obtain a radish regenerated plant.
(5) The formula of each culture medium in the steps (1) and (4) is as follows:
culture medium formulation of `NAU-LHZ` cotyledon with stalk:
pre-culture medium: MS solid culture medium
Co-culture medium: MS solid Medium+200. Mu. Mol/L As (acetosyringone)
Decarboxylation medium: MS solid culture medium +6-BA 6mg/L +NAA0.075mg/L +Carb 300mg/L
Rooting medium: 1/2MS solid culture medium+NAA 0.3mg/L
The time of the preculture in the step (1) is 2 to 4d, preferably 3d. The co-cultivation is carried out in the dark in the step (4) for 2-4 d, preferably 3d; the induced differentiation medium is subcultured once every 15d, preferably twice.
In the culture process, except the preculture, co-culture and rooting culture mediums, the addition ratio of 6-BA and NAA in the culture mediums of each step is higher, so that the explant continuously germinates new lateral buds. Each time of subculture, the small lateral buds that have been produced are isolated from the parent, and are cultured alone to obtain more buds. The rooting culture medium is added with auxin NAA, and after rooting, hardening seedlings and transplanting. The DNA of suspected transgenic radish leaves is extracted by a CTAB method, the pCAMBIA1300 universal primer is adopted for amplification and identification of positive transgenic radish seedlings, and then the differential expression condition of target genes is analyzed by RT-qPCR. In addition, the electrophoretically identified radish transgenic positive seedlings are selfed to obtain T 2 Extracting DNA after the generation of strain, and adopting universal primer amplification to identify radish transgenic positive seedlings, T 2 The transformation efficiency of the generation line reaches 46.15 percent.
According to the method, different target genes and plant expression vectors can be selected according to the needs in a specific operation process to carry out a radish transgenic experiment, a high-generation inbred line NAU-LHZ' cotyledon with a handle is used as a material, pCAMBIA1300 is used as a plant expression vector, an overexpression vector of pCAMBIA1300-RsGLK2.1 is constructed, and transgenic experiments are carried out by utilizing cotyledon infection with the handle.
The method specifically comprises the following steps:
1) The cotyledon with the handle of NAU-LHZ is used as an explant.
2) Selecting aseptic seedlings with the seedling age of about 4-5 d, removing stem tips, and placing selected explants on a pre-culture medium for 2-4 d; the pre-culture formula is MS solid culture medium.
3) And (3) streak activation, culture and infection liquid preparation of agrobacterium. Activating the agrobacterium tumefaciens liquid which is preserved at-80 ℃ and is transformed with the recombinant expression vector on a YEB solid culture medium, and culturing by shaking with the YEB liquid culture medium until OD 600 2.0, the cells were concentrated by centrifugation and resuspended to OD with 1/2MS solution 600 0.6.
4) The recombinant expression vector takes pCAMBIA1300 as a plant expression vector, and the constructed overexpression vector pCAMBIA1300-RsGLK2.1 of the RsGLK2.1 gene.
5) Infection of the `NAU-LHZ` cotyledons with stalks. And (3) picking out the cotyledon with the stalk from the preculture medium after 3d preculture in the step (2) and immersing the cotyledon with the stalk into a dye solution, and carrying out shaking culture for 20min at the temperature of 28 ℃ by a shaking table at 200rpm, wherein the bottom of all the cotyledon with the stalk is taken as the standard.
6) The infected cotyledon with the handle is fished out and spread on filter paper for blow drying, and is changed to an MS solid culture medium newly added with 200 mu M As for co-culture for 2-3 d. The medium is placed under dark conditions so that the Agrobacterium is able to invade the explant.
Carb 300mg/L was used to inhibit outbreaks of Agrobacterium using carbenicillin, the decarboxylation medium was MS solid medium +6-BA 6mg/L +NAA0.075 mg/L. The cotyledons with stalks after infestation were induced using decarboxylation medium, once every 15 d. The cotyledons with handles are transferred to a rooting medium for decarboxylation benzyl for culture when no agrobacterium is exploded after the cotyledons with handles bud, and the rooting medium is 1/2MS solid medium and IBA is 0.1 mg/L. In the secondary culture, the small lateral buds which are generated by the generation are separated from the parent, and the small lateral buds are cultured independently, so that the positive rate of the transgenic buds is increased. And (3) extracting DNA of suspected transgenic radish leaves by a CTAB method after rooting, amplifying and identifying positive transgenic radish seedlings by using pCAMBIA1300 universal primers, and analyzing the target gene expression difference by RT-qPCR. In addition, the electrophoretically identified radish transgenic positive seedlings are selfed to obtain T 2 And (3) the generation line is amplified by adopting a universal primer to identify the transgenic positive seedlings of the radishes. The method remarkably improves the transformation and screening efficiency of the radish transgenes, and greatly shortens the time for obtaining the transgenic positive seedlings.
The invention provides a convenient genetic transformation method aiming at the problems of long time consumption, low transformation and screening efficiency and the like of the genetic transformation system establishment of the radish flower dipping method, provides a new way for efficient transformation and gene function analysis of the radish, and also provides a new thought for transgenosis of other plants with difficult regeneration.
Drawings
FIG. 1 shows `NAU-LHZ` adventitious bud induction.
FIG. 2 shows PCR identification of transgenic positive plants transformed with pCAMBIA1300-RsGLK2.1 by `NAU-LHZ`. Wherein M: DL2000 molecular weight standard, the primer was pCAMBIA1300 universal primer.
FIG. 3 is a transgenic radish strain phenotype and chlorophyll content assay and RT-qPCR analysis. a. Transgenic radish strain phenotype; b. determining chlorophyll content of transgenic radish lines; RT-qPCR analysis.
FIG. 4 is T 2 And (5) carrying out PCR identification on the transgenic positive strain. Wherein M: DL2000 molecular weight standard, the primer was pCAMBIA1300 universal primer.
The present invention will be described in further detail below.
Detailed Description
The technical method of the present invention will be described in further detail with reference to specific embodiments. The following examples are illustrative only, and the present invention is not limited to these examples.
Application example 1 genetic transformation method of radish 'NAU-LHZ' cotyledon with stalk as explant and application thereof, specific implementation examples are as follows:
1. acquisition of aseptic seedlings
Selecting radish high-generation inbred line NAU-LHZ seeds with intact epidermis and full seeds, filling the seeds into a 10mL sterile centrifuge tube, and completing disinfection and inoculation on an ultra-clean workbench. Adding 75% ethanol for treatment for 45s, adding 0.2% sodium hypochlorite solution for treatment for 12-15 min, shaking for several times, and finally adding enough sterile water for rinsing for 4-5 times. After sterilization, the seeds were gripped with forceps and blotted dry on sterile filter paper before inoculation on MS solid medium (pH 5.8-6.0). And inoculating for 4-5 days to obtain the aseptic seedling.
2. Pre-culture
Taking aseptic seedlings with the seedling age of 4-5 d, removing stem tips, cutting into cotyledons with handles about 0.5cm to serve as explants for standby, and chamfering can increase wound area and ensure the infection efficiency of agrobacterium. Placing the selected explant on an MS solid culture medium for pre-culturing for 2-3 d.
3. Streak activation, culture and infection liquid preparation of agrobacterium
Taking out the agrobacterium liquid which is preserved at-80 ℃ and is transformed with the recombinant expression vector, streaking on a YEB+Kan100deg.C+Rif50mg/L solid culture medium after thawing, inversely culturing at 28 ℃ for 48 hours, picking up monoclonal and inoculating the monoclonal to the YEB+Kan100deg.C+Rif50mg/L liquid culture medium, and shaking culturing at 28 ℃ at 250rpm until the agrobacterium OD is reached 600 2.0, pouring the bacterial liquid into a 50mL sterile centrifuge tube, centrifuging at 5000rpm/min for 5min, enriching bacterial cells, and re-suspending to OD with sterilized 1/2MS liquid culture medium 600 For 0.6 standby. The pCAMBIA1300 is taken as a plant expression vector, and a pCAMBIA1300-RsGLK2.1 overexpression vector is constructed.
4. Culture medium formula
Pre-culture medium: MS solid culture medium
Co-culture medium: MS solid Medium+200. Mu. Mol/L As (acetosyringone)
Decarboxylation medium: MS solid culture medium +6-BA 6mg/L +NAA0.075mg/L +Carb 300mg/L
Rooting medium: 1/2MS solid culture medium+NAA 0.3mg/L
5. Specific culturing process
1) The precultured cotyledons with stalks are immersed in the immersion dye solution, and the seed is immersed for 20min at 28 ℃ and 200 rpm.
2) Spreading the infected cotyledon with the handle on filter paper, drying, inoculating to a new MS solid culture medium, and co-culturing for 2-3 d under dark condition.
3) The cotyledons with handles after co-culture are placed on an induced differentiation medium for differentiation culture, and the seed is subjected to secondary culture every 15 d. For each subculture, the meristematic small lateral buds should be isolated from the mother body and cultured separately (FIG. 1). The explants may be cultured on rooting medium until rooting.
6. Identification of transgenic lines
1) Identification of transgenic Positive seedlings at DNA level
And extracting new leaf DNA from the suspected transgenic positive single plant obtained by screening, and carrying out PCR identification by using pCAMBIA1300 universal primers (figure 2), wherein 3 strains in 6 strains have a band at 1400bp, and initially confirming that the target gene is successfully transferred into the radish, thus obtaining the transgenic positive single plant.
2) Determination of chlorophyll content
By analysis of the chlorophyll content, it was found that the chlorophyll content of lines 4,5 and 7 was significantly higher than that of the wild type, indicating that the rsglk2.1 gene was over expressed as a work (fig. 3).
3) RT-qPCR analysis of transgene expression
The relative expression levels of RsGLK2.1 genes of the strains 4,5 and 7 are found to be obviously increased compared with a control CK, and are 3.5-16 times higher than the control CK, which shows that the RsGLK2.1 genes are over-expressed successfully, and the over-expression vector is further proved to be successfully introduced into radish plants (figure 4).
3. Analysis of conversion efficiency
Selfing the transgenic positive seedlings of the radish identified by electrophoresis to obtain T 2 Extracting DNA after the generation of strain, and adopting universal primer amplification to identify radish transgenic positive seedlings, T 2 The transformation efficiency of the generation line reaches 46.15 percent (figure 4).
According to the statistics of test results, the time for obtaining positive transgenic plants through the infection and transformation of the 'NAU-LHZ' cotyledon with the handle provided by the invention is about 80-90d; the specific time varies depending on the genotype. Compared with the conventional flower dipping method for the radish, the method provided by the invention effectively improves the transgenic screening and transformation efficiency of the radish.
Claims (10)
1. An agrobacterium tumefaciens-mediated radish genetic transformation method and application thereof are characterized by comprising the following steps:
(1) Taking radish 'NAU-LHZ' cotyledon with stalk of 4-5 days old as explant to pre-culture on MS solid culture medium;
(2) Carrying out streak activation culture on agrobacterium tumefaciens bacteria solution transformed with recombinant expression vectors, collecting bacteria after activation, and re-suspending with a 1/2MS liquid culture medium to serve as an invader solution;
(3) Immersing the explant subjected to the preculture in the step (1) into an invasion dye solution for infection;
(4) Placing the infected explant on an MS solid co-culture medium, and performing co-culture for 3d under a dark condition;
(5) After co-culture, transferring the explant to a decarboxylation culture medium for induction and differentiation culture, carrying out subculture once a half month, and transferring to a rooting culture medium immediately after budding to obtain a radish regeneration plant;
(6) The DNA of suspected transgenic positive seedlings is extracted by a CTAB method, and pCAMBIA1300 universal primers are adopted for amplification and identification of positive seedlings;
(7) Extracting RNA of positive seedlings, reversely transcribing the RNA into cDNA, and analyzing the target gene expression difference of the common regenerated radish plants and the transgenic plants by adopting RT-qPCR.
(8) The transgenic strain is self-pollinated, T1 generation radish leaf DNA is extracted, and pCAMBIA1300 universal primer is adopted for amplification and identification of conversion efficiency.
2. The method and use according to claim 1, wherein the explant in step (1) is a cotyledon with a handle of radish 'NAU-LHZ'.
3. The method according to claim 1, wherein the explant pre-culture time in step (1) is 2-4 d, preferably 3d.
4. The method and use according to claim 1, wherein the activated and cultured agrobacteria liquid OD in step (2) 600 About 2.0, the infection liquid is adjusted to OD 600 From 0.5 to 0.8, preferably OD 600 0.6 to 0.7.
5. The method of claim 1, wherein the specific method of infestation of cotyledons with stalks in step (3) is: placing the pre-cultured explant and the infection liquid into a 250mL sterile conical flask, completely immersing the explant into 100-150 mL of the infection liquid, and oscillating and infecting the explant for 15-30 min, preferably 20min at a shaking table of 200rpm at 28 ℃.
6. The method according to claim 1 and the use thereof, wherein the co-cultivation in step (4) is carried out under dark conditions for a period of 2-4 d, preferably 3d; the decarboxylation culture time is 7-14 d; every 15 d.
7. The method of claim 1 and the use thereof, wherein the formulation of each solid medium in steps (1) and (4) is as follows:
pre-culture medium: MS culture medium
Co-culture medium: MS Medium+200. Mu. Mol/L As (acetosyringone)
Decarboxylation medium: MS+6-BA 6mg/L+NAA0.075mg/L+Carb 300mg/L
Rooting medium: 1/2MS+NAA 0.3mg/L.
8. The method and the application of the method according to claim 1, wherein the PCR-based amplification method is characterized in that the PCR-based amplification method is adopted to amplify and identify the transgenic positive seedlings of the radishes after extracting the DNA of the suspected transgenic positive strains of the radishes. On the basis, RNA of the transgenic positive seedling is extracted and reversely transcribed into cDNA, and the RT-qPCR is used for analyzing the expression condition of the target gene, so that the successful transfer of the target gene into the radish plant is further verified.
9. The method and the application according to claim 1, wherein the electrophoretically identified transgenic positive seedlings of the radish are selfed to obtain T 2 DNA is extracted after the generation strain, and pCAMBIA1300 universal primer is adopted to amplify and identify radish transgenic positive seedlings, T 2 The transformation efficiency of the generation strain can reach 46.15 percent.
10. The method of any one of claims 1 to 9 is suitable for the cultivation of transgenic radish plants.
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