CN112251460A

CN112251460A - Method for researching radish functional genome and verifying gene function

Info

Publication number: CN112251460A
Application number: CN202011169713.2A
Authority: CN
Inventors: 张晓辉; 张双双; 李锡香; 宋江萍; 王海平
Original assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Current assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Priority date: 2020-10-28
Filing date: 2020-10-28
Publication date: 2021-01-22

Abstract

The invention discloses a method for researching radish functional genome and verifying gene function, which takes stably inherited allotetraploid (RRCC genome) as receptor material after doubling distant hybrid of radish (raphivus) and cabbage (Brassica oleracea), researches the function by genetic transformation, over-expression or knocking out the target gene of radish, and knocks out the radish gene in the whole genome range to research the functional genome. The key genes for regulating and controlling somatic embryogenesis in the cabbage genome are utilized to drive the regeneration and genetic transformation of hybrid plants. The gene function of the radish is researched by transgenic overexpression, knockout or editing of the gene in the radish genome in the distant hybrid. Because the genetic transformation efficiency of the system is very high, the system is used for knocking out radish genes in large batch, and the research on radish functional genomes can be carried out.

Description

Method for researching radish functional genome and verifying gene function

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a method for researching a radish functional genome and verifying a gene function.

Background

Since the last century, scholars at home and abroad try to establish a regeneration system and a genetic transformation method aiming at radishes, but the efficiency is very low, the obtained transgenic plants are very limited, and the success of the transgenic is few and difficult to repeat. At present, no mature radish genetic transformation system exists. The failure to overexpress, knock out or edit radish genes in radish results in a lag in the research of radish functional genomes and the functional identification of genes. Therefore, the establishment of a method for researching radish functional genomes and verifying the gene functions is of great significance.

Radish is a typical recalcitrant plant which is difficult to genetically transform. The difficulty of genetic transformation of plants is genetically controlled. To overcome the difficult problem of radish genetic transformation, the early research is mostly focused on two directions: 1, research on genetic transformation methods such as adjusting the formula of a culture medium and the proportion of hormones. 2 screening radish germplasm resources, and searching radish varieties which are easy to regenerate and transform. Both of these directions have not met with ultimate success, although a great deal of labor, material, and time are invested. The possible reasons are that key genes in the radish genome that determine somatic embryogenesis are not expressed or cannot respond to induction by exogenous hormones. The cabbage has a variety which is regenerated efficiently and is easy to be transformed genetically. The results show that the key genes determining somatic embryogenesis in the cabbage genome are highly expressed and can effectively respond to the induction of exogenous hormones.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to establish a method for researching radish functional genome and verifying gene function.

The technical scheme of the invention is as follows: a method for researching radish functional genome and verifying gene function, which uses radish (Raphanus sativus) and cabbage (Brassica oleracea) distant hybrid doubled to stably inherit heterotetraploid (RRCC genome) as receptor material, and makes synthetic plant easy to genetically transform by introducing a set of cabbage genome. The functional genome research is carried out by knocking out radish genes in the whole genome range through genetic transformation, over-expression, knocking out or editing target genes of radish so as to research the functions of the radish genes.

Further, the radish includes all subspecies and varieties of RR genome, the cabbage includes all subspecies and varieties of CC genome, and the allotetraploid refers to a plant containing RRCC genome.

Further, the genetic transformation of the radish and cabbage distant hybrid is agrobacterium-mediated genetic transformation.

The genetic transformation method comprises the following steps:

preparing aseptic seedlings: selecting full seeds, cleaning, disinfecting and then uniformly sowing the seeds on a sowing culture medium, wherein the sowing culture medium comprises the following components: 1/2MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L, adjusting pH to 5.8 + -0.1, and sterilizing with high pressure steam;

pre-culturing: selecting seedlings with the seedling age of 6-10d, cutting off hypocotyls when the explants use the hypocotyls, laying a piece of sterile filter paper on a pre-culture medium, and flatly placing the hypocotyls on the filter paper; when the explant is used as a cotyledon disc, cutting off cotyledons, directly placing the cut cotyledons on a pre-culture medium, sealing the culture dish by using a sealing film, and pre-culturing for 2-4 d; the pre-culture medium comprises the following components: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1mg/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, adding AgNO₃To 5 mg/L;

shaking the bacteria: preparing LB, adding screening antibiotic (selected according to resistance genes on the vector, including but not limited to kanamycin) and rifampicin, picking agrobacterium containing the vector (the target gene overexpression or knockout or editing vector) from a plate or directly adding fresh bacterial liquid, shaking overnight under the shaking condition of 28 ℃ and 250 r/min;

preparing an agrobacterium suspension: shaking the bacteria until OD600 is 0.6-0.8, pouring the bacteria liquid into a sterile centrifuge tube, centrifuging, removing the supernatant, adding DM suspension, re-suspending, pouring into a sterile triangular flask, placing into a shaking table, shaking and culturing for 30min-1h under the shaking condition of 250r/min at 28 ℃, taking out the bacteria liquid, and diluting the bacteria liquid until OD600 is 0.5-0.6 by taking the DM suspension added with the Acetosyringone (AS) AS a background; the preparation method of the DM suspension comprises the following steps: MS + sucrose 30g/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, storing in refrigerator at 4 deg.C, adding Acetosyringone (AS) to 100 μmol/L before use;

co-culturing: placing the explants to be cultured for 2-4d in an agrobacterium tumefaciens suspension with OD600 of 0.5-0.6 for infection, continuously shaking the explants during the infection, then placing the explants on sterile filter paper, sucking the bacterial liquid to dry, laying a piece of sterile filter paper on a co-culture medium, horizontally placing hypocotyls on the filter paper, directly placing a cotyledon disc on the culture medium without filter paper, sealing the culture dish, and co-culturing for 2-4d, wherein the co-culture medium comprises the following components: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +2,4-D1mg/L + KT 0.3mg/L, adjusting pH to 5.8 +/-0.1, sterilizing with high pressure steam, and adding AS to 100 mu mol/L;

and (3) delayed culture, namely transferring the explants cultured together for 2-4d to a delayed culture medium to culture for 2-4d, wherein the delayed culture medium comprises the following components: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1mg/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, adding AgNO₃And Timentin to 5mg/L and 200mg/L, respectively;

screening and culturing: transferring the explants cultured for 2-4d to a screening culture medium, wherein the illumination intensity is 2000-4000lx, and bud differentiation begins to appear after 2-3 weeks of culture. Transferred to new medium for about 2-3 weeks of culture, the selection medium consisting of: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1mg/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, adding AgNO₃Timentin and hygromycin at final concentrations of 5mg/L, 200mg/L and 10mg/L, respectively;

rooting culture: cutting off the completely developed buds, transferring the buds to a rooting culture medium for rooting culture, wherein the rooting culture medium comprises the following components: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L + NAA0.1mg/L, adjusting pH to 5.8, sterilizing with high pressure steam, adding AgNO₃Timentin and hygromycin with final concentrations of 5mg/L, 200mg/L and 5mg/L respectively;

hardening seedlings: taking out the tissue-cultured rooted transgenic seedling, flushing the culture medium at the root with running water, transplanting the seedling into vermiculite, culturing in an incubator with a photoperiod of 20h illumination +4h darkness, watering every day to ensure humidity, pouring Hoagland nutrient solution after 3d, and transplanting the seedling into a greenhouse after 7d hardening.

Further, the genetic transformation is specifically:

preparing aseptic seedlings: selecting plump seeds, washing the seeds with sterile water for 3 times, disinfecting the seeds for 1min by using 75% alcohol, washing the seeds with sterile water for 3 times, diluting 84 disinfectant (the effective chlorine content is 34.0-46.0g/L) by one time, disinfecting the diluted disinfectant for 10min, washing the seeds with sterile water for 3-4 times, uniformly sowing the seeds on sowing culture media (1/2MS, 30g/L of cane sugar and 7.5g/L of agar or 2.5g/L of plant gel, adjusting the pH value to 5.8 +/-0.1, sterilizing the seeds by using high-pressure steam), and sowing about 30 seeds on each culture medium.

Pre-culturing: explants were taken with the hypocotyl or cotyledon disks. Selecting seedling of 6-10d, cutting hypocotyl with 0.5cm length with sharp surgical blade, adjusting pH to 5.8 + -0.1 in preculture medium (MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1 mg/L), sterilizing with high pressure steam, adding AgNO₃To 5mg/L) was laid a sterile filter paper, and the hypocotyl was laid flat on the filter paper; cotyledons were excised and placed directly on preculture medium. Sealing the culture dish with sealing film, and pre-culturing for 2-4 days.

Shaking the bacteria: preparing 50ml LB, adding screening antibiotic (selected according to resistance gene on the carrier, including but not limited to kanamycin) and rifampicin to 50mg/L respectively, picking agrobacterium colony containing carrier (target gene overexpression or knock-out or editing carrier) from the plate by using sterile toothpick or directly adding 1ml of fresh bacterial liquid, shaking overnight under the condition of 28 ℃ and 250r/min

Preparing an agrobacterium suspension: shaking the bacteria until OD600 is 0.6-0.8, pouring the bacteria liquid into a sterile centrifuge tube, rotating at 4000r/min, and centrifuging for 15 min. Discarding supernatant, adding DM suspension (MS + sucrose 30g/L, adjusting pH to 5.8 +/-0.1, sterilizing with high pressure steam, storing in a refrigerator at 4 ℃, adding Acetosyringone (AS) to 100 mu mol/L before use (injection: preventing AS from losing effect before use), re-suspending, pouring into a sterile triangular flask, placing into a shaking table for 30min-1h, shaking for 250r/min under the condition of 28 ℃, taking out bacterial liquid, and diluting the bacterial liquid to OD 600-0.6 by taking DM suspension added with AS AS background.

Co-culturing: explants of 2-4d preculture were infected for 10min with Agrobacterium suspension having OD600 ═ 0.5-0.6, with constant shaking. The explants were then placed on sterile filter paper and the broth was blotted dry. A piece of sterile filter paper is paved on a co-culture medium (MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +2,4-D1mg/L + KT 0.3mg/L, pH is adjusted to 5.8 +/-0.1, AS is added to 100 mu mol/L after high-pressure steam sterilization), the hypocotyl is horizontally placed on the filter paper, and a cotyledon tray is directly placed on the culture medium without filter paper. After the culture dish is sealed, co-culture is carried out for 2-4 d.

Delayed culture, transferring the explants co-cultured for 2-4 days to delayed culture medium (MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1mg/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, adding AgNO₃And Timentin to 5mg/L and 200mg/L, respectively), and culturing for 2-4 d.

Screening and culturing: transferring the explant after 2-4 days of delayed culture to a screening culture medium (MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1 mg/L), adjusting pH to 5.8 + -0.1, autoclaving, adding AgNO₃Timentin and hygromycin at final concentrations of 5mg/L, 200mg/L and 10mg/L, respectively), and illumination intensity of 2000-4000lx, and bud differentiation began to occur after 2-3 weeks of culture. Transferring to new culture medium every 2-3 weeks of culture to avoid the influence of nutrient reduction or antibiotic failure in the culture medium on explant growth.

Rooting culture: cutting completely developed bud, transferring to rooting culture medium (MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L + NAA0.1mg/L, adjusting pH to 5.8, sterilizing with high pressure steam, adding AgNO₃Timentin and hygromycin at final concentrations of 5mg/L, 200mg/L and 5mg/L, respectively).

Hardening seedlings: taking out the tissue culture rooted transgenic seedling, flushing the culture medium on the root with running water, transplanting to vermiculite, and culturing in an incubator. The photoperiod is 20h of light +4h of darkness, and watering is carried out every day to ensure the humidity. Pouring Hoagland nutrient solution after 3d, hardening seedlings for 7d, and transplanting the seedlings into a greenhouse.

Furthermore, the expressed or knocked-out radish target genes refer to coding genes and non-coding genes in a radish genome.

Further, the method for knocking out or editing the radish target gene utilizes CRISPR-Cas 9.

Further, transgenic seedling T of overproof radish gene₀PCR identification is carried out on the generation, and positive plants are selfed to obtain T₁And (5) plant generation. Q-PCR identifies the expression level of the target gene.

Further, the knockout plant utilizes PCR to amplify a DNA segment where a target site of the target gene is located, and sequencing verifies mutation.

Furthermore, phenotype observation is carried out on the over-expression or gene knockout plant, and the radish gene function is researched by comparing with a wild plant.

Compared with the prior art, the invention has the following beneficial effects:

the invention synthesizes intergeneric hybrid of radish and cabbage, which contains two sets of genomes of radish and cabbage. The key genes for regulating and controlling somatic embryogenesis in the cabbage genome are utilized to drive the regeneration and genetic transformation of hybrid plants. The gene function of the radish is researched by transgenic overexpression or knocking out the gene in the radish genome in the hybrid. The invention makes it possible to overexpress, knock out and edit radish genes and genomes for the first time. Because the genetic transformation efficiency of the system is very high, the system is used for knocking out radish genes in large batch, and the research on radish functional genomes can be carried out.

Drawings

FIG. 1 shows hypocotyl regenerated shoots of distant hybrids (RRCC) of radish and cabbage;

FIG. 2 shows regenerated buds of cotyledon disc of radish and cabbage distant hybrid (RRCC);

FIG. 3 shows genetically transformed regenerated shoots in rooting;

FIG. 4 shows roots from genetically transformed regenerated shoots;

FIG. 5 is an electrophoretogram of PCR products of regenerated plantlets. From left to right are 50bp DNA ladder, blank, colony PCR product and regenerated seedling PCR product (L1-L12), respectively;

FIG. 6 is an electrophoretogram of PCR products of regenerated plantlets. From left to right are 50bp DNA ladder, blank, colony PCR product and regenerated seedling PCR product (L1-L6), respectively;

FIG. 7 is the peak diagram of the PCR product sequencing of transgenic plant L1;

FIG. 8 is a diagram of the comparison of the PCR product sequence of transgenic plant L1 with the sequences of other plants;

FIG. 9 is the peak diagram of the PCR product sequencing of transgenic plant L4;

FIG. 10 is a diagram of the comparison of the PCR product sequence of transgenic plant L4 with the sequences of other plants;

FIG. 11 is the peak diagram of the PCR product sequencing of transgenic plant L11;

FIG. 12 is a diagram of the comparison of the PCR product sequence of transgenic plant L11 with the sequences of other plants.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

1. Creation of distant hybrid of radish and cabbage

Radish (Raphanus sativus) is used as a female parent, cabbage (Brassica oleracea) is used as a male parent, embryo rescue is carried out after pollination and hybridization, colchicine is added into a culture medium to promote chromosome doubling, and distant hybrid (RRCC genome) of the radish and the cabbage is artificially synthesized.

2. Radish gene expression or editing vector construction

Cloning radish target genes and loading the radish target genes onto a plant expression vector, or designing gRNA according to a target gene sequence to construct a CRISPR-Cas9 gene editing vector. And transferring into agrobacterium.

3. Genetic transformation

Sowing: selecting full seeds, washing the seeds with sterile water for 3 times, disinfecting the seeds with 75% alcohol for 1min, washing the seeds with sterile water for 3 times, diluting 84 disinfectant (with the effective chlorine content of 34.0-46.0g/L) by one time, disinfecting the diluted disinfectant for 10min, washing the seeds with sterile water for 3-4 times, uniformly sowing the diluted disinfectant on a sowing culture medium (1/2MS + sucrose 30g/L + agar 7.5g/L, adjusting the pH value to 5.8 +/-0.1, sterilizing the diluted disinfectant with high-pressure steam), and sowing about 30 seeds on each bottle of culture medium.

Pre-culturing: explants were taken with the hypocotyl or cotyledon disks. Selecting seedling of 6-10d, cutting hypocotyl with sharp surgical blade, and growingThe concentration is 0.5cm, the pH is adjusted to 5.8 + -0.1 in a pre-culture medium (MS + sucrose 30g/L + agar 7.5g/L +6-BA 2mg/L + NAA0.1mg/L), and AgNO is added after autoclaving₃To 5mg/L, poured into a sterile petri dish for solidification) is laid with a piece of sterile filter paper, and the hypocotyl is laid on the filter paper; cotyledons were excised and placed directly on preculture medium. Sealing the culture dish with sealing film, and pre-culturing for 2-4 days.

The transformation method is an agrobacterium-mediated transformation method

Shaking the bacteria:

preparing 50ml LB, adding kanamycin (Kan) and rifampicin (Rif) to 50mg/L, picking Agrobacterium containing vector (target gene overexpression or knock-out or editing vector) from plate with aseptic toothpick or directly adding fresh bacterial liquid 1ml, shaking overnight at 28 deg.C and 250r/min

Preparing an agrobacterium suspension: shaking the bacteria until OD600 is 0.6-0.8, pouring the bacteria liquid into a sterile centrifuge tube, rotating at 4000r/min, and centrifuging for 15 min. Discarding the supernatant, adding DM suspension (MS + sucrose 30g/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, storing in refrigerator at 4 deg.C, adding acetosyringone AS to 100 μmol/L before use, injecting, and preventing AS from losing effect before use). After heavy suspension, pouring the suspension into a sterile triangular flask, placing the flask into a shaking table for shaking culture for 30min-1h under the shaking condition of 250r/min at 28 ℃. Then, the bacterial suspension was taken out and diluted to an OD600 of 0.5 to 0.6 against a background of DM suspension added with AS.

Co-culturing: explants of 2-4d preculture were infected for 10min with Agrobacterium suspension having OD600 ═ 0.5-0.6, with constant shaking. The explants were then placed on sterile filter paper and the broth was blotted dry. A piece of sterile filter paper is paved on a co-culture medium (MS + sucrose 30g/L + agar 7.5g/L +2,4-D1mg/L + KT 0.3mg/L, pH is adjusted to 5.8 +/-0.1, AS is added to 100 mu mol/L after high-pressure steam sterilization, and the mixture is poured into a sterile culture dish for solidification), the hypocotyl is horizontally placed on the filter paper, and a cotyledon tray is directly placed on the culture medium without filter paper. After the culture dish is sealed, co-culture is carried out for 2-4 d.

Delayed culture, transferring the explants co-cultured for 2-4d to delayed culture medium (MS + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1mg/L, adjusting pH to 5.8 ±)0.1, adding AgNO after high-pressure steam sterilization₃And Timentin to 5mg/L and 200mg/L, respectively, poured into a sterile petri dish for coagulation, and cultured for 2-4 d.

Screening and culturing: transferring the explant after 2-4 days of delayed culture to a screening culture medium (MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1 mg/L), adjusting pH to 5.8 + -0.1, autoclaving, adding AgNO₃Timentin and hygromycin with final concentrations of 5mg/L, 200mg/L and 10mg/L respectively, poured into a sterile culture dish for solidification), with illumination intensity of 2000 and 4000lx, and bud differentiation begins to appear after 2-3 weeks of culture. Transferring to new culture medium every 2-3 weeks of culture to avoid the influence of nutrient reduction or antibiotic failure in the culture medium on explant growth.

Rooting culture: cutting completely developed bud, transferring to rooting culture medium (MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L + NAA0.1mg/L, adjusting pH to 5.8, sterilizing with high pressure steam, adding AgNO₃Timentin and hygromycin with final concentrations of 5mg/L, 200mg/L and 5mg/L respectively, poured into a sterile triangular flask for solidification and standby).

4. Transgenic seedling T₀PCR identification is carried out on the generation, and positive plants are selfed. The gene knockout plant utilizes PCR to amplify a DNA segment where a target site of a target gene is located, and sequencing verifies mutation.

5. And (3) carrying out phenotype observation on the over-expressed or gene-knocked-out plant, comparing with a wild plant, and researching the gene function of the radish.

6. The target gene CLV is exemplified. CLV can promote the development of ovary and has the potential of improving the yield of crop seeds.

The specific method comprises the following steps: the nucleic acid sequence of Arabidopsis CLV2 gene is found in NCBI, and is compared with RRCC whole genome sequence to find out the corresponding homologous gene in RRCC. gRNA is designed aiming at CLV2 homologous genes from radish chromosomes, and loaded onto a plant expression vector preloaded with a Cas9 gene to transform agrobacterium. The radish and cabbage distant hybrid is genetically transformed using agrobacterium as a medium, and the regenerated shoots are cultured for rooting to obtain transformed shoots (see fig. 1-4) (the specific process is the same as the genetic transformation step mentioned in section 3 above). Positive seedlings were confirmed by PCR detection of the transformed seedlings (see fig. 5 and 6), and the target fragment of CLV2 gene of the obtained positive plants was PCR amplified and sequenced, among which nested peaks were found in L1, L4 and L11 plants (see fig. 7, 9 and 11). Cloning the PCR product into T1 vector, sequencing to identify its mutation type, 1 adenine insertion in the target site of No. L1 (see figure 8), 13bp deletion in the target site of No. L4 (see figure 10), 12bp deletion and 1 cytosine insertion in the target site of No. L11 (see figure 12). The above mutations are close to the PAM site, so that the coding frame of the CLV2 gene is mutated, and translation is terminated early. The phenomenon that pistil development of mutant plants is influenced and the carpel expands and cracks is found, and the fact that the development of pistil and carpel is regulated and controlled by the radish CLV2 gene is shown.

Claims

1. A method for researching radish functional genome and verifying gene function is characterized in that the method uses stably inherited allotetraploid after doubling radish and cabbage distant hybrids as receptor material, researches the function of radish by genetic transformation, over-expression, knockout or editing of radish target gene, and knocks out radish gene in the whole genome range to research functional genome.

2. The method of claim 1, wherein the radish comprises all subspecies, varieties of the RR genome, the cabbage comprises all subspecies, varieties of the CC genome, and the allotetraploid is a plant comprising the RRCC genome.

3. The method of claim 1, wherein the genetic transformation is agrobacterium-mediated genetic transformation.

4. The method according to claim 1, wherein the genetic transformation step is:

shaking the bacteria: preparing LB, adding screening antibiotic and rifampicin, picking agrobacterium containing a target gene overexpression or knockout or editing vector from a flat plate or directly adding fresh bacterial liquid, and oscillating overnight under the oscillation condition of 28 ℃ and 250 r/min;

preparing an agrobacterium suspension: shaking the bacteria until OD600 is 0.6-0.8, pouring the bacteria liquid into a sterile centrifuge tube, centrifuging, removing the supernatant, adding DM suspension, re-suspending, pouring into a sterile triangular flask, placing into a shaking table, shaking and culturing for 30min-1h under the shaking condition of 250r/min at 28 ℃, taking out the bacteria liquid, and diluting the bacteria liquid until OD600 is 0.5-0.6 by taking the DM suspension added with the acetosyringone as a background; the preparation method of the DM suspension comprises the following steps: MS + sucrose 30g/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, storing in refrigerator at 4 deg.C, and adding acetosyringone to 100 μmol/L before use;

co-culturing: placing the explants to be cultured for 2-4d in an agrobacterium tumefaciens suspension with OD600 of 0.5-0.6 for infection, continuously shaking the explants during the infection, then placing the explants on sterile filter paper, sucking the bacterial liquid to dry, laying a piece of sterile filter paper on a co-culture medium, horizontally placing hypocotyls on the filter paper, directly placing a cotyledon disc on the culture medium without filter paper, sealing the culture dish, and co-culturing for 2-4d, wherein the co-culture medium comprises the following components: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +2,4-D1mg/L + KT 0.3mg/L, adjusting pH to 5.8 +/-0.1, sterilizing with high pressure steam, and adding acetosyringone to 100 μmol/L;

screening and culturing: transferring the explants cultured for 2-4d to a screening culture medium with illumination intensity of 2000-4000lx, starting bud differentiation after 2-3 weeks of culture, and transferring to a new screening culture medium every 2-3 weeks of culture, wherein the screening culture medium comprises the following components: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1mg/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, adding AgNO₃Timentin and hygromycin at final concentrations of 5mg/L, 200mg/L and 10mg/L, respectively;

5. The method according to claim 1, wherein the genetic transformation is in particular:

preparing aseptic seedlings: selecting full seeds, cleaning with sterile water for 3 times, sterilizing with 75% alcohol for 1min, cleaning with sterile water for 3 times, diluting 84 disinfectant for one time, sterilizing for 10min, cleaning with sterile water for 3-4 times, and uniformly sowing on sowing culture medium with about 30 seeds on each culture medium; the seeding culture medium comprises the following components: 1/2MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L, adjusting pH to 5.8 + -0.1, and sterilizing with high pressure steam;

pre-culturing: explants were hypocotyl or cotyledon disks; selecting seedlings with the seedling age of 6-10d, cutting hypocotyls with the length of 0.5cm by using a sharp surgical blade, spreading a piece of sterile filter paper on a pre-culture medium, and flatly placing the hypocotyls on the filter paper; cutting cotyledon, directly placing on pre-culture medium, sealing the culture dish with sealing film, and pre-culturing for 2-4 d; the pre-culture medium comprises the following components: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1mg/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, adding AgNO₃To 5 mg/L;

shaking the bacteria: preparing 50ml LB, adding screening antibiotic and rifampicin to 50mg/L, picking agrobacterium colony containing target gene over-expression or knock-out or editing carrier from the plate with aseptic toothpick or directly adding 1ml fresh bacterial liquid, shaking overnight at 28 deg.C and 250 r/min;

preparing an agrobacterium suspension: shaking the bacteria until OD600 is 0.6-0.8, pouring the bacteria liquid into a sterile centrifuge tube, centrifuging for 15min at the rotation speed of 4000r/min, removing the supernatant, adding DM suspension, re-suspending, pouring into a sterile triangular flask, putting into a shaker for 30min-1h, shaking at the temperature of 28 ℃, taking out the bacteria liquid, and diluting the bacteria liquid until OD600 is 0.5-0.6 by taking the DM suspension added with the acetosyringone as background; the preparation method of the DM suspension comprises the following steps: MS + sucrose 30g/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, storing in refrigerator at 4 deg.C, and adding acetosyringone to 100 μmol/L before use;

co-culturing: placing the explants to be pre-cultured for 2-4d in an agrobacterium tumefaciens suspension with OD600 of 0.5-0.6 for infection for 10min, continuously shaking the suspension, then placing the explants on sterile filter paper, sucking the bacterial liquid to dry, laying a piece of sterile filter paper on a co-culture medium, horizontally placing hypocotyls on the filter paper, directly placing cotyledon discs on the culture medium without the filter paper, sealing the culture dish, and co-culturing for 2-4d, wherein the co-culture medium comprises the following components: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +2,4-D1mg/L, KT 0.3mg/L, adjusting pH to 5.8 +/-0.1, sterilizing with high pressure steam, and adding acetosyringone to 100 μmol/L;

screening and culturing: transferring the explants cultured for 2-4d in a delayed way to a screening culture medium, wherein the illumination intensity is 2000-: MS + sucrose 30g/L + agar 7.5g/L or plant gel 2.5g/L +6-BA 2mg/L + NAA0.1mg/L, adjusting pH to 5.8 + -0.1, sterilizing with high pressure steam, adding AgNO₃Timentin and hygromycin at final concentrations of 5mg/L, 200mg/L and 10mg/L, respectively;

6. The method of claim 1, wherein the overexpressed or knocked-out radish target gene is a coding gene and a non-coding gene in a radish genome.

7. The method of claim 1, wherein the method for knocking out or editing the radish target gene is through CRISPR-Cas 9.

8. The method of claim 1, wherein transgenic T-shoots overexpressing the radish gene₀PCR identification is carried out on the generation, and positive plants are selfed to obtain T₁And (4) plant generation, and Q-PCR identification of the expression quantity of the target gene.

9. The method of claim 1, wherein the knockout plant is subjected to PCR amplification of a DNA fragment containing a target site of the target gene, and sequencing to verify the mutation.

10. The method of claim 1, wherein the overexpressing or knockout plant is phenotypically observed to study radish gene function as compared to a wild-type plant.