CN110699378A - Genetic transformation method of a short-lived plant Arabidopsis thaliana from Xinjiang - Google Patents

Abstract

The invention discloses a genetic transformation method of the short-lived plant Arabidopsis thaliana in Xinjiang. The genetic transformation method comprises the following steps: 1) using the Agrobacterium infection solution containing the target gene expression vector to infect the petioles of Arabidopsis thaliana, and culturing the infected petioles of Arabidopsis thaliana in a symbiotic medium to obtain The petiole after the symbiotic culture; 2) the petiole after the symbiotic culture is cultivated in a screening medium to obtain adventitious buds; 3) the adventitious buds are cultivated in a rooting medium to obtain complete Arabidopsis thaliana transgenic plants. The invention successfully establishes a genetic transformation method of Arabidopsis thaliana thaliana thaliana thaliana thaliana thaliana thaliana petiole as an explant mediated by Agrobacterium tumefaciens, and the establishment of the genetic transformation method can lay a foundation for subsequent research on the function of important genes of Arabidopsis thaliana thaliana. Base.

Description

Genetic transformation method of a short-lived plant Arabidopsis thaliana from Xinjiang

technical field

The invention belongs to the field of biotechnology, and in particular relates to a genetic transformation method for the short-lived plant Arabidopsis thaliana in Xinjiang.

Background technique

Arabidopsis pumila is a typical early spring short-lived plant that is widely distributed in extreme environments in Xinjiang. In the second volume of the Flora of Xinjiang and the 33rd volume of the Flora of China, it is named Arabidopsis mouse, commonly known as Arabidopsis thaliana. Arabidopsis thaliana is a relative species of the model plant Arabidopsis thaliana. Compared with Arabidopsis thaliana, Arabidopsis thaliana has stronger photosynthetic efficiency, larger seed yield, and more salt tolerance. Therefore, Arabidopsis thaliana is a good material to study the mechanism of adaptation between plants and the environment.

In the early stage, the applicant of the present invention mainly carried out research on the gene expression level of Arabidopsis thaliana in response to salt stress, and cloned a number of salt tolerance-related genes. However, since the genetic transformation system of Arabidopsis thaliana has not been established, the gene transformation is mainly transformed into model plants Arabidopsis thaliana, thus limiting the in-depth study of gene function. Therefore, in order to better analyze the salt tolerance mechanism of Arabidopsis thaliana and explore the important gene functions of this species, it is particularly important to establish a genetic transformation system of Arabidopsis thaliana.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a genetic transformation method of Arabidopsis thaliana.

The method for genetic transformation of Arabidopsis thaliana provided by the present invention includes the step of genetic transformation using the petioles of Arabidopsis thaliana as explants.

The above method may include the following steps:

1) using the Agrobacterium infection solution containing the target gene expression vector to infect the petioles of Arabidopsis thaliana, and carrying out symbiotic culture on the infected Arabidopsis thaliana petioles in a symbiotic medium to obtain the petioles after the symbiotic culture;

2) subculture the petiole after the symbiotic culture in the screening medium to obtain adventitious buds;

3) rooting the adventitious shoots in a rooting medium to obtain a complete transgenic Arabidopsis thaliana plant.

In the above method, in step 1), the OD ₆₀₀ of the infection solution is 0.6, and the infection time is 10 min.

In the above method, the symbiotic medium comprises 0.5mg/L of 6-benzylaminoadenine, 0.1mg/L of naphthalene acetic acid and 20mg/L of acetosyringone;

The screening medium comprises 0.5 mg/L 6-benzylaminoadenine, 0.1 mg/L naphthaleneacetic acid, 10 mg/L screening agent and 500 mg/L bacteriostatic agent;

The rooting medium includes 0.1 mg/L naphthalene acetic acid, 10 mg/L screening agent and 500 mg/L bacteriostatic agent.

Further, the screening agent may be hygromycin B; the bacteriostatic agent may be carbenicillin.

Further, the pH of the symbiotic medium can be 5.7-5.9, specifically 5.8;

The pH of the screening medium can be 5.7-5.9, specifically 5.8;

The pH of the rooting medium may be 5.7-5.9, specifically 5.8.

In specific embodiments of the present invention,

The symbiotic medium is composed of MS solid medium, 6-benzylaminoadenine, naphthaleneacetic acid and acetosyringone;

The screening medium is composed of MS solid medium, 6-benzylaminoadenine, naphthaleneacetic acid, hygromycin B and carbenicillin;

The rooting medium consisted of MS solid medium, naphthalene acetic acid, hygromycin B and carbenicillin.

The MS solid medium (1 liter) is made by dissolving 4.4g MS basal medium, 30g sucrose, 0.5g 2-(N-morpholine)ethanesulfonic acid with distilled water, and adjusting the pH to 5.8, then adding 1.5g of plant gel, dilute to 1L, and the medium obtained after autoclaving at 121°C for 15min.

In the above method, in step 1), the culture conditions can be aseptic, 22°C, and cultured under dark conditions for 24h;

In step 2), the culturing conditions can be aseptic, 22° C., 16h light/8h dark, to obtain adventitious buds containing at least 4 leaves; during the culturing process, the screening medium is replaced once a week;

In step 3), the culture conditions may be aseptic, 22° C., 16h light/8h dark conditions for 2 months.

In the above method, step 1) further includes the following steps: blotting the excess bacterial liquid on the surface of the infected Arabidopsis thaliana petiole with sterile filter paper, and then placing it in a symbiotic medium for symbiotic culture.

In the above method, the length of the petiole may be 0.8-1.2 cm, specifically 1 cm.

The preparation method of the petiole can be specifically carried out according to the following steps: sterilizing the Arabidopsis thaliana seeds to obtain the sterilized Arabidopsis thaliana seeds; and sowing the sterilized Arabidopsis thaliana seeds on-demand in MS solid medium , first vernalize at 4°C for 3 days, and then cultivate for 7-8 days in a light incubator with 16h light/8h darkness and a temperature of 22°C to obtain Arabidopsis thaliana seedlings; the Arabidopsis thaliana The seedlings were transplanted into sterile jars containing MS solid medium, and continued to cultivate for about 4 weeks in a light incubator with 16h light/8h darkness and a temperature of 22°C, and obtained about five-week-old seedlings that grew many rosettes. Arabidopsis thaliana plants of leaves; the sturdy petioles obtained from the Arabidopsis thaliana plants are the petioles.

In the above method, the expression vector containing the target gene may be an expression vector containing the Arabidopsis thaliana ApP5CS1 gene (sequence 1). The expression vector containing the Arabidopsis thaliana ApP5CS1 gene (sequence 1) may specifically be a 35S::ApP5CS1 expression vector. The 35S::ApP5CS1 expression vector is obtained by inserting the Arabidopsis thaliana ApP5CS1 gene (sequence 1) between the NcoI and Bst EII restriction sites of the pCAMBIA1301 vector (with hygromycin resistance).

In the above method, the Arabidopsis thaliana is Arabidopsis thaliana (Arabidopsispumila) derived from Xinjiang (Changji).

Still another object of the present invention is to provide a kit for genetic transformation of Arabidopsis thaliana.

The kit for genetic transformation of Arabidopsis provided by the present invention includes the above-mentioned symbiotic medium and/or screening medium and/or rooting medium.

The application of the above-mentioned kit in the genetic transformation of Arabidopsis thaliana or the preparation of genetically transformed products of Arabidopsis thaliana also belongs to the protection scope of the present invention.

The application of the above method or kit in the breeding of Arabidopsis thaliana also belongs to the protection scope of the present invention.

The present invention successfully establishes the Arabidopsis thaliana genetic transformation method mediated by Agrobacterium tumefaciens by using the leaves and petioles of Arabidopsis thaliana as explants. Features lay the foundation.

Description of drawings

Figure 1 is a flow chart of the genetic transformation of Arabidopsis thaliana petiole and leaf explants.

Figure 2 is a schematic diagram of the structure of the 35S::ApP5CS1 expression vector containing the target gene fragment with a size of 2.154 kb.

Figure 3 shows the process of Agrobacterium tumefaciens-mediated genetic transformation of Arabidopsis thaliana petiole explants. A is 5-week-old Arabidopsis thaliana petiole infected with Agrobacterium with an OD ₆₀₀ value of 0.6 for 10 min; B is the co-culture of Arabidopsis thaliana petiole explants after infection for two days; C is the infected Arabidopsis thaliana petiole Mustard petiole explants were selected and cultured for 7 days; D is callus formed by Arabidopsis thaliana petiole explants after 35 days of infection; E is callus of Arabidopsis thaliana petiole explants after 53 days of infection; F For the small Arabidopsis thaliana petiole explants form adventitious buds in about 3 months; G is that adventitious buds are transferred into rooting medium; H is that adventitious buds are induced to form a large number of roots in 2 months and 15 days; I is the observation of the roots of transgenic tissue culture seedlings; J means transplanting to nutrient soil for one month; K means T1 generation transgenic seedlings bear a lot of fruit. The bar line of the scale bar in the figure AJ represents 1 cm, and the bar line of the scale bar in the figure K represents 5 cm.

Figure 4 shows the tissue culture process of Arabidopsis thaliana leaf explants mediated by Agrobacterium tumefaciens. A: 5-week-old Arabidopsis thaliana leaves were infected with Agrobacterium with an OD ₆₀₀ value of 1.0 for 5 min; B: Infected Arabidopsis thaliana leaf explants were co-cultured for two days; C: Infected Arabidopsis thaliana leaf explants The callus status of mustard leaf explants at 53 days; D is about 3 months and 20 days for leaf explants to form adventitious buds; E is for adventitious buds transferred to rooting medium; F is for adventitious buds induced to form a large number of buds in 2 months and 15 days root.

Figure 5 shows the PCR verification of the genomic DNA of some transgenic tissue culture seedlings. M is Marker 5000, "1" is the positive control of plasmid 35S::ApP5CS1, "2" is the negative control with ddH ₂ O as template, "3" is the control of wild-type plant DNA PCR results, and "4-8" are all Identify the correct transgenic plants.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

The experimental materials and sources involved in the following examples: 6-BA (6-Benzylaminopurine, Fluka), hygromycin B (Hygromycin B, Roche), kanamycin sulfate (Kanamycin sulfate, Amresco), rifampicin (Rifampicin, Sigma), gentamicin sulfate (Gentamicin sulfate, Amresco), carbenicillin (CarbeniClllin disodium salt, Germany), acetosyringone (3',5'-Dimethoxy-4'-hydroxyacetophenone, domestic), pancreatic Peptone (Tryptone, OXOID), yeast extract (Yeast extract, OXOID), Silwet L-77 (Pharmacia), D5000 DNA Ladder (Biomiga), sodium hypochlorite, sucrose, sodium chloride and ethanol are all domestic reagent grades, naphthalene acetic acid ( 1-Naphthaleneacetic acid, Solarbio), 2-(N-morpholine)ethanesulfonic acid (MES, Amresco), Phytogel (Sigma).

The culture medium and formula involved in the following examples are as follows:

MS liquid medium (1 liter) is a mixture of 4.4g MS basal medium (MS basal medium is a product of Phytotech, product number M519), 30g sucrose, 0.5g 2-(N-morpholine)ethanesulfonic acid, distilled water Dissolve and mix well, adjust the pH to 5.8, dilute the volume to 1L, and sterilize the medium by autoclaving at 121°C for 15min.

MS solid medium (1 liter) is made by dissolving 4.4g MS basal medium, 30g sucrose, 0.5g 2-(N-morpholine)ethanesulfonic acid in distilled water, adjusting the pH to 5.8, and adding 1.5g Plant gel, dilute to 1L, and sterilize the medium by autoclaving at 121°C for 15min.

The symbiotic medium is sterilized MS solid medium + 0.5 mg/L 6-benzylaminoadenine (6-BA) + 0.1 mg/L naphthalene acetic acid (NAA) + 20 mg/L acetosyringone (AS), pH is 5.8. Antibiotics were added and mixed well before the MS solid medium started to solidify.

The screening or subculture medium is sterilized MS solid medium + 0.5 mg/L 6-benzylaminoadenine (6-BA) + 0.1 mg/L naphthalene acetic acid (NAA) + 10 mg/L hygromycin B (Hygromycin B) B) + 500 mg/L carbenicillin (Carb), pH 5.8.

The rooting medium was sterilized MS solid medium + 0.1 mg/L naphthalene acetic acid (NAA) + 10 mg/L hygromycin B (Hygromycin B) + 500 mg/L carbenicillin (Carb), pH 5.8.

The tissue culture infection solution was MS liquid medium + 20 mg/L acetosyringone (AS), pH 5.8.

LB solid medium (1 L): 10 g tryptone (Tryptone) + 5 g yeast extract (Yeast extract) + 10 g sodium chloride + 8 g agar powder, pH 7.0.

LB liquid medium (1 L): 10 g tryptone (Tryptone) + 5 g yeast extract (Yeast extract) + 10 g sodium chloride, pH 7.0.

All hormones and antibiotics were filtered and sterilized. After the MS medium was sterilized and before it started to solidify, it was added and mixed well.

The pCAMBIA1301 vector (with hygromycin resistance) in the following examples is a product of CAMBIA, Canberra, ACT, Australia, described in the literature "Li, C., Zhang, Y., Zhang, K., Guo, D. , Cui, B., Wang, X., & Huang, X. (2015). Promoting flowering, lateral shoot outgrowth, leaf development, and flower abscission in tobacco plants overexpressing cotton FLOWERING LOCUST(FT)-like gene GhFT1. Frontiers in plant science, 6, 454." in.

The seeds of Arabidopsis pumila in the following examples are from Changji, Xinjiang, and are recorded in the document "Yang, L., Jin, Y., Huang, W., Sun, Q., Liu, F., & Huang , X. Full-length transcriptomesequences of ephemeral plant Arabidopsis pumila provides insight into geneexpression dynamics during continuous salt stress. BMC genomics, 2018, 19(1):717.”, publicly available from the applicant, this biological material is only for It is used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

Agrobacterium GV3101 in the following examples is described in the literature "Li, C., Zhang, Y., Zhang, K., Guo, D., Cui, B., Wang, X., & Huang, X.. Promoting flowering , lateral shoot outgrowth, leaf development, and flower abscission in tobacco plants overexpressing cotton FLOWERING LOCUS T(FT)-like gene GhFT1. Frontiers in plant science, 2015, 6, 454.”, publicly available from the applicant, the biological material is only for It is used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

Embodiment 1, the genetic transformation method of Arabidopsis thaliana

All Arabidopsis thaliana explants required in this example are prepared and carried out according to the flow chart of the genetic transformation of different Arabidopsis thaliana explants shown in FIG. 1 . The detailed operation method is carried out according to the following steps.

1. Sterilization and cultivation of seeds

1. Sterilization of seeds

In the ultra-clean workbench, first wash the seeds of Arabidopsis thaliana with 75% (volume fraction) ethanol solution for 1min, then shake and wash with 20% sodium hypochlorite solution with available chlorine for 8-10min, and finally wash with sterile water for 6-7 minutes times, fully shaken and mixed each time to obtain sterilized Arabidopsis thaliana seeds.

2. Cultivation of Arabidopsis thaliana seedlings

The sterilized Arabidopsis thaliana seeds were sown on-demand in petri dishes containing MS solid medium, placed at 4°C for vernalization for 3 days, and then placed in a light incubator with 16h light/8h darkness and a temperature of 22°C. Cultivated in medium for 7-8 days to obtain Arabidopsis thaliana seedlings.

3. Cultivation of Arabidopsis thaliana plants

The small Arabidopsis thaliana seedlings in good growth condition were transplanted into sterile jars containing MS solid medium, sealed to ensure sterility, and then placed in a light incubator with 16h light/8h darkness and a temperature of 22 °C The cultivation was continued for about 4 weeks, and an Arabidopsis thaliana plant growing with many rosette leaves was obtained at the age of about 5 weeks. Large and thick bright green leaves and sturdy petioles were obtained from Arabidopsis thaliana plants as explants.

2. Activation culture of Agrobacterium tumefaciens

Pyrroline-5-carboxylic acid synthase (P5CS) is a bifunctional enzyme with both pyrroline-5-carboxylic acid synthase and pyrroline-5-carboxylic acid reductase activities, which can catalyze The first two steps of amino acid anabolism are the rate-limiting enzymes in the process of proline synthesis.

1. Construction of recombinant bacteria

The 35S::ApP5CS1 expression vector was transformed into Agrobacterium tumefaciens GV3101, and positive recombinant bacteria were obtained after verification by bacterial liquid PCR was correct, which was stored in a -80°C refrigerator for later use.

The 35S::ApP5CS1 expression vector is a vector obtained by inserting the Arabidopsis thaliana ApP5CS1 gene (sequence 1) between the NcoI and Bst EII restriction sites of the pCAMBIA1301 vector (with hygromycin resistance). A schematic diagram of the structure of the 35S::ApP5CS1 expression vector is shown in Figure 2.

2. Culture of recombinant bacteria

Take the preserved recombinant bacteria (GV3101 Agrobacterium strain containing 35S::ApP5CS1) and streak it into LB solid medium containing 50mg/L Kan, 25mg/L Rif and 50mg/L Gen, and invert in a biochemical incubator at 28°C Incubate for 2 days until a single colony grows. Pick a single colony into a 10 mL sterile round-bottom tube containing 3 mL of LB liquid medium containing 50 mg/L Kan, 25 mg/L Rif and 50 mg/L Gen, and incubate at 28°C with shaking at 180 rpm until the OD ₆₀₀ reaches 0.8- 1.0 bacterial solution, spare.

3. Identification of recombinant bacteria

The specific primer ApP5CS1 (forward primer: GC CCATGG ATGGAGATTCTTCCGATTC; reverse primer: TA GGTGACC TTAGCTTTGTGAGAGCTCG, the underlined part indicates the restriction site, the base before the underline belongs to the restriction site), at 94°C for 2 min; 94°C 30s, 56°C 30s, 72°C 2min 20s, 35 cycles; amplify the bacterial solution shaken in step 2 at 72°C for 10min, and use the 35S::ApP5CS1 plasmid diluted 50 times as a positive control template for amplification, and the identification is correct. of Agrobacterium.

4. Preparation of infection solution

Take 500 uL of correctly identified Agrobacterium in 50 mL of LB liquid medium containing 50 mg/L Kan, 25 mg/L Rif and 50 mg/L Gen at 28°C, under shaking conditions of 180 rpm shaker, until the OD ₆₀₀ of the bacterial solution reaches 0.8. Then, the bacterial liquid with an OD ₆₀₀ of 0.8 was centrifuged in a refrigerated centrifuge at 4°C and 5000 rpm for 10 min, the liquid was discarded, and the bacterial precipitate was left, and then diluted and mixed with MS liquid medium to obtain an OD ₆₀₀ of 0.6 The bacterial liquid and the bacterial liquid with an OD ₆₀₀ of 1.0 were added to AS to a final concentration of 20 mg/L, and the seal was sealed to ensure sterility. Finally, it was shaken and cultured for 05 h at 28 °C and 180 rpm for activation to obtain an infection solution, which was used for later use.

3. Agrobacterium transformation of Arabidopsis thaliana

(1) Agrobacterium transformation of Arabidopsis thaliana petioles

1. Co-cultivation

Cut the 35-day-old sterile Arabidopsis thaliana stout petiole into a length of about 1 cm on the ultra-clean workbench, and infect the cut petioles in the infection solution of OD ₆₀₀ = 0.6 prepared in the above step 2 for 5 min respectively. and 10min, and infect 5min and 10min respectively in the infection solution of OD ₆₀₀ =1.0. There are 4 different treatment methods in this experiment (Table 1 and Table 2), and each group of treatment methods is set to 3 replicates. After infection The material was taken out and placed on sterile filter paper to blot dry, and finally transferred to a symbiotic medium covered with a layer of filter paper (Figure 3A, Figure 3B), sealed to ensure sterility at 22 °C, and cultivated in the dark for 24 hours. petiole.

2. Induction culture of callus

The co-cultured petioles were transferred to the screening medium for screening and culture, sealed after marking the date to ensure sterility, and cultured in a light incubator with a temperature of 22°C for 16h light/8h dark. The callus was obtained by changing fresh screening medium once a week. From the time the explants were transferred to the screening medium for cultivation, the calculation formulas for the percentage of callus formation (%) of petiole explants and the percentage of hygromycin-resistant callus (%) were calculated as follows: percentage of callus formation (%)=(number of explants growing callus/total number of explants)×100%; percentage of hygromycin-resistant callus (%)=(number of hygromycin-resistant callus/ total number of explants) × 100%. Statistical data were analyzed with SPSS19.0 software. Significance Analysis The statistical data were analyzed using one-way ANOVA and Duncan's test to determine the significant difference between the data of each group, and P<0.05 was considered as a significant difference.

The results showed that the petiole explants gradually appeared differentiation after one week of infection (Fig. 3C). The infected Arabidopsis thaliana petiole explants formed obvious callus around 35 days after infection (Fig. 3D); the infected Arabidopsis thaliana petiole explants were induced to form a lot of green at 53 days after infection. Dense callus (Fig. 3E).

The results of infection of petiole explants by different combinations are shown in Table 1 and Table 2. The results showed that the percentage of callus formation of Arabidopsis _thaliana petiole was different with different concentrations of infection solution and different time. The percentage of callus formation was 17.91±1.33; the concentration of infection solution was OD ₆₀₀ =0.6, and the infection time was 10min, the percentage of callus formation was 34.23±2.30; the concentration of infection solution was OD ₆₀₀ =1.0, and the infection time was 5min , the percentage of callus formation was 23.04±1.5; when the concentration of infection solution was OD ₆₀₀ =1.0, and the infection time was 10 min, the percentage of callus formation was 21.61±2.44. This shows that for petiole explants, the infection solution concentration is OD ₆₀₀ =0.6, and the percentage of callus formation when the infection time is 10min is significantly higher than that of the other three combinations (significance analysis results with the other three groups P <0.05).

Table 1. Callus formation of petioles and leaf explants of Arabidopsis thaliana infected with different concentrations of infection solution at different times

Table 2. Callus formation of petioles and leaf explants of Arabidopsis thaliana at different concentrations of infecting solution at different times

The statistical results of callus formation percentage (%) and hygromycin-resistant callus percentage (%) of petiole explants under the condition that the infection solution concentration is OD ₆₀₀ =0.6 and the infection time is 10 min are shown in Table 3. The results showed that the percentage of callus formation of petiole was significantly higher than that of leaf (P<0.05), and the percentage of hygromycin-tolerant callus of petiole explant was also significantly higher than that of leaf explant hygromycin-resistant callus percentage (P<0.05).

Table 3. Statistics of genetic transformation data of different explants of Arabidopsis thaliana

3. Rooting induction culture

The callus is continued to be cultivated in the screening medium, and the fresh screening medium is replaced once a week. When the adventitious bud to be grown grows to have 4 leaves, the adventitious bud (the length of the adventitious bud is 2cm) is cut off and the Transfer to rooting medium and conduct rooting induction again in a light incubator with 16h light/8h dark and a temperature of 22°C until the regenerated seedlings develop a large number of strong roots.

The results showed that the petiole explants were induced to form many adventitious buds in about 3 months (Fig. 3F, Table 3). The adventitious shoots that formed 4 leaves or more were transferred into the rooting medium (Fig. 3G), and a large number of lateral roots were formed after induction in the rooting medium for about 2 months (Fig. 3H, I). The petiole explants successfully obtained 5 seedlings.

4. Plant culture and identification

After generating a large number of lateral roots, open the sealing film of the sterile seedlings, add double-distilled water to the bottle, and the amount of water just covers the medium. Seedlings were extracted for genomic DNA and PCR identification (forward primer for amplifying the ApP5CS1 gene: GC CCATGG ATGGAGATTCTTCCGATTC; reverse primer: TA GGTGACC TTAGCTTTGTGAGAGCTCG, the underlined part represents the restriction site, and the bases before the underline belong to restriction sites point; Arabidopsis thaliana with a 2.154kb fragment obtained by PCR amplification is the positive seedling). The PCR amplification identification of genomic DNA proved that the obtained 5 seedlings were all positive ApP5CS1 transgenic Arabidopsis thaliana seedlings, and the transformation rate was 100% (Table 4).

Table 4. Statistics of the acquisition of transgenic plants and the positive transformation rate

Explant name get seedlings Positive Plant Identification Conversion rate(%) petiole 5 5 100 blade 7 6 85.71

Five positive ApP5CS1 transgenic Arabidopsis thaliana seedlings were transplanted and routinely cultured, and successfully obtained transgenic plants with healthy growth and dark green leaves (Figure 3J). T1 generation transgenic plants (Fig. 3K).

(2) Agrobacterium-transformed Arabidopsis thaliana leaves

1. Co-cultivation

In the ultra-clean workbench, the 35-day-old sterile Arabidopsis thaliana wide and thick bright green leaves are cut into 1cm ² (length 1cm, about 1cm wide), and the cut leaves are respectively prepared in the above step 2 _OD600 5min and 10min respectively in the infection solution with OD 600 = 0.6 and 5min and 10min respectively in the infection solution with OD ₆₀₀ = 1.0 (Fig. 4A), a total of 4 different treatment methods were used in this experiment (Table 1 and Table 2). ), each group of treatment methods was set to 3 repetitions, after infection, the material was taken out and placed on sterile filter paper to blot dry, and finally transferred to a symbiotic medium covered with a layer of filter paper (Figure 4B), sealed to ensure sterility, at 22 ℃, cultured in the dark for 24 h, and obtained the co-cultured leaves.

2. Induction culture of callus

The co-cultured leaves were transferred to the screening medium for screening and culture, sealed after marking the date to ensure sterility, and cultured in a light incubator with a temperature of 22°C for 16h light/8h dark. The callus was obtained by changing fresh screening medium once a week. The percentage (%) of callus formation of leaf explants and the percentage (%) of hygromycin-resistant callus were counted every day since the explants were transferred to the selection medium for culture. The calculation formulas are as follows: percentage of callus formation (%)=(number of explants growing callus/total number of explants)×100%; percentage of hygromycin-resistant callus (%)=(hygromycin The number of callus resistant to venom/the total number of explants) × 100%.

The results showed that the infected Arabidopsis thaliana leaf explants also formed callus at about 35 days, and a large number of green dense callus were induced at about 53 days (Fig. 4C).

The infection results of different combinations on leaf explants are shown in Tables 1 and 2. The results showed that the percentages of callus formation of Arabidopsis thaliana leaves were different with different concentrations of infecting liquid and different time. The leaf explants were OD ₆₀₀ = 0.6 when the infecting liquid concentration was 0.6 and the infection time was 5 min. The percentage of callus formation was 10.84±1.46; the concentration of infection solution was OD ₆₀₀ =0.6, and the infection time was 10min, the percentage of callus formation was 17.89±1.65; the concentration of infection solution was OD ₆₀₀ =1.0, and the infection time was 5min , the percentage of callus formation was 31.23±2.67; when the concentration of infection solution was OD ₆₀₀ =1.0, and the infection time was 10 min, the percentage of callus formation was 19.98±4.35. This shows that for leaf explants, the percentage of callus formation was significantly higher than that of the other three combinations when the infection solution concentration was OD ₆₀₀ = 1.0 and the infection time was 5 min (significance analysis with the other three groups P <0.05).

The statistical results of callus formation percentage (%) and hygromycin-resistant callus percentage (%) of leaf explants under the condition that the infection solution concentration is OD ₆₀₀ = 1.0 and the infection time is 5 min are shown in Table 3. The percentage of leaf callus formation was significantly lower than that of petiole callus formation (P<0.05), and the percentage of hygromycin-tolerant callus of leaf explant was also significantly lower than that of petiole explant hygromycin-tolerant callus. percentage (P<0.05).

3. Rooting induction culture

The callus is continued to be cultivated in the screening medium, and the fresh screening medium is replaced once a week. When the adventitious buds to be grown grow to have 4 or more leaves, cut the adventitious buds (the length of the adventitious buds is 2cm) And transferred to the rooting medium again in 16h light/8h dark, the temperature is 22 ℃ in the light incubator for rooting induction, until the regenerated seedlings cultivated a large number of strong roots.

The results showed that the leaf explants formed adventitious buds in about 112 days (Fig. 4D), which was longer than that of petiole explants (Table 3). The adventitious shoots that formed 4 leaves or more were transferred into the rooting medium (Fig. 4E), and a large number of lateral roots were formed after induction with the rooting medium for about 75 days (Fig. 4F). Seven seedlings were obtained from leaf explants.

4. Plant culture and identification

After generating a large number of lateral roots, open the sealing film of the sterile seedlings, add double-distilled water to the bottle, and the amount of water just covers the medium. Seedlings were extracted for genomic DNA and PCR identification (forward primer for amplifying the ApP5CS1 gene: GC CCATGG ATGGAGATTCTTCCGATTC; reverse primer: TA GGTGACC TTAGCTTTGTGAGAGCTCG, the underlined part represents the restriction site, and the bases before the underline belong to restriction sites The 2.154kb Arabidopsis thaliana seedling obtained by PCR amplification is the positive seedling). The PCR amplification identification of genomic DNA showed that 6 of the 7 seedlings obtained were positive ApP5CS1 transgenic Arabidopsis seedlings, and the transformation rate was 85.71% (Table 4).

Six positive ApP5CS1 transgenic Arabidopsis thaliana seedlings were transplanted and routinely cultured, and successfully obtained transgenic plants with healthy growth and dark green leaves.

The results of genetic transformation of the petiole and leaf explants of Arabidopsis thaliana were compared and analyzed, and it was found that the optimum Agrobacterium concentration and infection time were different for petioles and leaf explants. When the OD ₆₀₀ value of Agrobacterium tumefaciens was 0.6 and the infection time of petiole explants was 0.6 and the infection time was 10 min, the infection effect was better, and the results of significant analysis with the other three groups showed P<0.05 (Table 1 and Table 2); The OD ₆₀₀ value of Agrobacterium was 1.0, and the effect was better when the infection time was 5 minutes, and the results of significant analysis with the other three groups were P<0.05 (Table 1 and Table 2). When the number of petiole explants infected was 117, the number of callus-forming explants reached 40, accounting for 34.19% of the total number of explants (Table 3). When the number is 99, 31 explants can form callus, accounting for 31.31% of the total number of explants (Table 3); and the percentage of callus formation of petiole explants after Agrobacterium infection is higher on leaves (Table 3). Among the callus formed by petiole explants, 37 callus formed were hygromycin-resistant, and the number of hygromycin-resistant callus accounted for 31.62% of the total number of explants. The results were also higher than the ratio of leaf explants with hygromycin resistance (Table 3). The callus formation time of petiole explants and leaf explants is almost the same time, but for the induction time of adventitious buds, after the petiole explants are infected with Agrobacterium, it needs to be screened and cultured for 91 days to induce the formation of adventitious buds. However, after the leaf explants are infected with Agrobacterium, it takes about 112 days of screening and culture to induce the formation of adventitious buds. The time to screen out adventitious buds from petiole explants is much shorter than that of leaf explants (Table 3). . Based on the above results, it is the best choice for the genetic transformation of Arabidopsis thaliana using petioles as explants.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the technical principles of the present invention, several improvements and modifications can be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

sequence listing

<110> Shihezi University

<120> Genetic transformation method of a short-lived plant Arabidopsis thaliana from Xinjiang

<160>1

<170>PatentIn version 3.5

<210>1

<211>2154

<212> DNA

<213>Artificial Sequence

<400>1

atggaggagc tagatcgttc acgtgctttt gccagagacg ttaagcgtat cgtcgtcaag 60

gttgggaccg cagttgtgac tggaaaaggt ggaagattgg ctcttggtcg cttgggagca 120

ctgtgtgaac agcttgcgga attaaactcg gatggatttg aggtgatatt ggtgtcatct 180

ggtgcggttg gccttggccg tcaaaggctt cgatatcgac aattagtcaa tagcagcttt 240

gcggatcttc agaagcctca gactgaactt gatggcaagg cttgtgctgg tgttgggcaa 300

agcagtctta tggcttatta cgagactatg ttcgaccagc tggatgtgac cgctgctcag 360

cttctggtga atgacagtag ttttagagac aaggacttca ggaagcaact taatgaaact 420

gtcaagtcca tgcttgattt gagggttatt ccaattttca atgagaatga tgctataagt 480

acccgtagag ccccatatca ggattcttct ggtattttct gggacaatga cagcctagct 540

gctctgctgg cgctggaact caaagctgat cttctgattc tcctgagtga tgttgaaggt 600

ctttaacacag gccctccaag tgatcctaac tcaaagttaa tccacacttt tgttaaagaa 660

aaacatcaag acgagattac attcggcgac aaatcaagat tagggagagg gggtatgacc 720

gcaaaagtca aagctgcagt gaatgcagct tatgctggga ttcctgtcat cataaccagt 780

gggtattcag ctgagaacat tgacaaagtc ctcagaggac tgcgtgttgg aaccttattc 840

catcaagatg ctcgtttatg ggctccgatc acagattcta gcgctcgtga catggctgtt 900

gcggcaaggg aaagttccag aaagcttcag gccctatctt cagaagatag gaaaaaaatt 960

ctgcttgata ttgctgatgc tcttgaagca aatgaaacaa caatcaaagc tgagaatgag 1020

ttagatgtag ctgcagcaca agaggctggg ttggaagaat cgttggtggc tcgcttagtt 1080

atgacgcctg ggaagatctc aagccttgca gcttcagttc gtaagctagc tgaaatggaa 1140

gatccaatcg gccgtgtttt aaagaaaaca gaggtggctg atggtcttgt cttagagaag 1200

acctcatcgc cattaggcgt acttctgatt gtttttgaat ctcgacctga tgcacttgta 1260

cagatagctt cgcttgccat ccggagtgga aatggtctct tactgaaggg tggaaaggag 1320

gcccggcgat caaatgctat cttacacaag gtgatcactg atgcaattcc agagactgtc 1380

gggggtaaac tcattggact tgtgacttca agagaagaga ttcctgattt gctcaagctt 1440

gatgacgtta tcgatcttgt gatcccaaga ggcagcaaca agcttgttac tcagataaaa 1500

aatactacaa aaatccctgt gctgggtcat gctgatggaa tctgtcatgt atatgtcgac 1560

aagtcttgtg acacggatat ggcaaagcgc atagtctctg atgcaaagtt agactatcca 1620

gcagcctgta atgcgatgga aacccttctt gtgcataagg atctagagca gaatgccgtg 1680

ctcaatgagc ttatttttgc tctgcagagc aatggagtca ctttatatgg tggcccaagg 1740

gcaagtgcaa tactgaacat accagaagca cggtcgttta accatgaata ctgttccaag 1800

gcttgcaccg ttgaagttgt agaagacgtt tatggtgcta tagatcacat tcaccaacat 1860

gggagtgcac acacagactg cattgtgaca gaggattccg aagttgcaga gcttttcctt 1920

cgccaagtgg acagcgctgc tgttttccac aacgcaagca caaggttctc agatggcttt 1980

cgatttggac ttggtgctga ggtgggagta agcacgggca ggatccatgc tcgtggtcca 2040

gttggggttg aaggattact cacaacaaga tggataatga gaggaaaagg acaagttgtg 2100

gacggagata atggaattgt ttacacccat caagacattc ccatccaagc ctaa 2154

Claims (10)

1. A genetic transformation method of arabidopsis thaliana comprises the step of carrying out genetic transformation by taking the petiole of arabidopsis thaliana as an explant.

2. The method of claim 1, wherein: the method comprises the following steps:

1) infecting the petioles of the arabidopsis thaliana by using an agrobacterium infection solution containing a target gene expression vector, and carrying out symbiotic culture on the infected petioles of the arabidopsis thaliana in a symbiotic culture medium to obtain the petioles after symbiotic culture;

2) carrying out subculture on the petioles subjected to symbiotic culture in a screening culture medium to obtain adventitious buds;

3) and (3) carrying out rooting culture on the adventitious bud in a rooting culture medium to obtain a complete small arabidopsis transgenic plant.

3. The method of claim 2, wherein: in step 1), OD of the staining solution₆₀₀0.6, the time of infection is 10 min.

4. A method according to any one of claims 1 to 3, wherein: the symbiotic culture medium comprises 0.5mg/L of 6-benzylamino adenine, 0.1mg/L of naphthylacetic acid and 20mg/L of acetosyringone;

or the screening culture medium comprises 0.5mg/L of 6-benzylamino adenine, 0.1mg/L of naphthylacetic acid, 10mg/L of screening agent and 500mg/L of bacteriostatic agent;

or the rooting culture medium comprises 0.1mg/L of naphthylacetic acid, 10mg/L of screening agent and 500mg/L of bacteriostatic agent.

5. The method according to any one of claims 1 to 4, wherein:

the pH value of the symbiotic culture medium is 5.7-5.9, specifically 5.8;

or, the pH of the screening medium is 5.7-5.9, specifically 5.8;

or, the pH of the rooting medium is 5.7-5.9, specifically 5.8;

or, the screening agent is hygromycin B;

or the bacteriostatic agent is carbenicillin.

6. The method according to any one of claims 1 to 5, wherein:

in the step 1), the culture condition is aseptic, 22 ℃ and 24 hours under the dark condition;

or, in the step 2), culturing under the sterile condition at 22 ℃ for 16h of light/8 h of dark condition to obtain adventitious buds at least containing 4 leaves; the screening culture medium is replaced once a week in the culture process;

or, in the step 3), the culture condition is aseptic, the temperature is 22 ℃, and the culture is carried out under 16h of light/8 h of dark condition for 2 months.

7. The method according to any one of claims 1 to 6, wherein: the length of the petiole is 0.8-1.2 cm.

8. The method according to any one of claims 1 to 7, wherein: the step 1) also comprises the following steps: and (4) sucking the redundant bacterial liquid on the surface of the infected petiole of the small arabidopsis thaliana by using sterile filter paper, and then placing the infected petiole of the small arabidopsis thaliana in a symbiotic culture medium for symbiotic culture.

9. A kit for the genetic transformation of small arabidopsis thaliana comprising the symbiotic medium and/or the screening medium and/or the rooting medium according to any one of claims 1 to 8.

10. Use of the kit of claim 9 in the genetic transformation of small arabidopsis;

or, the use of the kit of claim 9 for the preparation of a product of the genetic transformation of small arabidopsis;

or, the use of the method of any one of claims 1 to 8 or the kit of claim 9 in the breeding of small arabidopsis thaliana.

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