CN103882054A - Method for transient expression of introducing exogenous gene into chrysanthemum or related species - Google Patents

Abstract

The invention provides a method for transiently expressing an exogenous gene into chrysanthemums or related species through the Agrobacterium injection infiltration method. The exogenous gene is the GUS gene, which is less than 10kb overexpressed by carrying a 35s promoter and a terminator. Vector plasmid carrying. The main steps include (1) plant acquisition (2) construction of plant expression vector pORER1-2×35s:gus (3) preparation of infection solution (4) preparation of chrysanthemum leaves (5) leaf transformation (6) cultivation of leaves after transformation (7) Detection of positively transformed leaves. In the present invention, by selecting suitable explants and using Agrobacterium strains as susceptible strains, rifampicin and kanamycin are used to select and infect the bacteria; at the same time, P19 is mixed with Agrobacterium to make an infection solution, and P19 can Effectively inhibit the silencing effect of the plant on the expression of the foreign import vector, increase the expression level, and rationally optimize the co-cultivation conditions, so that the transformation time of this method is shortened to about 45 days, and the transformation efficiency reaches more than 60%. The method of the invention provides the possibility for more effectively screening and analyzing the gene function and protein localization of the chrysanthemum and further obtaining stable transformants.

Description

A method for transiently expressing exogenous genes into chrysanthemums or related species

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a method for transiently expressing exogenous genes into chrysanthemums or related species through the injection infiltration method of agrobacterium.

Background technique

Chrysanthemum (Chrysanthemum morifolium Ramat.), native to China, belongs to the perennial herbaceous flowers of the genus Chrysanthemum in the family Asteraceae. It has a cultivation history of more than 3,000 years in my country and is one of the top ten famous flowers in China. 30% of the world's cut flowers (Hong Bo et al., 2009). Chrysanthemum has high ornamental value and has been used more and more in landscaping and beautification in recent years. It is a kind of mass consumption flower, and it is urgent to increase the progress of chrysanthemum variety breeding and innovation work (Zhu Mingtao et al., 2011), so as to provide good economic benefits and social benefits. However, limited by the species' own genes, traditional breeding methods are difficult to succeed. Although distant hybridization has achieved some results, it is impossible to introduce the required genes at will. Transgenic technology provides a new idea and approach for chrysanthemum breeding.

Agrobacterium tumefaciens-mediated transgenesis is one of the most commonly used means to study the function of chrysanthemum genes and obtain genetically modified organisms. As the research progressed, it was found that due to the complexity of the genetic background of chrysanthemums, a large number of foreign genes were not expressed as expected in chrysanthemums, and the application of this traditional genetic transformation method in chrysanthemums was greatly limited (Yu Xin et al., 2009 ).

The Agrobacterium-mediated transient expression method is a fast and effective method for analyzing protein expression developed in recent years. Compared with the traditional transgenic process, it has the advantages of short cycle, safety and high efficiency, simple operation, relatively low cost, and high transformation efficiency. High, the advantages of being able to infect multiple species at the same time. At present, this method has achieved success in many plant tissues, such as tobacco leaves, Arabidopsis leaves, rose petals, pinellia sterile leaves, tomato leaves and fruits, grape leaves, etc. However, the expression in chrysanthemum has problems such as low transformation efficiency, low expression level of exogenous genes, high chimera strain rate and gene silencing during regeneration; selection of explants, determination of antibiotics, selection of susceptible strains, and co-cultivation Conditions, etc. play a decisive role in the transformation efficiency, and there are problems of cumbersome operation and long cycle time, which usually takes 4-6 months. Integrating the use of different strains in the successful cases of chrysanthemum transgenics, the genetic transformation rate is low, generally between 5-6 months. 40% (Teixeira et al., 2013).

Contents of the invention

The invention provides a method for transiently expressing foreign genes into chrysanthemums and related species. Mediated by Agrobacterium, the plant expression vector pORER1-2×35s:gus is constructed so that the foreign gene GUS can be expressed in chrysanthemums and chrysanthemums. Transient expression of related species solves the problem of long transformation period and low efficiency of Agrobacterium-transformed chrysanthemum genetic transformation, and provides the possibility for more effective screening and analysis of chrysanthemum gene function, protein localization and further obtaining stable transformants.

To achieve the above object, the present invention adopts the following technical means:

A method for transiently expressing exogenous genes into chrysanthemums or chrysanthemum relatives. The transient expression vector containing the GUS gene is transferred into the mesophyll cells of chrysanthemums and chrysanthemums relatives through the Agrobacterium injection infiltration method. GUS genes are expressed in leaves.

A method for transiently expressing exogenous genes into chrysanthemums or chrysanthemum relatives. Preferably, the transient expression vector is an overexpression vector plasmid of less than 10 kb carrying a 35s promoter and a terminator.

A method for transiently expressing exogenous genes into chrysanthemums or chrysanthemum close relatives, comprising the steps of:

(1) Plant acquisition: use chrysanthemum cutting seedlings with 10-14 fully expanded leaves 30d-50d after rooting; select the middle and upper part of the cutting seedlings to fully expand the leaves and mark them for injection;

(2) Construction of the plant expression vector pORER1-2×35s:gus: The designed primers are upstream primer 2×35S-Sac-F:SEQ ID NO.2, downstream primer 2×35S-Nhe-R:SEQ ID NO.3, and pCAMBIA1301 Plasmid DNA was used as a template, and high-fidelity enzymes were used for PCR amplification reaction, and SacⅡ and NheI restriction sites were introduced into the upstream and downstream of pCAMBIA1301 plasmid DNA, respectively, and the PCR product was recovered; the PCR product was ligated with the pORER1 vector after SacⅡ and NheI double digestion , transformed TOP10 competent cells, extracted positive plasmids, and successfully constructed the plant expression vector pORER1-2×35s:gus;

(3) Preparation of infection solution: transfer pORER1-2×35s:gus into Agrobacterium; pick a single clone, detect by PCR electrophoresis, and select a positive Agrobacterium clone with the correct plasmid construction and transfer it into YEB liquid medium for overnight Gently shake and cultivate for 16-20 hours, and shake P19 at the same time; centrifuge the shaken two bacterial solutions at room temperature, remove the supernatant, collect the bacterial cells, and resuspend them with infection buffer respectively; Mix the bacterial solution with equal volumes of P19, then shake the mixed bacterial solution at room temperature for at least 3 hours;

(4) Preparation of chrysanthemum leaves: Point or cross wounds on both sides of the main vein at the proximal end of the leaf without veins, the wound only needs to penetrate the leaf epidermis, and cannot penetrate the leaf;

(5) Leaf transformation: use the syringe with the needle removed, preferably a 2ml syringe, to absorb the infection solution prepared in step 3), aim the water outlet at the incision, slowly push the plunger of the syringe, and at the same time lightly press the leaf farthest corresponding to the incision with your fingers Position the shaft end so that the infecting liquid penetrates into the back of the leaf, repeat several times so that the infecting liquid spreads to the entire leaf as much as possible, and mark the corresponding area of the injected leaf;

(6) Post-transformation cultivation of leaves: Seal and moisturize the transformed cuttings with plastic wrap, place them in the dark and cultivate them for 1.5-2 days, then turn them into light and cultivate them for 2-3 days to obtain transformed living plants;

(7) Detection of positively transformed leaves: Submerge the marked leaves with X-Gluc staining solution, decolorize overnight after staining until transparent, then observe the GUS staining status of the leaves; design GUS gene primers: upstream primer GUS-RT-F: SEQ ID NO.4 , downstream primer GUS-RT-R:SEQ ID NO.5, select marked leaves to extract RNA, and reverse transcribe into cDNA, use cDNA as template PCR amplification to detect the expression of GUS in positively transformed leaves.

A method for transiently expressing exogenous genes into chrysanthemums or chrysanthemum close relatives. As a preference, the high-fidelity enzyme in the above reaction step 2) uses TaKaRa's PrimeSTAR ^TM HSDNA Polymerase; the PCR product is recovered with a gel recovery kit, using Transform TOP10 competent cells by heat shock method; PCR reaction system, 50 μL containing: 10×HSRCRBuffer 5.0 μL, 2×35S-Sac-F, 20 μmol L-1.0 μL each ^of 2×35S-Nhe-R primers, 2.5mmol· L ^-1 dNTPmix 4.0 μL, PrimeSTAR ^TM HSDNAPolymerase 0.4 μL, DNA template 1 μL, ddH ₂ O 37.6 μL; reaction program: pre-denaturation at 95°C for 4min, then melting at 94°C for 30sec, annealing at 55°C for 30sec, extension at 72°C for 1min30sec, The reaction was performed for 30 cycles and extended at 72°C for 10 min.

A method for transiently expressing exogenous genes into chrysanthemums or chrysanthemum relatives. As a preference, the plant expression vector pORER1-2×35s:gus in step 3) is transformed into competent Agrobacterium by freeze-thawing method, and the used Agrobacterium The bacterial strain is EHA105, which is cultured in YEB medium, and the YEB medium is supplemented with rifampicin and kanamycin; Agrobacterium is cultured overnight in YEB liquid medium to make OD600 reach 1.0-1.8, and shaking strain P19 also makes OD600 reach 1.0 -1.8; set the speed of the centrifuge to 4000rpm and centrifuge for 10min when the bacteria are collected and centrifuged; the formula of the infection buffer is 10mMMES, 100μMAS, 50mMMgCl ₂ aqueous solution, pH=5.6; Or 0.6 or 0.8 or 1.0; after adding P19, culture at 200 rpm at 28°C.

A method for transiently expressing an exogenous gene into a chrysanthemum, preferably, the chrysanthemum variety used is Youxiang or Shenma or a closely related species of chrysanthemum.

The present invention selects suitable explants, uses Agrobacterium strains as susceptible strains, and selects susceptible strains through rifampicin and kanamycin; meanwhile, the mixed culture of P19 and Agrobacterium is used to make an infection solution, which can effectively inhibit The silencing effect of the plant on the expression of the foreign import vector increases the expression level, and rationally optimizes the co-cultivation conditions, so that the transformation time of this method is shortened to about 45 days, and the transformation efficiency reaches more than 60%.

Description of drawings

Figure 1 is the electrophoresis detection of pORER1-2×35s:gus vector double enzyme digestion verification.

M: DL15000Marker, DL2000Marker. 1, 2: pORER1-2×35s:gus vector double digestion.

Figure 2 is the electrophoresis of PCR amplification detection of Agrobacterium liquid containing pORER1-2×35s:gus.

M: DL2000Marker; 1-6: Agrobacterium culture liquid; 7: pORER1-2×35s: gus vector positive control; 8: pORER1 empty vector negative control.

Figure 3 is the cutting seedlings of the closely related species Chrysanthemum Nao, 'Youxiang' and 'Shenma'. a: Chrysanthemum chinensis, a closely related species; b: 'Youxiang'; c: 'Shenma'.

Figure 4 shows the GUS staining status of pORER1-2×35s:gus transformed leaves.

y1-y5: The 1st to 5th slices of 'Youxiang' from the top to the bottom of the morphology are fully unfolded;

s1-s5: The 1st to 5th fully expanded leaves of 'Shenma' from the top to the bottom of the morphology;

j1-j5: The 1st to 5th fully expanded leaves of the closely related species Chrysanthemum chinensis from the morphological upper end to the morphological lower end.

Figure 5 is the detection of GUS expression in pORER1-2×35s:gus positive transformed leaves by RT-PCR.

A: Chrysanthemum chinensis, a closely related species; B: 'Youxiang'; C: 'Shenma'. M: DL2000 Marker.

1-4: Negative control of the 2nd-5th fully expanded leaves transformed with pORER1; 5: Positive control of pORER1-2×35s: gus vector; 6-9: pORER1-2×35s: gus positively transformed 2nd-5th fully expanded leaves leaf.

Fig. 6 is a map of the plant expression vector pORER1-2×35s:gus.

Detailed ways

The present invention will be further described below in conjunction with the examples, all of which are implemented on the premise of the technical solution of the present invention.

1. Plant acquisition: 30d-50d after rooting, cutting seedlings of 'Shenma', 'Youxiang', and related species Chrysanthemum Brain with 10-14 fully expanded leaves were used (Figure 3); Tender, well-grown, middle-upper fully expanded 4-5 leaves are marked for injection.

2. Construction of plant expression vector pORER1-2×35s:gus

Design primers: the upstream primer is 2×35S-Sac-F: SEQIDNO.2, the downstream primer is 2×35S-Nhe-R: SEQIDNO.3, using the pCAMBIA1301 plasmid DNA as a template, using TaKaRa’s PrimeSTARTMHSDNAPolymerase (a high-fidelity enzyme) Perform PCR amplification reaction; among them, 50 μL reaction system during PCR: 10×HSRCRBuffer 5.0 μL, 20 μmol L-1 2×35S-Sac-F, 2×35S-Nhe-R primers 1.0 μL each, 2.5 mmol·L-1 L-1 dNTPmix 4.0 μL, PrimeSTARTMHSDNAPolymerase 0.4 μL, DNA template 1 μL, ddH2O 37.6 μL; reaction program: pre-denaturation at 95°C for 4 min, then melting at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min and 30 sec, and 30 cycles of reaction , and extended at 72°C for 10 min; the PCR product was recovered with a gel extraction kit produced by AXYGEN in the United States to recover 2×35S fragments, and sent to Invitrogen for sequencing verification. The PCR product was ligated with the SacⅡ and NheI double digested pORER1 vector with TaKaRa T4 DNA ligase overnight at 16°C for 16 hours. The heat shock method was used to transform TOP10 competent cells. The specific steps were: (1) 10 μL of the ligation product was added to 200 μL of competent cells, and ice-bathed for 30 minutes; (2) After heat shock at 42°C for 90 seconds, immediately ice-bathed for 2 minutes; (3) Added 500 μL LB liquid culture (4) Centrifuge at 6000rpm at room temperature for 5min, leave 40-60μL of supernatant, smear LB or Amp or X-gal or IPTG plates, pick positive single clones for expansion; use Fermentas SacⅡ and NheI double digestion, 1% agarose gel electrophoresis detection (Figure 1) and sequencing verification. The recombinant plasmid was extracted with AxyPrep plasmid DNA extraction kit, and the plant expression vector pORER1-2×35s:gus (Figure 6) was constructed.

3. Transformation of Agrobacterium with expression vector pORER1-2×35s:gus

The plant expression vector pORER1-2×35s:gus constructed above was transformed into competent Agrobacterium by freeze-thaw method, the Agrobacterium strain used was EHA105, and single clones were picked for PCR electrophoresis detection, the results were shown in Figure 3, and positive clones were selected at 28 ℃, 220rpm shaking culture, the bacterial solution was added to the final concentration of 15% sterile glycerol and stored in a -80 ℃ refrigerator for use in the transformation of chrysanthemum leaves. At the same time, the empty pORER1 was transformed into Agrobacterium EHA105 by the same method as a negative control for the transformed leaves.

In the above steps, the preparation process of competent Agrobacterium includes the following steps: (1) Pick a single colony of Agrobacterium from the YEB plate containing 50 μg/ml rifampicin, and inoculate it in 5 ml of YEB liquid containing 50 μg/ml rifampicin Culture medium, 200rpm, 28°C overnight culture, 1-2d; (2) Take 2ml overnight culture solution and inoculate in 50ml YEB liquid medium containing the same antibiotic, 200rpm, 28°C culture until OD600 reaches 0.5-0.6. (3) Bacterial solution was placed in an ice bath for 30 minutes, and transferred to a 50ml pre-cooled centrifuge tube. (4) 4°C, 5000rpm, 10min, collect the bacteria, and resuspend the bacteria in 2ml of 20mM sterile CaCl2 solution pre-cooled by ice. Divide the bacterial liquid into 200 μl/tube, freeze in liquid nitrogen for 1 min, and store at -80°C.

The plant expression vector pORER1-2×35s:gus is transformed into competent Agrobacterium EHA105 by freeze-thaw method, including the following steps: (1) Add 1 μl of pORER1-2×35s:gus plasmid with a content of about 1ng to 100 μl of competent cells, Ice bath for 30 minutes; (2) Quick freezing in liquid nitrogen for 5 minutes, and thawing in a water bath at 37°C for 5 minutes. (3) Add 1ml of YEB liquid medium (with 50mg/L rifampicin and 50mg/L kanamycin added), and culture at 28°C and 200rpm for 4-5h. (4) Centrifuge at 6000 rpm for 1 min at room temperature, and discard 900 μl of the supernatant. (5) Resuspend the pellet, smear it on a YEB solid plate containing 50 μg/ml rifampicin and 50 μg/ml kanamycin, and culture it upside down at 28°C for 2-3 days.

4. PCR amplification detection of Agrobacterium liquid containing pORER1-2×35s:gus

Select a single colony and inoculate it in 50ml of YEB liquid medium containing rifampicin 50μg/ml and kanamycin 50μg/ml, shake the bacteria at 28°C and 200rpm, and use the bacterial solution as a template for PCR detection. The 25ul reaction system is as follows: 10 ×PCRBuffer 2.5μl; Mg ²⁺ 1.5μl; 2.5mM dNTP mixture 2.0μl; 10μM 2×35S-Sac-F 1.0μl; 10μM 2×35S-Nhe-R 1.0μl; Agrobacterium broth 1.0μl; 5U /μl Taq enzyme 0.2μl; ddH2O 15.8μl.

The reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 45 s, and 35 cycles; extension at 72°C for 10 min, and then 5 μl of PCR reaction solution was taken for agarose gel electrophoresis detection, and at the same time, the PCR reaction solution was detected by agarose gel electrophoresis. Agrobacterium EHA105 transformed with pORER1 was tested as a negative control (see Figure 2). The single clone of Agrobacterium positive by PCR is the pORER1-2×35s:gus strain.

5. Preparation of Infection Solution

Pick the positive Agrobacterium clones containing the correctly constructed plasmid pORER1-2×35s:gus from the YEB medium containing 50 μg/mL rifampin and 50 μg/mL kanamycin, and also pick the clones containing empty pORER1 Each positive Agrobacterium clone was cultured overnight in 6 mL YEB liquid medium containing 50 μg/mL rifampicin and 50 μg/mL kanamycin for 16-20 hours; at the same time, shake 6 mL P192 in equal volume, P19 can effectively inhibit the exogenous import of plants The silencing effect of vector expression increases the expression level. Then transfer each of the four bacterial solutions into 100mL YEB, 50μg/mL Rif5mg, 50μg/mL Kan5mg liquid medium, culture overnight for 16-20h and shake until OD600 is about 1.0-1.8. Shaking is beneficial to prevent the production of dead bacteria, to maintain the vitality of bacteria, and to increase the conversion rate. Then centrifuge the shaken bacterial solution at 4000rpm for 10min at room temperature, remove the supernatant, collect the bacterial cells, and use 10-16mL infection buffer respectively. The OD600 value was measured by ultraviolet spectrophotometer, and the OD600 of each bacterial solution was adjusted to be 0.8±0.02. Then, in each bacterial solution containing different expression vectors, that is, the positive Agrobacterium containing the correctly constructed plasmid pORER1-2×35s:gus and the positive Agrobacterium containing empty pORER1 were respectively added with an equal volume of P19, and then the mixed bacteria After incubating the liquid at 28° C. at a speed of 200 rpm for no less than 3 hours, it was prepared to inject chrysanthemum leaves of appropriate size.

6. Agrobacterium EHA105-mediated transient transformation of chrysanthemum leaves and post-transformation culture

Before injecting the brain leaves of 'Youxiang', 'Shenma', and related species of chrysanthemums, make a spot wound on both sides of the main vein at the proximal end of the leaf without veins. The size of the wound is 0.1cm×0.1cm, and the wound only needs to penetrate the The leaf epidermis cannot penetrate the leaves; the leaves are injected immediately to prevent the juice from clogging the wound and impeding penetration. Use a 2mL syringe with the needle removed to absorb the infection liquid, align the water outlet with the incision, slowly push the plunger of the syringe, and at the same time lightly press the distal end of the leaf corresponding to the incision with your fingers, so that the infection liquid can penetrate into the back of the leaf, possibly Multiple operations are required to infiltrate and moisten the entire leaf, inject 8-12 leaves at each leaf position, and mark the corresponding area of the injected leaf. The transformed cutting seedlings are sealed and kept moist with plastic wrap, placed in the dark for 2 days, and then transferred to the light for 2 days to obtain transformed live plants.

7. Detection of positively transformed leaves

1) GUS staining

Select the marked leaves, wash and dry them, and place them in the X-Gluc staining reaction solution according to the leaf position. The X-Gluc staining reaction solution includes: 50mMNaH2PO4, pH7.0; 0.5mMK4[Fe(CN)6]; 0.1%TritonX -100; 20% methanol; 0.5mg/mlX-Gluc, vacuum pumping for 10min, 37°C overnight in the dark, after staining, decolorize with 70% ethanol overnight, then decolorize with 95% ethanol, after the leaves are decolorized to transparent, observe Leaf GUS staining status (as shown in Figure 4).

It can be seen from Figure 4 that the transient expression transformation effect of the cut chrysanthemum 'Youxiang' is the best, and the expression efficiency of 'Shenma' is the lowest; in general, the same plant, from the morphological upper end to the morphological lower end, counts from the 1st to the 5th piece The expression efficiency of fully expanded leaves increased first and then decreased, and it was better to select the fourth fully expanded leaf for injection of transformed leaves. From the GUS staining of the leaves, it can be seen that the staining depths of the leaves of the same leaf position of the same plant are inconsistent, indicating that the plant's own condition and the penetration of the infection solution have an impact on the instantaneous transformation. As can be seen from Table 1 and Figure 4, the leaf conditions and transformation rates after transformation are: 'Youxiang' is the best, and the closely related species Chrysanthemum Brain is the worst, reflecting the differences in the transformation rates of plant species and varieties; The GUS staining status of the later leaves, the younger leaves of the same plant are more severely damaged, and the more closely related species of chrysanthemum have more damaged leaves of the three plants, indicating the maturity of the leaves, the growth status of the leaves, the thickness of the leaves, and the smoothness of the leaf epidermis etc. have a certain influence on the instantaneous conversion efficiency.

Table 1 Transient transformation of leaves of 'Youxiang', 'Shenma', and Chrysanthemum Brain

2) RT-PCR detection

Select 0.1 g of marked leaves after cleaning and drying, extract total RNA from leaves with RNAisoReagent produced by TaKaRa, and reverse transcribe it into cDNA. Using cDNA as a template and untransformed leaves as a negative control, design GUS gene primers: upstream primer GUS- RT-F: SEQIDNO.4, downstream primer GUS-RT-R: SEQIDNO.5 for PCR, PCR detection positive expression of GUS in transformed leaves, the results are shown in Figure 5.

The cDNA synthesis steps in the above RT-PCR detection process are as follows:

(1) Preparation of reaction mixture: 2 μl of TotalRNA; 2 μl of 100 μM Oligo (dT) ₁₈ Primer; 8 μl of RNaseFreedH ₂ O. (2) Keep warm at 70°C for 10 minutes, and ice for 2 minutes. (3) Microcentrifuge to aggregate the reactants at the bottom of the PCR tube. (4) Configure the following reverse transcription reaction solution in the above tube: 12 μl of the above-mentioned template RNA/primer denaturation solution; 4 μl of 5×M-MLV Buffer; 0.5 μl of 10 mM dNTPsMixture; 0.5 μl of 40 U/μl RNase Inhibitor; 200 U/μl of RTaseM-MLV ( RHaseH-) 0.8 μl; RNaseFreedH ₂ O 1.7 μl; (5) 42°C extension for 60 minutes; (6) 70°C incubation for 15 minutes and cooling on ice, the obtained cDNA can be used directly or stored at -20°C.

GUS gene primers for PCR amplification reaction system are as follows: 10×PCRBuffer 2.5μl; Mg ²⁺ 1.5μl; 2.5mM dNTPmixture 2.0μl; 10μM GUS-RT-F 1.0μl; 10μM GUS-RT-R 1.0μl; cDNA 1.0 μl; 5 U/μl Taq enzyme 0.2 μl; ddH ₂ O 15.8 μl.

The reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and 30 cycles; extension at 72°C for 10 min, and electrophoresis detection with 1% agarose gel after the reaction was completed.

It can be known that the above-mentioned embodiments are only exemplary implementations adopted to illustrate the principles of the invention, but the present invention is not limited thereto, and those skilled in the art can make various improvements and changes without departing from the essence of the present invention. These improvements And changes also belong to the protection scope of the present invention.

Claims (6)

1. A method for carrying out transient expression of exogenous gene into chrysanthemum or chrysanthemum close relatives, characterized in that: the transient expression vector containing GUS gene is transferred into chrysanthemum and chrysanthemum close relatives mesophyll cells by Agrobacterium injection infiltration In , the GUS gene was transiently expressed in leaves after culture. 2. the method for transiently expressing exogenous gene into chrysanthemum or chrysanthemum relatives according to claim 1, characterized in that: the transient expression vector is an overexpression vector plasmid carrying 35s promoter and terminator, less than 10kb. 3. the described exogenous gene of claim 2 is transferred into the method for transient expression of chrysanthemum, it is characterized in that, the method comprises the steps: (1) Plant acquisition: use chrysanthemum cutting seedlings with 10-14 fully expanded leaves 30d-50d after rooting; select the middle and upper part of the cutting seedlings to fully expand the leaves and mark them for injection; (2) Construction of the plant expression vector pORER1-2×35s:gus: The designed primers are upstream primer 2×35S-Sac-F:SEQ ID NO.2, downstream primer 2×35S-Nhe-R:SEQ ID NO.3, and pCAMBIA1301 Plasmid DNA was used as a template, and high-fidelity enzymes were used for PCR amplification reaction. SacⅡ and NheI restriction sites were introduced into the upstream and downstream of pCAMBIA1301 plasmid DNA, respectively, and the PCR product was recovered; Connection, transformation of TOP10 competent cells, extraction of positive plasmids, and successful construction of the plant expression vector pORER1-2×35s:gus; (3) Preparation of infection solution: transfer pORER1-2×35s:gus into Agrobacterium; pick a single clone, detect by PCR electrophoresis, and select a positive Agrobacterium clone with the correct plasmid construction and transfer it into YEB liquid medium for overnight Gently shake and cultivate for 16-20 hours, and shake P19 at the same time; centrifuge the shaken two bacterial solutions at room temperature, remove the supernatant, collect the bacterial cells, and resuspend them with infection buffer respectively; Mix the bacterial solution with equal volumes of P19, then shake the mixed bacterial solution at room temperature for at least 3 hours; (4) Preparation of chrysanthemum leaves: Point or cross wounds on both sides of the main vein at the proximal end of the leaf without veins, the wound only needs to penetrate the leaf epidermis, and cannot penetrate the leaf; (5) Leaf transformation: use the syringe with the needle removed, preferably a 2ml syringe, to absorb the infection solution prepared in step 3), aim the water outlet at the incision, slowly push the plunger of the syringe, and at the same time lightly press the leaf farthest corresponding to the incision with your fingers Position the shaft end so that the infecting liquid penetrates into the back of the leaf, repeat several times so that the infecting liquid spreads to the entire leaf as much as possible, and mark the corresponding area of the injected leaf; (6) Post-transformation cultivation of leaves: Seal and moisturize the transformed cuttings with plastic wrap, place them in the dark and cultivate them for 1.5-2 days, then turn them into light and cultivate them for 2-3 days to obtain transformed living plants; (7) Detection of positively transformed leaves: Submerge the marked leaves with X-Gluc staining solution, decolorize overnight after staining until transparent, then observe the GUS staining status of the leaves; design GUS gene primers: upstream primer GUS-RT-F: SEQ ID NO.4 , downstream primer GUS-RT-R:SEQ ID NO.5, select marked leaves to extract RNA, and reverse transcribe into cDNA, use cDNA as template PCR amplification to detect the expression of GUS in positively transformed leaves. 4. the method for transiently expressing exogenous gene into chrysanthemum or chrysanthemum close relatives according to claim 3, is characterized in that, high-fidelity enzyme adopts TaKaRa's ^PrimeSTARTM HSDNAPolymerasePCR in above-mentioned reaction step 2); Recovery kit recovery, transform TOP10 competent cells by heat shock method; PCR reaction system, 50 μL containing: 10×HSRCRBuffer 5.0 μL, 2×35S-Sac-F, 20 μmol ^{L- 1} 2×35S-Nhe-R primers 1.0 μL each, 2.5 mmol L ^-1 dNTPmix 4.0 μL, PrimeSTAR ^TM HSDNA Polymerase 0.4 μL, DNA template 1 μL, ddH ₂ O 37.6 μL; reaction program: pre-denaturation at 95°C for 4 min, then melting at 94°C for 30 sec, annealing at 55°C 30sec, 72°C extension for 1min30sec, 30 cycles of reaction, 72°C extension for 10min. 5. The method for transiently expressing exogenous genes into chrysanthemums or chrysanthemum relatives according to claim 4, characterized in that, in step 3), the plant expression vector pORER1-2×35s:gus is transformed by freeze-thaw method Agrobacterium in state, the Agrobacterium strain used is EHA105, cultivated on 50ml YEB liquid medium; YEB medium is added with 50mg/L rifampicin and 50mg/L kanamycin; Agrobacterium is cultured in YEB liquid Cultivate overnight in the medium to make the OD600 reach 1.0-1.8, and shake the bacteria P19 to make the OD600 also reach 1.0-1.8; set the speed of the centrifuge to 4000rpm and centrifuge for 10min; the formula of the infection buffer is 10mMMES, 100μMAS, 50mMMgCl ₂ Aqueous solution, pH=5.6; Infection buffer adjusts the concentration of the bacterial solution so that the OD600 is 0.4 or 0.6 or 0.8 or 1.0; The positive Agrobacterium bacterial solution is mixed with P19 in equal volumes and cultured at 200 rpm at 28°C. 6. The method for transiently expressing foreign genes into chrysanthemums or chrysanthemum relatives according to any one of claims 1-5, characterized in that the material used is chrysanthemum 'Youxiang' or 'Shenma' or A closely related species is Chrysanthemumnankinginse.

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