Agrobacterium rhizogenes-mediated sunflower instantaneous transformation system and construction method thereof

Abstract

The invention relates to the technical field of plant genetic engineering, and provides an agrobacterium rhizogenes-mediated sunflower transient transformation system and a construction method thereof, sterile conditions and tissue culture are not needed, a vector to be transformed is transferred into agrobacterium rhizogenes, positive strains are cultured and screened, and a dip dyeing solution is prepared; cutting a stem section of the sunflower, and pricking holes near a cut; immersing the stem segments in the dip dyeing liquid for a period of time; taking out and inserting into sterilized vermiculite soil mixed with orobanche coerulescens for rooting to obtain an instantly transformed sunflower plant; the method is easy and convenient to operate and short in period, the expression level of the HaCERK1 gene can be effectively improved, the resistance of sunflowers to orobanche coerulescens infection is enhanced, and the gene function research period is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and more specifically to a sunflower transient transformation system mediated by Agrobacterium rhizogenes and a construction method thereof. Background Art

[0002] Sunflower (Helianthus annuus) is an important economic crop that is planted on a large scale. It has the characteristics of strong adaptability, drought resistance, salt tolerance, and barrenness resistance, and can be used for soil improvement. Sunflower seeds are the main source of edible oil in the world, and the consumption of sunflower seeds is increasing every year. Sunflower broom (Orobanche cumana Wallr) is a parasitic weed that extracts water and nutrients from the host sunflower, resulting in slow growth of sunflowers, a sharp drop in yield, and serious impact on oil and protein content. At present, the main control measures for sunflower broom are chemical, biological control and induced resistance, but the results are not satisfactory. For a period of time, breeding varieties resistant to broom was considered an economical, environmentally friendly and effective control method. However, this resistance was quickly overcome by the evolution of sunflower broom. Therefore, accelerating resistance breeding is an urgent problem to be solved.

[0003] The cut-dip-budding (CDB) system is an instantaneous transformation system similar to the Arabidopsis floral dip method, which does not require tissue culture and is easy to operate. It can quickly acquire phenotypes, does not rely on instantaneous plant transformation, avoids lethal mutations, and can study the functions of complex redundant genes and quickly compare gene functions between different species. Therefore, it has been widely used in some species with complex genetic backgrounds, difficult instantaneous transformation, and strong regeneration ability.

[0004] Public document 1 (Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture (2022). Cell Discovery, 8(1), Article 100345) studies the method of achieving instantaneous transformation of plants through the CDB (cut-dip-budding) system. The document focuses on the principles and applications of the CDB system, which uses Agrobacterium rhizogenes to mediate instantaneous transformation without relying on tissue culture, thereby significantly simplifying the complexity and technical barriers of the traditional transformation process. In the study, this system was successfully used to genetically modify a variety of plants such as rubber grass, sweet potato and Ailanthus, proving that it has extremely high applicability in plants with poor regeneration ability or complex genetic background. The advantages of the CDB system are that it is easy to operate, efficient, and has low requirements for the tissue regeneration ability of the target plant to be transformed. It is suitable for widespread use in plant functional gene research and genetic modification engineering.

[0005] In practical applications, especially for the transient transformation needs of sunflower, a specific species, the methods in existing literature have not fully solved technical difficulties such as stem segment rooting conditions and antibiotic screening efficiency, which may limit the wide application of this system in sunflower transient transformation. In addition, the research direction of existing literature mainly focuses on universal verification and basic transient transformation, and does not discuss in detail the transformation of certain specific functional genes, such as the study of receptor kinase genes related to sunflower resistance to parasitic weeds. Summary of the invention

[0006] In order to overcome the above-mentioned defects of the prior art, the present invention provides a sunflower transient transformation system mediated by Agrobacterium rhizogenes and a construction method thereof, wherein the pRI101-GFP-HaCERK1 overexpression vector is introduced into the sunflower by using Agrobacterium rhizogenes K599 as a medium, and efficient transformation of the receptor-like kinase gene is achieved through the steps of cutting stem segments, piercing, immersing and rooting. The problems of low transient transformation efficiency, difficulty in rooting and poor antibiotic screening effect of sunflower in the prior art are solved. The method of the present invention does not require tissue culture, is easy to operate, and can accelerate the process of breeding for resistance to Orobancha.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The invention discloses a sunflower transient transformation system mediated by Agrobacterium rhizogenes. The transient transformation system comprises a receptor-like kinase gene vector pRI101-GFP-HaCERK1 constructed by taking Agrobacterium rhizogenes K599 as a mediator and taking pRI101-GFP as an overexpression vector.

[0009] A method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes comprises the following steps:

[0010] Step S1, transferring the gene vector to be transferred into competent cells of Agrobacterium rhizogenes K599, and obtaining positive strains through resistance screening;

[0011] Step S2, inoculating the selected positive strains into liquid culture medium for cultivation and sequencing;

[0012] Step S3, the successfully sequenced bacteria are propagated into a new culture medium, cultured overnight until the absorbance at a wavelength of 600 nm reaches 0.8-1.0, and the culture medium is used as the staining solution;

[0013] Step S4, simultaneously spreading the positive strains on LB solid medium containing 50 mg / L kanamycin and 50 mg / L streptomycin;

[0014] Step S5, selecting a sunflower that has been cultivated for about 5 days, and cutting a stem segment about 5 cm below the cotyledon of the sunflower as an experimental material;

[0015] Step S6, using a sterile needle to evenly pierce 7-8 small holes 1 cm from the cut stem segment;

[0016] Step S7, completely immersing the stem segment after the hole treatment into the prepared impregnation solution, so that the surface of the stem segment is fully in contact with the bacterial solution, and infiltrating for about 1 hour to obtain the infiltrated stem segment;

[0017] Step S8, taking out the infiltrated stem segment from the bacterial solution, and using a bent gun tip to take the bacterial cells on the plate and smear them around the incision and the puncture hole;

[0018] Step S9, inserting the treated stem segments into sterilized vermiculite soil mixed with Orobancha for rooting to obtain instantaneously transformed sunflower plants.

[0019] As a further solution of the present invention, the gene vector to be transferred includes a pRI101-GFP plasmid for gene editing.

[0020] As a further embodiment of the present invention, the pRI101-GFP plasmid includes pRI101-GFP-HaCERK1.

[0021] As a further solution of the present invention, the positive strains include strains that are screened by two antibiotics, kanamycin and streptomycin, and are positive in both bacterial liquid polymerase chain reaction and sequencing.

[0022] As a further embodiment of the present invention, in step S2, the liquid culture medium is LB culture medium containing 50 mg / L kanamycin and 50 mg / L streptomycin.

[0023] As a further embodiment of the present invention, the branch of the sunflower is 3 to 7 cm in length and carries 2 cotyledons and 1 to 2 new leaves.

[0024] As a further solution of the present invention, in step S9, the substrate for stem segment insertion includes sterilized vermiculite, and the culture temperature is 24-26°C.

[0025] As a further solution of the present invention, after the rooting of the stem segments is completed, transient transformation detection is further performed on the rooted sunflower plants.

[0026] Compared with the prior art, the method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes of the present invention has the following beneficial effects:

[0027] The present invention provides a transient transformation system that directly performs transient transformation on tissues above the stem of a sunflower plant without the need for sterile conditions or tissue culture. The transient transformation system of the present invention is used to recombinantly transform the sunflower HaCERK1 gene with a vector pRI101, and gene editing for overexpression of the HaCERK1 gene can effectively increase the expression level of the sunflower HaCERK1 gene, thereby effectively promoting the resistance of the sunflower to infection by Orobanche.

[0028] The instantaneous transformation system of the present invention has a cycle of 7 to 28 days, which saves time and effectively shortens the research cycle of the sunflower anti-broomrape gene function; the instantaneous transformation rate is high; the sunflower instantaneous transformation system and method provided by the present invention eliminate the process of callus induction and tissue culture, simplify experimental operations, and reduce experimental costs, which will greatly improve the progress of sunflower gene function research and greatly save time and costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an electrophoresis detection diagram of the positive strain of the sunflower transient transformation vector pRI101-GFP-HaCERK1 of the present invention.

[0030] Figure 2 This is a schematic diagram of the structure of the exogenous sunflower transient transformation vector mediated by Agrobacterium rhizogenes of the present invention.

[0031] Figure 3 The figure shows the transmembrane region and probability distribution of the HaCERK1 protein of the present invention.

[0032] Figure 4 This is a protein sequence alignment of receptor kinase 1 from different species.

[0033] Figure 5 This is the predicted secondary structure of HaCERK1.

[0034] Figure 6 This is an operation flow chart of the sunflower instantaneous transformation system of the cut-immersion-regeneration root system of the present invention.

[0035] Figure 7 This is a comparison chart of the root regeneration rate of the sunflower transient transformation system mediated by Agrobacterium rhizogenes of the present invention.

[0036] Figure 8 This is a comparison chart of the detection rate of the instantaneous conversion of sunflower into green fluorescence by the handheld UV light (365nm) of the present invention.

[0037] Fig. 9 This is a fluorescence microscope image of the root transformation status of sunflower 12 days after instantaneous transformation of the present invention.

[0038] Fig.10 This is a comparison chart of the growth phenotypes of sunflower plants in different treatment groups of the present invention.

[0039] Fig.11 This is a statistical comparison chart of the number of parasitic Broomrape in sunflower plants in different treatment groups of the present invention.

[0040] Fig.12 This is the subcellular localization map of the HaCERK1 protein of the present invention.

[0041] In the figure, pRI101: pRI101 vector; M: Marker, molecular weight standard; mark: marker; K-1: sample K-1; K-2: sample K-2; K-3: sample K-3; CE-3: sample CE-3; CE-4: sample CE-4; CE-5: sample CE-5; pRI101-EV: pRI101 empty vector; CaMV35S: CaMV35S promoter; eGFP: enhanced green fluorescent protein; NOS terminator: NOS terminator; pRI101-HaCERK1: pRI101 vector with HaCERK1 gene; test-F: forward primer; test-R: reverse primer; TMHMM: transmembrane helix hidden Markov model; SVM H / L Raw: SVM high / low raw data; SVM iL / oLRaw: SVM inner / outer raw data; SVM RE / IRE Raw: SVM transmembrane region / non-transmembrane region raw data; SVM SP / ISPRaw: SVM signal peptide / non-signal peptide raw data; SVM: support vector machine; Signal peptide: signal peptide; Cytoplasmic: cytoplasmic; Extracellular: extracellular; Re-entrant Helix: reentrant helix; Transmembrane Helix: transmembrane helix; Disordered: disordered; Alpha helix: alpha helix; Beta strand: beta strand; TM helix: TM helix; query: query; HaCERK1: sunflower receptor kinase 1; AtCERK1: Arabidopsis receptor kinase 1; OsCERK1: rice receptor kinase 1; ZmCERK1: maize receptor kinase 1; NaCERK1: tobacco receptor kinase 1; GmCERK1: soybean receptor kinase 1; LcCERK1: Zoysia receptor kinase 1; WmCERK1: wheat receptor kinase 1; AaCERK1: Artemisia argyi receptor kinase 1; Sequence: sequence; Secondary structure: secondary structure; SSconfidence: secondary structure confidence; Disorder: disordered region; Disorder confidence: disordered region confidence; Confidence Key: confidence key; High: high; Low: low; Root: root system; Transformation: transformation; Efficiency: efficiency; Control: control; pRI101-GFP: pRI101 enhanced green fluorescent protein vector; GFP: green fluorescent protein; Detection: detection; Percentage (%): percentage; K599: Agrobacterium rhizogenes K599; Normal: normal;Fluorescence: fluorescence; 12dpi: 12 days after inoculation; Orobanche cumana: sunflower, number: number. ; DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The invention discloses an Agrobacterium rhizogenes-mediated sunflower transient transformation system. The transient transformation system uses Agrobacterium rhizogenes K599 as a mediator, pRI101-GFP as an overexpression vector, and constructs a receptor-like kinase gene vector pRI101-GFP-HaCERK1.

[0044] A method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes comprises the following steps:

[0045] Step S1, transferring the gene vector to be transferred into competent cells of Agrobacterium rhizogenes K599, and obtaining positive strains through resistance screening;

[0046] Step S2, inoculating the selected positive strain into a liquid culture medium and culturing until the bacterial solution becomes uniformly turbid;

[0047] Step S3, further expanding the culture in a conical flask, and the absorbance at a wavelength of 600 nm reaches 0.8-1.0 after overnight culture, and using it as a staining solution;

[0048] Step S4, simultaneously spreading the positive strains on LB solid medium containing 50 mg / L kanamycin and 50 mg / L streptomycin;

[0049] Step S5, selecting a sunflower that has been cultivated for about 5 days, cutting a stem segment about 5 cm below the cotyledon of the sunflower as an experimental material, and ensuring that the cut remains fresh and free of contamination;

[0050] Step S6, using a sterile needle to evenly pierce 7-8 small holes 1 cm from the cut stem segment, avoiding holes that are too deep to maintain the integrity of the stem segment tissue;

[0051] Step S7, completely immersing the stem segment after the hole treatment into the prepared impregnation solution, so that the surface of the stem segment is fully in contact with the bacterial solution, and infiltrating for about 1 hour to obtain the infiltrated stem segment;

[0052] Step S8, taking out the infiltrated stem segment from the bacterial solution, and using a bent gun tip to take the bacterial cells on the plate and smear them around the incision and the puncture hole;

[0053] Step S9, inserting the treated stem segments into sterilized vermiculite soil mixed with Orobancha for rooting to obtain instantaneously transformed sunflower plants.

[0054] The gene vector to be transferred includes a pRI101-GFP plasmid for gene editing.

[0055] The pRI101-GFP plasmid includes pRI101-GFP-HaCERK1.

[0056] The positive strains include strains that are screened by two antibiotics, kanamycin and streptomycin, and are positive in both bacterial liquid polymerase chain reaction and sequencing.

[0057] In step S2, the liquid culture medium is LB culture medium containing 50 mg / L kanamycin and 50 mg / L streptomycin.

[0058] The branches of the sunflower are 3 to 7 cm in length and carry 2 cotyledons and 1 to 2 new leaves.

[0059] In step S9, the substrate for stem segment insertion includes sterilized vermiculite, and the culture temperature is 24-26°C.

[0060] After the rooting of the stem segments was completed, the rooted sunflower plants were further tested for transient transformation.

[0061] Example 1

[0062] In the embodiment of the present invention, a pRI101-GFP vector for overexpressing the HaCERK1 gene is constructed. The pRI101-GFP vector includes a target sequence for overexpressing the HaCERK1 gene. The target sequence is preferably located between the restriction sites of Sal Ⅰ and Kpn Ⅰ of the pRI101-GFP vector.

[0063] Example 2

[0064] In the embodiment of the present invention, Agrobacterium for overexpressing the HaCERK1 gene is constructed, wherein the Agrobacterium includes the pRI101-GFP overexpression vector, and the initial strain of the Agrobacterium preferably includes Agrobacterium rhizogenes, and more preferably Agrobacterium rhizogenes K599.

[0065] Example 3

[0066] In the embodiment of the present invention, a sunflower transient transformation system mediated by Agrobacterium rhizogenes is used to implement overexpression of the HaCERK1 gene, and the specific steps are as follows:

[0067] Step 1: Transform the vector containing the target gene into Agrobacterium rhizogenes strain K599;

[0068] Step 2: Pick the positive strains and extract the bacteria for overnight culture;

[0069] Step 3: Take 300 μL of the overnight cultured strain and add it to 100 mL of LB medium containing kanamycin and streptomycin, culture until the OD600 value reaches 1, and use it directly for infection;

[0070] Step 4: Take 300 μL of the overnight cultured strain and spread it on LB medium containing kanamycin and streptomycin, and culture until the whole plate is covered;

[0071] Step 5: Cut the seedlings that have germinated for about 5 days, use a needle to poke seven or eight small holes within 1 cm above the cut to increase the infection area, and soak them in the bacterial solution of step 3 for about 1 hour;

[0072] Step 6: Use a coating stick or a bent gun tip to apply the Agrobacterium obtained in step 4 to the cut ends of the sunflower seedlings and plant them in vermiculite;

[0073] Step 7: Next, take 3-5 mL of the Agrobacterium from step 3 and drip it into the vermiculite along the stem;

[0074] Step 8, placing the planted seedlings in a culture room, culturing for 1-2 weeks at a temperature of 25°C, a light period of 14 hours, and a dark period of 10 hours, and detecting the expression of green fluorescence in the hairy roots with a handheld scanner;

[0075] Step nine, remove the fluorescent roots, further use PCR to detect whether the target gene has been successfully transferred, and use qRT-PCR to verify whether the target gene is highly expressed;

[0076] Step 10. After 4 weeks of cultivation, the soil was cut open to count the number of parasitic Orobancha in the control group and the experimental group to clarify that the HaCERK1 gene plays a positive regulatory role in the defense of sunflower Orobancha.

[0077] Example 4

[0078] like Figure 1The figure shows the electrophoresis detection diagram of the positive strain of the sunflower transient transformation vector pRI101-GFP-HaCERK1 of the present invention. The lanes are labeled with pRI101-1, pRI101-2, and pRI101-3 (respectively labeled as K-1, K-2, and K-3), which are used to detect the positive strain of the empty vector pRI101; and the pRI101-HaCERK1-1, pRI101-HaCERK1-2, and pRI101-HaCERK1-3 (respectively labeled as CE-3, CE-4, and CE-5) lanes detect the positive strain of the recombinant overexpression vector carrying the HaCERK1 gene, proving that the HaCERK1 gene has been successfully inserted into the vector and successfully transformed in Agrobacterium. These results verify the reliability of the vector construction and gene transformation used in the Agrobacterium rhizogenes-mediated sunflower transient transformation system of the present invention.

[0079] Example 5

[0080] like Figure 2 Shown is a schematic diagram of the structure of the exogenous sunflower transient transformation vector mediated by rhizogenes of the present invention, showing two vector structures used in the rhizogenes-mediated sunflower transient transformation system: pRI101-EV (empty vector) and pRI101-HaCERK1 (overexpression vector with HaCERK1 gene). The pRI101-EV vector in the figure contains the CaMV35S promoter, the eGFP reporter gene, and the NOS terminator, which are used to verify the basic functions of the transformation system and as a control group. The pRI101-HaCERK1 vector adds the HaCERK1 gene on the basis of pRI101-EV, which is located between the CaMV35S promoter and eGFP, and is driven to express by CaMV35S. The marked test-F and test-R are the positions of the forward primer and the reverse primer, respectively, which are used to verify whether the HaCERK1 gene is successfully inserted into the vector and whether the expression system works properly through PCR later.

[0081] Example 6

[0082] Figure 3-Figure 5 This is the bioinformatics analysis of the HaCERK1 protein of the present invention. Figure 3 The figure is a transmembrane region and probability distribution diagram of the HaCERK1 protein of the present invention, showing the transmembrane topological structure and posterior probability distribution of the HaCERK1 protein predicted by the TMHMM model. According to the curve, it can be observed that at the 200-300 amino acid positions, HaCERK1 has obvious transmembrane characteristics, which is consistent with the characteristic that receptor-like kinases usually have a single transmembrane domain. This figure verifies that the encoded product of the HaCERK1 gene has the structural characteristics of a receptor kinase, and HaCERK1 is a plasma membrane protein containing a signal peptide, which provides a theoretical basis for its function in sunflower. Figure 4This is the result of sequence alignment of the receptor kinase-like 1 protein in nine different species, including sunflower, Arabidopsis, rice, corn, tobacco, soybean, Zoysia japonica, wheat and Artemisia selengensis. The white part is the highly conserved region.

[0083] Figure 5 It is the secondary structure prediction of HaCERK1. The first row is the sequence position, and the amino acids are colored based on their properties: polar amino acids include A, S, T, G, and P; hydrophobic amino acids include M, I, L, and V; charged amino acids include K, R, E, N, D, H, and Q; aromatic amino acids include W, Y, F, and C; the confidence assessment row uses a numerical rating scale from 0 to 9 to represent the confidence level, where the closer the value is to 9, the higher the confidence, and the closer the value is to 0, the lower the confidence.

[0084] Example 7

[0085] like Figure 6 The present invention shows a cut-immersion-regeneration root system sunflower instantaneous transformation system operation flow chart, including: a, cutting off the sunflower from the stem segment after 5 days of growth; b, using a needle to make a hole 1 cm from the incision; c, immersing in the corresponding Agrobacterium solution for about 1 hour; d, further applying the bacteria on the plate to the vicinity of the incision with a sterile gun head; e, vertically inserting into vermiculite; f, injecting the soaked bacteria solution with a gun head. Finally, the plant is placed on a moisturizing cover and cultured in a culture room for one week to observe the rooting situation.

[0086] like Figure 7 The figure shows a comparison of the root regeneration rate of the instantaneous transformation system of sunflower of the present invention, showing the statistical results of the root regeneration rate of sunflower in different treatment groups, including the root regeneration rate data of the control group (Control), the pRI101-GFP empty vector group and the pRI101-HaCERK1 vector group. It can be seen from the box plot that the root regeneration rate of the control group and the experimental group is between 70% and 80%, with no significant difference, indicating that the transient transformation system mediated by Agrobacterium rhizogenes by the PRI101-GFP vector of the exogenous HaCERK1 gene has no significant effect on the root regeneration rate.

[0087] Example 8

[0088] like Figure 8The figure shows the comparison of the green fluorescence detection of instantaneous transformation of sunflower by the handheld UV light (365nm) of the present invention. The box plot shows that the median of the green fluorescence detection rate of pRI101-HaCERK1 is 60%, and the range of the upper and lower quartiles is between 50% and 80%, showing a high transformation efficiency, indicating that the vector can effectively transform the HaCERK1 gene into the target tissue. The K599 group only achieved the purpose of rooting, and the pRI101-GFP vector was not transferred, so naturally no green fluorescence was detected, and it was used as a control group. The figure can verify the effectiveness of the designed vector in the sunflower genetic transformation system.

[0089] like Fig. 9 Shown are fluorescence microscope images of the root transformation status of the sunflower 12 days after transient transformation of the present invention. The Normal group is fluorescence microscope imaging of the sunflower root that has not been transiently transformed, and its fluorescence signal is not obvious; the Fluorescence group is fluorescence microscope imaging of the sunflower root that has been transiently transformed. In the transient transformation system, the pRI101-GFP-HaCERK1 plasmid contains green fluorescent protein (GFP). Successful transient transformation causes the root cells to express GFP, and the green fluorescence signal can be clearly observed under a fluorescence microscope.

[0090] Example 9

[0091] like Fig.10 The figure shows the growth phenotype comparison of sunflower plants in different treatment groups of the present invention, indicating that overexpression of the HaCERK1 gene can promote the height of sunflower plants; Fig.11 As shown, the number of sunflower broomrape parasites can be reduced, indicating that the HaCERK1 gene has certain potential in the defense against broomrape parasitism. Fig.12 The subcellular localization of HaCERK1 protein was shown, and it was mainly localized on the cell membrane.

[0092] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

[0093] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A transient transformation system of sunflower mediated by Agrobacterium rhizogenes, characterized in that: The transient transformation system comprises a receptor-like kinase gene vector pRI 101-GFP-HaCERK1 constructed by using Agrobacterium rhizogenes K599 as a mediating bacterium and pRI 101-GFP as an overexpression vector.

2. A method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes, characterized in that: The following steps are involved: Step S1, transferring the gene vector to be transferred into competent cells of Agrobacterium rhizogenes K599, and obtaining positive strains through resistance screening; Step S2, inoculating the selected positive strain into a liquid culture medium for cultivation and extracting the bacteria; Step S3, inoculating the extracted bacteria into the culture medium, culturing overnight until the absorbance at a wavelength of 600 nm reaches 0.8-1.0, and using it as the impregnation solution; Step S4, simultaneously spreading the positive strains on LB solid medium containing 50 mg / L kanamycin and 50 mg / L streptomycin; Step S5, selecting a sunflower that has been cultivated for about 5 days, and cutting a stem segment about 5 cm below the cotyledon of the sunflower as an experimental material; Step S6, using a sterile needle to evenly pierce 7-8 small holes 1 cm from the cut stem segment; Step S7, completely immersing the stem segment after the hole treatment into the prepared impregnation solution, so that the surface of the stem segment is fully in contact with the bacterial solution, and infiltrating for about 1 hour to obtain the infiltrated stem segment; Step S8, taking out the infiltrated stem segment from the bacterial solution, and using a bent gun tip to take the bacterial cells on the plate and smear them around the incision and the puncture hole; Step S9, inserting the treated stem segments into sterilized vermiculite soil mixed with Orobancha for rooting to obtain instantaneously transformed sunflower plants.

3. The method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes according to claim 2, characterized in that: The gene vector to be transferred includes a pRI 101-GFP plasmid for gene editing.

4. The method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes according to claim 2 or 3, characterized in that: The pRI 101-GFP plasmid includes pRI 101-GFP-HaCERK1.

5. The method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes according to claim 2, characterized in that: The positive strains include strains that are screened by two antibiotics, kanamycin and streptomycin, and are positive in both bacterial liquid polymerase chain reaction and sequencing.

6. The method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes according to claim 2 or 5, characterized in that: In step S2, the liquid culture medium is LB culture medium containing 50 mg / L kanamycin and 50 mg / L streptomycin.

7. The method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes according to claim 2, characterized in that: The branches of the sunflower are 3 to 7 cm in length and carry 2 cotyledons and 1 to 2 new leaves.

8. The method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes according to claim 2, characterized in that: In step S9, the substrate for stem segment insertion includes sterilized vermiculite, and the culture temperature is 24-26°C.

9. The method for constructing a sunflower transient transformation system mediated by Agrobacterium rhizogenes according to claim 2, characterized in that: After the rooting of the stem segments was completed, the rooted sunflower plants were further tested for transient transformation.

Images