Agrobacterium tumefaciens-mediated method for realizing transient expression of target gene at hypocotyl and root of mung bean
Through the Agrobacterium-mediated vacuum infection method, the problem of efficiently achieving transient expression of target genes in the hypocotyl and roots of mung bean seedlings was solved, and efficient gene expression and transformation were achieved, providing new methods for mung bean biobreeding and research.
Patent Information
- Application Number
- CN202510034725.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-08
AI Technical Summary
How to efficiently achieve transient expression of target genes in the hypocotyl and roots of mung bean seedlings? At present, research on mung bean genetic transformation is still shallow, and many research systems have not been established, which seriously restricts the research on mung bean biological breeding, disease resistance and stress resistance mechanisms.
Agrobacterium-mediated vacuum infection method was adopted to activate and expand the culture of Agrobacterium, and Agrobacterium infectious solution was prepared, and mung bean seedlings were placed in the infecting solution for vacuum penetration, which prompted Agrobacterium to enter the hypocotyl and root tissue to achieve transient expression of the target gene.
The efficient transient expression of the target gene in the hypocotyl and roots of mung beans was achieved, and the efficiency of Agrobacterium infection and transformation efficiency was improved, laying the foundation for the research of mung bean transient expression system and molecular breeding.
Smart Images
Figure HDA0005235221880000011 
Figure HDA0005235221880000012
Abstract
Description
Technical Field
[0001] The invention relates to the field of plant molecular biotechnology and genetic engineering, and in particular to a method for realizing transient expression of target genes in mung bean hypocotyls and roots mediated by Agrobacterium. Background Art
[0002] Transient expression technology is a technology that achieves short-term high-level expression of target genes. Compared with stable expression, transient expression takes a shorter time, but the target gene is not integrated into the host plant gamete chromosome and cannot be stably inherited to the next generation. Due to its advantages such as simple operation, low cost, short cycle and good transformation effect, it has been widely used in biological research. Among them, Agrobacterium-mediated transient expression is the most common.
[0003] Mung bean (Vigna radiata L.) is an important edible legume crop in my country. It has the advantages of balanced nutrition, short growth period, long sowing period, drought resistance, barrenness tolerance, nitrogen fixation and soil conservation, and is suitable for intercropping with other crops. Mung bean has great flexibility in sowing period and is a good crop for disaster relief and replanting. At present, research on transgenic mung bean is still shallow, and many research systems have not been established, which seriously restricts the research on biological breeding, disease resistance and stress resistance mechanism of mung bean. Summary of the invention
[0004] The technical problem to be solved by the present invention is how to efficiently realize the transient expression of target genes in the hypocotyls and roots of mung bean seedlings.
[0005] In order to solve the above technical problems, the present invention first provides a method for realizing transient expression of target genes in hypocotyls and roots of mung bean seedlings mediated by Agrobacterium.
[0006] The method for realizing transient expression of target gene in hypocotyl and root of mung bean seedlings provided by the present invention comprises the following steps:
[0007] 1) activating and culturing Agrobacterium containing the target gene expression vector to obtain an activated bacterial solution; expanding and culturing the activated bacterial solution to obtain an Agrobacterium bacterial solution; centrifuging the Agrobacterium bacterial solution to collect Agrobacterium cells;
[0008] 2) Resuspend the Agrobacterium cells in an Agrobacterium suspension until the OD of the bacterial solution is 600nm 0.6-0.8, after standing for 2-3 hours, the Agrobacterium infection solution was obtained;
[0009] 3) placing the mung bean seedlings in the Agrobacterium infection solution so that the liquid level of the Agrobacterium infection solution covers the cotyledons of the mung bean seedlings; then vacuum-infecting the mung bean seedlings so that the Agrobacterium infection solution infiltrates into the hypocotyls and root tissues of the mung bean seedlings to obtain infected mung bean seedlings; and culturing the infected mung bean seedlings;
[0010] 4) After culturing for 2-3 days, detecting the expression of the target gene in the hypocotyls and roots of the mung bean seedlings.
[0011] In the above method, in 1), the target gene includes endogenous genes and exogenous genes. The endogenous gene includes genes from mung beans; the exogenous gene includes genes from pathogens or other organisms, and reporter genes.
[0012] In some embodiments, the target gene is the GFP gene (SEQ ID NO: 1).
[0013] In some embodiments, the target gene is the GUS gene (SEQ ID NO: 2).
[0014] In the above method, in 1), the target gene expression vector is a vector obtained by connecting the target gene to an expression vector.
[0015] Wherein, the expression vector may be a plasmid, a cosmid, a phage or a viral vector. The expression vector containing the target gene may be a vector containing a target gene expression cassette constructed using an existing plant expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector may also contain the 3'-end non-translated region of the exogenous gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the non-translated region transcribed at the 3' end of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions.
[0016] When constructing the expression vector containing the target gene, an enhancer can also be used, including a translation enhancer or a transcription enhancer. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) that can encode enzymes or luminescent compounds that can produce color changes that can be expressed in plants, antibiotic marker genes (such as nptII genes that confer resistance to kanamycin and related antibiotics, bar genes that confer resistance to the herbicide phosphinothricin, hph genes that confer resistance to the antibiotic hygromycin, and dhfr genes that confer resistance to methotrexate, EPSPS genes that confer resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose.
[0017] In some embodiments, the target gene expression vector is a GFP gene expression vector. The GFP gene expression vector is a pBI121-GFP plasmid.
[0018] In some embodiments, the target gene expression vector is a GUS gene expression vector. The GUS gene expression vector is a pBI121 plasmid.
[0019] In the above method, in 1), the Agrobacterium is Agrobacterium tumefaciens, such as Agrobacterium tumefaciens GV3101.
[0020] In the above method, in 1), the activation culture is cultured until the bacterial solution becomes turbid and has no white flocs.
[0021] The expansion culture is to culture the bacterial solution until OD 600nm The value is 0.6-1.0.
[0022] The culture medium used for the activation culture and the expansion culture is YEP culture medium. The solvent of the YEP culture medium is water, and the solutes and their concentrations are 10g / L yeast extract, 10g / L tryptone, and 5g / L sodium chloride.
[0023] In some embodiments, in 1), the culture medium used for activation culture and expansion culture is YEP culture medium containing 50 mg / L kanamycin (Kan) and 50 mg / L rifampicin (Rif). Kanamycin (Kan) plays a role in inhibiting bacteria and screening cells containing recombinant vectors in YEP culture medium.
[0024] In the above method, in 1), the conditions for the activation culture and the expansion culture can be 26-30° C. and 180-220 rpm, specifically 28° C. and 200 rpm.
[0025] The centrifugal condition may be 4000-6000 rpm for 10-15 min, specifically 6000 rpm for 10 min.
[0026] The 1) specifically comprises the following steps: inoculating Agrobacterium containing the target gene expression vector into 2 mL of YEP medium, shaking and culturing at 200 rpm in a 28° C. shaker for 12 h to obtain an activated bacterial solution; then inoculating 1 mL of the activated bacterial solution into 50 mL of YEP medium, shaking and culturing at 200 rpm in a 28° C. shaker until the OD 600nm The value is 0.6-1.0, and Agrobacterium bacterial liquid is obtained; the Agrobacterium bacterial liquid is centrifuged at 6000 rpm for 10 min to collect Agrobacterium cells.
[0027] In the above method, in 2), the solute of the Agrobacterium suspension includes 2-morpholineethanesulfonic acid, Na2HPO4, NaOH, glucose, acetosyringone, cysteine and Tween-20.
[0028] Furthermore, the concentration of the 2-morpholineethanesulfonic acid in the Agrobacterium suspension is 50 mM.
[0029] The concentration of Na2HPO4 in the Agrobacterium suspension was 2 mM.
[0030] The concentration of NaOH in the Agrobacterium suspension was 2 mM.
[0031] The concentration of glucose in the Agrobacterium suspension was 28 mM.
[0032] The concentration of acetosyringone in the Agrobacterium suspension was 0.1 mM.
[0033] The concentration of cysteine in the Agrobacterium suspension was 4.1 mM.
[0034] The volume fraction of Tween-20 in the Agrobacterium suspension is 0.1%.
[0035] In some embodiments, the Agrobacterium suspension is a solution obtained by mixing 2-morpholineethanesulfonic acid, Na2HPO4, NaOH, glucose, acetosyringone, cysteine and Tween-20 with double distilled water, and the final concentrations of the 2-morpholineethanesulfonic acid, the Na2HPO4, the NaOH, the glucose, the acetosyringone, the cysteine and the Tween-20 in the Agrobacterium suspension are 50mM, 2mM, 2mM, 28mM, 0.1mM, 4.1mM and 0.1% (volume fraction), respectively.
[0036] In the above method, in 3), the mung bean seedlings can be seedlings obtained by culturing mung bean seeds in vermiculite for 12-16 days.
[0037] In some embodiments, the mung bean seedlings are seedlings obtained by culturing mung bean seeds in vermiculite for 14 days.
[0038] In the above method, in 3), the vacuum infection condition may be vacuum penetration at (-75)-(-60) kPa for 13-17 min, specifically vacuum penetration at -70 kPa for 15 min.
[0039] In the above method, in 3), the culturing method comprises the following steps: planting the infected mung bean seedlings in vermiculite and watering them.
[0040] The culture conditions are 16 hours light / 8 hours dark, 22-28°C; specifically 16 hours light / 8 hours dark, 25°C.
[0041] In the above method, in 4), the method for detecting the expression of the target gene in the hypocotyl and root of mung bean seedlings includes but is not limited to qRT-PCR, RT-PCR, staining (such as GUS staining), fluorescence microscopy, and Western Blotting. When the target gene is an endogenous gene of mung bean, qRT-PCR or Western Blotting is preferably used; when the target gene is an exogenous gene, RT-PCR, qRT-PCR, and Western Blotting are preferably used; when the target gene is a fluorescent protein gene, qRT-PCR, fluorescence microscopy, or Western Blotting is preferably used.
[0042] In order to solve the above technical problems, the present invention also provides a product for realizing transient expression of target genes in the hypocotyls and roots of mung bean seedlings.
[0043] The product provided by the invention for realizing transient expression of target genes in hypocotyls and roots of mung bean seedlings comprises the above-mentioned Agrobacterium infection solution.
[0044] Furthermore, the product for achieving transient expression of the target gene in the hypocotyls and roots of mung bean seedlings also includes the above-mentioned expression vector, the above-mentioned Agrobacterium and the above-mentioned YEP culture medium.
[0045] In order to solve the above technical problems, the present invention finally provides any one of the following applications a1)-a3):
[0046] a1) Use of the above product in achieving transient expression of target genes in hypocotyls and roots of mung bean seedlings;
[0047] a2) Use of the above method or product in screening or assisting screening of genes (such as pathogen resistance genes) that infect mung bean hypocotyls and roots;
[0048] a3) Use of the above method or product in studying or assisting in studying the functions of genes that infect mung bean hypocotyls and roots (such as pathogen resistance genes).
[0049] Any of the mung beans mentioned above includes not only various mung bean resources and their closely related species, varieties, strains, and breeding materials, but also their variants, cultivated variants, and their offspring.
[0050] In a specific embodiment of the present invention, the mung bean is Jilu No. 7.
[0051] The present invention provides a method for realizing transient expression of target genes in hypocotyls and roots of mung bean mediated by Agrobacterium. The method comprises the following steps: culturing and collecting Agrobacterium containing target genes, preparing Agrobacterium infection solution, infecting mung bean seedlings by vacuum infection method mediated by Agrobacterium, recovering culture of mung bean seedlings and detecting expression of target genes. The method provided by the present invention optimizes Agrobacterium bacterial solution and Agrobacterium infection solution, and simultaneously performs vacuum infection treatment on hypocotyls and roots of mung bean seedlings, thereby promoting Agrobacterium to enter cells of hypocotyls and roots of mung bean, and realizing transient expression of target genes in hypocotyls and roots of mung bean seedlings for the first time. The method lays a foundation for the research of transient expression system of mung bean and molecular breeding of mung bean, and has the advantages of high infection efficiency and transformation efficiency compared with the infection method using only Agrobacterium immersion.
[0052] The beneficial effects of the present invention are as follows:
[0053] 1) The present invention provides a method for realizing transient expression of a target gene in the hypocotyl and root of mung bean mediated by Agrobacterium, which can realize efficient transient expression of the target gene in the hypocotyl and root of mung bean.
[0054] 2) The target gene in the present invention is efficiently and transiently expressed in the hypocotyl and root, which lays a foundation for the screening and identification of disease resistance genes of pathogens infecting the hypocotyl and root.
[0055] 3) The present invention utilizes a vacuum environment to greatly improve the efficiency of Agrobacterium infection of mung bean hypocotyls and root tissues, facilitating subsequent detection of target gene expression levels.
[0056] 4) The Agrobacterium suspension of the present invention enhances the infectivity of Agrobacterium to the hypocotyls and roots of mung bean seedlings, and the surfactant promotes the infection of Agrobacterium and improves the penetration ability of the Agrobacterium suspension in plant tissues. BRIEF DESCRIPTION OF THE DRAWINGS
[0057] Figure 1 These are the results of staining the hypocotyls and roots of mung beans infected with Agrobacterium containing the pBI121 plasmid using different methods and then stained with GUS staining solution.
[0058] Figure 2 This is the fluorescence imaging result of mung bean root tip infected with Agrobacterium containing pBI121-GFP plasmid under confocal laser scanning microscope. DETAILED DESCRIPTION
[0059] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0060] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0061] Jilu No. 7 in the following examples is recorded in the document "Fan Baojie et al., Breeding of a new high-yield and early-maturing mung bean variety Jilu No. 7, Crop Magazine, 2009.2".
[0062] The solvent of the YEP medium in the following examples is water, and the solutes and their concentrations are 10 g / L yeast extract, 10 g / L tryptone, and 5 g / L sodium chloride, respectively.
[0063] The pBI121 plasmid in the following examples is a product of Hunan Keai Medical Instrument Co., Ltd. (Ubao Biotech), with the product number VT1388. The pBI121 plasmid is a plasmid expressing the GUS gene.
[0064] The pBI121-GFP plasmid in the following examples is a product of Hunan Keai Medical Instrument Co., Ltd. (Ubao Biotech), with the product number VT2117. The pBI121-GFP plasmid is a plasmid expressing the GFP gene.
[0065] Example 1: A method for transiently expressing the target gene GUS in mung bean hypocotyls and roots mediated by Agrobacterium
[0066] In this example, Jilu No. 7 was used as the mung bean plant material, and GUS was used as the target gene to achieve transient expression of the target gene in the hypocotyl and root of mung bean. Specifically, the following steps were included:
[0067] 1. Activate and collect Agrobacterium containing the plasmid expressing the GUS gene
[0068] 1. The pBI121 plasmid was transformed into Agrobacterium tumefaciens GV3101, and after identification, Agrobacterium containing the pBI121 plasmid was obtained. Then the Agrobacterium containing the pBI121 plasmid was streaked and cultured, and a single clone was picked into 2 mL of YEP medium containing 50 mg / L kanamycin (Kan) and 50 mg / L rifampicin (Rif), and cultured in a shaker at 28°C and 200 rpm for 12 hours until it became turbid and free of white flocs, to obtain an activated bacterial solution.
[0069] 2. Take 1 mL of activated bacterial solution and transfer it to 50 mL of YEP liquid medium containing 50 mg / L kanamycin (Kan) and 50 mg / L rifampicin (Rif), and culture it in a shaker at 28°C and 200 rpm until OD 600nm The value is between 0.6-1.0, and Agrobacterium liquid is obtained.
[0070] 3. Pour 50 mL of Agrobacterium culture solution into a centrifuge tube and centrifuge at 6000 rpm for 10 min. Discard the supernatant and collect the Agrobacterium cells (containing the pBI121 plasmid) for later use.
[0071] According to the above method, the Agrobacterium containing the pBI121 plasmid was replaced with Agrobacterium tumefaciens GV3101, and the other steps remained unchanged to obtain Agrobacterium cells (control).
[0072] 2. Preparation of Agrobacterium infection solution
[0073] 1. Mix 2-morpholineethanesulfonic acid, Na2HPO4, NaOH, glucose, acetosyringone, cysteine and Tween-20 with double distilled water to make the final concentrations of 50mM, 2mM, 2mM, 28mM, 0.1mM, 4.1mM and 0.1% (volume fraction), respectively, to obtain an Agrobacterium suspension.
[0074] 2. Take 3 mL of Agrobacterium suspension and add it to the centrifuge tube containing Agrobacterium cells (containing pBI121 plasmid). Use a pipette to blow the cells adhering to the bottom of the centrifuge tube until the cells are completely resuspended. Then use the Agrobacterium suspension to dilute the bacterial solution and use a spectrophotometer to measure the OD of the resuspended bacterial solution. 600nm Value, until OD 600nm=0.6-0.8, and obtain Agrobacterium infection solution (containing pBI121 plasmid).
[0075] According to the above method, the Agrobacterium cells (containing the pBI121 plasmid) were replaced with the Agrobacterium cells (control), and the other steps remained unchanged to obtain the Agrobacterium infection solution (control).
[0076] 3. Place the Agrobacterium infection solution in a 100 mL conical flask and let it stand at room temperature for 2-3 hours.
[0077] 3. Different methods of infecting mung bean seedlings
[0078] 1. Vacuum immersion infection
[0079] Mung bean seeds (Ji Lu No. 7) were planted in vermiculite for 14 days to obtain mung bean seedlings, the roots of which were cleaned and then placed in conical bottles containing Agrobacterium infection solution (containing pBI121 plasmid) so that the Agrobacterium infection solution (containing pBI121 plasmid) covered the mung bean cotyledons. At the same time, the Agrobacterium infection solution (control) was used as a control.
[0080] The conical flask containing the mung bean seedlings was placed in a vacuum device and vacuum-infiltrated for 15 minutes under a vacuum environment of -70 kPa; the vacuum-treated mung bean seedlings were then replanted in vermiculite, and tap water was used to irrigate the planted mung bean seedlings, and then the seedlings were placed in an environment with a photoperiod of 16 hours light / 8 hours dark and a temperature of 25°C for cultivation.
[0081] 2. Soaking and infection
[0082] After the Agrobacterium infection solution (containing the pBI121 plasmid) covers the mung bean cotyledons, soak for 3 hours; then replant the soaked mung bean seedlings in vermiculite, use tap water to irrigate the transplanted mung bean seedlings, and then place them in an environment with a photoperiod of 16 hours light / 8 hours dark and a temperature of 25°C for cultivation.
[0083] IV. GUS gene expression detection
[0084] After 48-72 hours of vacuum infection, the roots of the mung bean seedlings were washed again, and the hypocotyls and roots of the mung bean seedlings were immersed in GUS staining solution (Beijing Bio-Ray Biotechnology Co., Ltd., BN20175). After incubation at 37°C for 16 hours, the staining results were observed.
[0085] The results are as follows Figure 1As shown, the results showed that after the mung bean seedlings were vacuum-immersed and infected with Agrobacterium infection solution (containing pBI121 plasmid), the hypocotyls and roots appeared blue; while the mung bean seedlings that were only immersed in Agrobacterium infection solution (containing pBI121 plasmid) only showed blue at the junction of the root and stem; at the same time, the mung bean seedlings did not show blue after being vacuum-immersed in Agrobacterium infection solution (control). These results show that the plasmid pBI121 expressing the GUS gene successfully entered the hypocotyls and root tissues of Jilu No. 7 through vacuum-immersion infection with Agrobacterium and was successfully transcribed and translated; vacuum-immersion infection can effectively promote Agrobacterium infection of mung bean hypocotyls and roots.
[0086] Example 2: A method for transiently expressing the target gene GFP in mung bean hypocotyls and roots mediated by Agrobacterium
[0087] In this example, Jilu No. 7 was used as the mung bean plant material, and GFP was used as the target gene to achieve transient expression of the target gene in the hypocotyl and root of mung bean. Specifically, the following steps were included:
[0088] 1. Activate and collect Agrobacterium containing plasmid expressing GFP gene
[0089] 1. Transform Agrobacterium tumefaciens GV3101 with the plasmid pBI121-GFP expressing the GFP gene, and obtain Agrobacterium containing the pBI121-GFP plasmid after identification. Then, streak the Agrobacterium containing the pBI121-GFP plasmid, pick a single clone and transfer it to 2 mL of YEP medium containing 50 mg / L kanamycin (Kan) and 50 mg / L rifampicin (Rif), and culture it in a shaker at 28°C and 200 rpm for 12 hours until it becomes turbid and free of white flocs, to obtain an activated bacterial solution.
[0090] 2. Same as step 2 of Example 1, step 1.
[0091] 3. Same as step 3 of Example 1, step 1.
[0092] 2. Preparation of Agrobacterium infection solution
[0093] Same as step 2 of Example 1.
[0094] 3. Vacuum infection of mung bean seedlings
[0095] Same as step 1 in step 3 of Example 1.
[0096] IV. GFP gene expression detection
[0097] After 48-72 hours of vacuum infection, the roots of the mung bean seedlings were washed again, and the root tips were taken to observe the expression of GFP protein in the roots under a laser confocal microscope. At the same time, the mung bean seedlings vacuum-infected with Agrobacterium without pBI121-GFP plasmid were used as blank controls.
[0098] The results are as follows Figure 1 As shown, the results showed that after the Agrobacterium containing the pBI121-GFP plasmid infected the mung bean seedlings, a green fluorescent signal appeared at the root tip, while no green fluorescent signal was detected in the blank control. These results indicate that the plasmid pBI121-GFP expressing the GFP gene successfully entered the root tissue of Jilu No. 7 through vacuum immersion infection by Agrobacterium and was successfully transcribed and translated.
[0099] The above results show that the present invention promotes Agrobacterium infection, improves the penetration ability of Agrobacterium suspension in plant tissues, can achieve efficient transient expression of target genes in mung bean hypocotyls and roots, facilitates subsequent detection of target gene expression levels, and lays a foundation for the screening and identification of disease resistance genes for pathogens infecting hypocotyls and roots.
[0100] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.
Claims
1. A method for achieving transient expression of a target gene in the hypocotyl and root of mung bean seedlings mediated by Agrobacterium, the method comprising the following steps: 1) activating and culturing Agrobacterium containing the target gene expression vector to obtain an activated bacterial solution; expanding and culturing the activated bacterial solution to obtain an Agrobacterium bacterial solution; centrifuging the Agrobacterium bacterial solution to collect Agrobacterium cells; 2) Resuspend the Agrobacterium cells in an Agrobacterium suspension until the OD of the bacterial solution is 600nm 0.6-0.8, after standing for 2-3 hours, the Agrobacterium infection solution was obtained; 3) placing the mung bean seedlings in the Agrobacterium infection solution so that the liquid level of the Agrobacterium infection solution covers the cotyledons of the mung bean seedlings; then vacuum-infecting the mung bean seedlings so that the Agrobacterium infection solution infiltrates into the hypocotyls and root tissues of the mung bean seedlings to obtain infected mung bean seedlings; and culturing the infected mung bean seedlings; 4) After culturing for 2-3 days, detecting the expression of the target gene in the hypocotyls and roots of the mung bean seedlings.
2. The method according to claim 1, characterized in that: In 1), the target gene expression vector is a vector obtained by connecting the target gene to an expression vector; Alternatively, the Agrobacterium is Agrobacterium tumefaciens.
3. The method according to claim 1 or 2, characterized in that: In 1), the activation culture is cultured until the bacterial solution is turbid and has no white flocs; Or, the expanded culture is cultured until the bacterial liquid OD 600nm The value is 0.6-1.0; Or, the culture medium used for the activation culture and the expansion culture is YEP culture medium; Or, the conditions of the activation culture and the expansion culture are both 26-30° C. and 180-220 rpm; Alternatively, the centrifugation condition is 4000-6000 rpm for 10-15 min.
4. The method according to any one of claims 1 to 3, characterized in that: In the above 2), the solutes of the Agrobacterium suspension include 2-morpholineethanesulfonic acid, Na2HPO4, NaOH, glucose, acetosyringone, cysteine and Tween-20.
5. The method according to claim 4, characterized in that: The concentration of 2-morpholineethanesulfonic acid in the Agrobacterium suspension is 50 mM; The concentration of Na2HPO4 in the Agrobacterium suspension is 2 mM; The concentration of NaOH in the Agrobacterium suspension is 2 mM; The concentration of glucose in the Agrobacterium suspension is 28 mM; The concentration of acetosyringone in the Agrobacterium suspension is 0.1 mM; The concentration of cysteine in the Agrobacterium suspension is 4.1 mM; The volume fraction of Tween-20 in the Agrobacterium suspension is 0.1%.
6. The method according to any one of claims 1 to 5, characterized in that: In the above 3), the mung bean seedlings are obtained by culturing mung bean seeds in vermiculite for 12-16 days.
7. The method according to any one of claims 1 to 6, characterized in that: In the above 3), the vacuum infection condition is (-75)-(-60) kPa vacuum penetration for 13-17 min; Or, the culturing method comprises the following steps: planting the infected mung bean seedlings in vermiculite and watering them; Alternatively, the culture conditions are 16 hours light / 8 hours dark, 22-28°C.
8. The method according to any one of claims 1 to 7, characterized in that: In the above 4), the methods for detecting the expression of the target gene in the hypocotyls and roots of the mung bean seedlings include qRT-PCR method, RT-PCR method, staining method, fluorescence microscopy detection method, and Western Blotting method.
9. A product for achieving transient expression of target genes in the hypocotyls and roots of mung bean seedlings, comprising the Agrobacterium infection solution of claims 1-8.
10. Any application of a1) to a3) below: a1) Use of the product of claim 9 in achieving transient expression of target genes in hypocotyls and roots of mung bean seedlings; a2) Use of the method according to any one of claims 1 to 8 or the product according to claim 9 in screening or assisting in screening genes that infect mung bean hypocotyls and roots; a3) Use of the method according to any one of claims 1 to 8 or the product according to claim 9 in studying or assisting in studying the function of genes that infect mung bean hypocotyls and roots.