Agrobacterium tumefaciens-mediated pea leaf injection instantaneous transformation method and application thereof

Abstract

The invention relates to the field of transformation methods and application thereof, in particular to an agrobacterium tumefaciens-mediated pea leaf injection transient transformation method and application thereof, which comprises the following steps: introducing an exogenous gene into pea leaf cells through an injection method by using an agrobacterium tumefaciens-mediated gene transformation system in pea leaves, and realizing transient expression of the exogenous gene; according to the pea leaf injection transient transformation system based on agrobacterium tumefaciens mediation provided by the invention, through an optimized technical scheme, the problems of low transformation efficiency, unstable exogenous gene expression, high operation complexity and the like in the prior art are solved, and remarkable beneficial effects are brought to plant gene function research and commercial application; through an efficient, stable, simple and convenient pea leaf instantaneous transformation technology, many problems in the prior art are solved, the efficiency of gene function research and commercial application is improved, and a powerful technical support is provided for further development of plant molecular biology and genetic engineering technologies.

Description

Technical Field

[0001] The invention relates to a transformation method and an application field thereof, and in particular to a pea leaf injection transient transformation method mediated by Agrobacterium tumefaciens and an application thereof. Background Art

[0002] Plant genetic engineering technology occupies an important position in modern agriculture and biotechnology. Its core lies in introducing exogenous genes into plant cells and stably expressing them for crop improvement, resistance enhancement and optimization of breeding strategies. Through transgenic means, researchers can achieve genetic modification of plants and develop crops with characteristics such as disease resistance, drought resistance, and increased nutritional value. The widespread application of these technologies has not only improved agricultural productivity, but also provided an important way to solve the problem of global food security.

[0003] Among the many plant gene transformation methods, Agrobacterium tumefaciens-mediated gene transformation technology is the most common one. Agrobacterium tumefaciens is a naturally occurring soil pathogen that can insert T-DNA fragments into the host plant genome to achieve gene introduction. This technology is widely used in transgenic research of dicotyledons and some monocotyledons due to its high efficiency, stability and low cost, especially in model plants such as Arabidopsis and tobacco. The stable transformation technology mediated by Agrobacterium tumefaciens is already very mature and has even been successful in commercial applications.

[0004] However, stable transformation technology has some limitations, especially that it is time-consuming, usually taking months or even years to screen out stable transformed plants. In addition, the stable transformation steps are cumbersome, and the required experimental conditions and costs are high, which is very inconvenient when it is necessary to quickly verify the function of exogenous genes or screen promoter activity. Therefore, the scientific community has begun to pay extensive attention to transient expression systems. Transient expression technology can express exogenous genes in a short period of time and quickly obtain experimental data, thereby greatly shortening the experimental cycle. Compared with stable transformation, transient transformation operations are simpler and do not rely on long-term screening of transgenic plants.

[0005] Existing transient expression technologies mainly focus on two methods: leaf injection transformation mediated by Agrobacterium tumefaciens and particle bombardment transformation. Among them, leaf injection transformation mediated by Agrobacterium tumefaciens is considered to be a relatively simple and effective method, which is widely used in model plants such as tobacco, Arabidopsis and some crops. This method injects Agrobacterium resuspension on plant leaves to express foreign genes in a short time, and detects them by enzyme labeling or fluorescence. Transient expression technology is particularly suitable for experiments such as promoter function verification and protein interaction analysis.

[0006] Although the plant gene transformation technology mediated by Agrobacterium tumefaciens has been widely used in the field of plant molecular biology, there are still some obvious defects and deficiencies in the existing technology, especially in the transient transformation of specific plants such as peas. These defects are mainly manifested in the following aspects:

[0007] (1) Stable transformation takes a long time

[0008] Although traditional stable transformation technology can achieve long-term expression of exogenous genes, its operation steps are complicated and time-consuming. Usually, it takes several months or even years of screening and phenotypic identification to obtain stably transformed plants. This is too long for research on rapid verification of gene function or screening of important promoter activity, and it is difficult to meet the needs of scientific research and industrial development.

[0009] (2) Instantaneous conversion technology is not efficient

[0010] Although the current transient transformation technology provides a faster option than stable transformation, its efficiency is still low in some plants, especially crops such as peas. Existing transient transformation research is more focused on model plants such as tobacco and Arabidopsis, while the transient transformation efficiency of peas is low, resulting in insufficient expression of exogenous genes, which limits its application in gene function research in plants such as peas.

[0011] (3) The universality of the transformation method is low

[0012] Many transient transformation methods have been optimized for specific plants such as tobacco or Arabidopsis, but these methods often have poor effects on plants such as peas. Due to differences in gene introduction, transformation efficiency, gene expression, etc. among different plant species, the universality of existing methods is low, resulting in unstable transformation effects and difficulty in promoting their application to a wider range of crops.

[0013] (4) Unstable expression of exogenous genes

[0014] Although the existing transient expression system can quickly express exogenous genes, due to the limitations of transformation efficiency and stability, the expression of exogenous genes in leaves is usually not stable enough and the expression level is low. Especially when conducting large-scale screening experiments, consistent results are often not obtained. This is particularly evident in experiments that require precise data, such as promoter function verification and gene expression analysis.

[0015] (5) Operational complexity and cost issues

[0016] Among the existing plant transient transformation methods, many techniques require relatively complex operating steps, such as particle bombardment, which not only requires professional equipment but also requires high-cost reagent investment, limiting its use in resource-limited laboratories and small biological companies. At the same time, the reagents or techniques used in some transformation methods are difficult to store and operate for a long time, which increases the complexity and cost of the experiment.

[0017] (6) Research on transient transformation of pea is relatively scarce

[0018] Compared with other crops such as tobacco and Arabidopsis, there are relatively few studies on transient transformation of pea, especially in biotechnology companies and breeding development. As an important leguminous crop, transient transformation technology of pea urgently needs to be more deeply studied and optimized to meet the needs of rapid screening of exogenous genes and verification of functions. However, existing research is often limited to model plants and fails to provide efficient transient transformation solutions for pea.

[0019] In summary, the application of existing Agrobacterium tumefaciens-mediated transient transformation technology in peas still faces problems such as low transformation efficiency, unstable exogenous gene expression, and high operational complexity. These technical defects limit the promotion and use of transient transformation technology in pea gene function research and commercial applications. Therefore, it is urgent to develop an efficient, simple and stable pea leaf transient transformation method to meet the research and development needs of biological companies and research institutes.

[0020] Therefore, in order to address the above-mentioned problems of low transformation efficiency, unstable exogenous gene expression and high operational complexity of pea, a pea leaf injection transient transformation method mediated by Agrobacterium tumefaciens and its application can be designed. Summary of the invention

[0021] In order to overcome the problems of low transformation efficiency, unstable expression of exogenous genes and high operational complexity in pea.

[0022] The technical solution of the present invention is: a method for transient transformation of pea leaves mediated by Agrobacterium tumefaciens injection, characterized in that the gene transformation system mediated by Agrobacterium tumefaciens is used in pea leaves to introduce exogenous genes into pea leaf cells by injection and achieve their transient expression.

[0023] An application of a method for transient transformation of pea leaves mediated by Agrobacterium tumefaciens, comprising the above-mentioned method for transient transformation of pea leaves mediated by Agrobacterium tumefaciens, and the steps are as follows:

[0024] (1) Pea seed preparation and cultivation: Select plump and healthy pea seeds for sowing and wait until they grow to 4 compound leaves and unfold as experimental materials;

[0025] (2) Preparation of infection solution: Prepare Agrobacterium tumefaciens competent cell suspension and transformation working solution through optimized formula to ensure maximum transformation efficiency;

[0026] (3) Leaf injection: Use a disposable syringe to inject the resuspended Agrobacterium tumefaciens solution into the back of pea leaves to ensure that the exogenous gene can fully enter the leaf cells;

[0027] (4) Transient expression: Through optimized culture conditions, the transient expression of exogenous genes in pea leaves is ensured to be stable and efficient;

[0028] (5) GUS staining and analysis: The expression of exogenous genes is detected and verified through the GUS reporter gene system to quickly obtain experimental results.

[0029] Beneficial effects of the present invention:

[0030] The pea leaf injection transient transformation system based on Agrobacterium tumefaciens mediation provided by the present invention overcomes the problems of low transformation efficiency, unstable exogenous gene expression, high operation complexity, etc. in the prior art through an optimized technical solution, and brings significant beneficial effects to plant gene function research and commercial application, which are specifically embodied in the following aspects:

[0031] 1. Significantly improve conversion efficiency

[0032] By optimizing the preparation of Agrobacterium tumefaciens bacterial solution, the formula of the infection solution and the injection conditions, the present invention greatly improves the instantaneous transformation efficiency of pea leaves, so that exogenous genes can be more efficiently introduced into pea cells and expressed rapidly. This efficient transformation method can achieve significant gene expression effects in a relatively short period of time, significantly shortening the experimental cycle.

[0033] 2. Enhance the stability of exogenous gene expression

[0034] The present invention ensures that the expression of exogenous genes in pea leaves is more stable by optimizing the injection method, culture conditions and bacterial liquid concentration. The experimental results show that the expression of exogenous genes is high and the results are highly consistent when the transient transformation method of the present invention is used. This is of great significance for research such as gene function screening and promoter verification that require high-precision and high-stability experimental results.

[0035] 3. Simplify the operation process and reduce experimental costs

[0036] The present invention reduces the complexity of experimental operation by simplifying the injection operation and culture steps. Compared with transient transformation methods such as particle bombardment, the present invention does not require expensive equipment and complex experimental conditions, and only requires conventional laboratory equipment to achieve efficient transformation. Its simple operation method reduces experimental costs and is particularly suitable for large-scale gene screening and verification by scientific research institutions and biological companies with limited resources.

[0037] 4. Accelerate the process of gene function screening and promoter verification

[0038] The transient transformation system of the present invention can obtain the expression results of exogenous genes within just a few days, greatly accelerating the progress of experiments such as gene function screening and promoter verification. This rapid expression system is suitable for research projects that require rapid verification of gene functions, and is particularly suitable for use by biological companies when developing new crop varieties or gene function products.

[0039] 5. Strong universality, suitable for a variety of plant research

[0040] Although the present invention focuses on the transient transformation of pea leaves, the technical system has strong universality and can be applied to transient expression research of other crops or model plants through similar optimization. Its versatility provides technical support for gene function research and transgenic technology development of other crops and has broad application prospects.

[0041] 6. Improve the repeatability and reliability of experimental data

[0042] By optimizing the experimental conditions and bacterial liquid concentration, the present invention ensures that the results of each transformation experiment have high repeatability and reliability, the expression results of the exogenous gene are stable and consistent, and the accidental errors and uncertainties in the experiment are reduced. This provides scientific research institutions and biological companies with a reliable experimental method and facilitates subsequent application development.

[0043] 7. Suitable for commercial development and application

[0044] The efficient and simple instantaneous transformation method provided by the present invention is particularly suitable for commercial applications of biological companies in the field of plant genetic engineering. Its efficient gene screening capability can help companies develop products and verify functions more quickly, shorten product launch time, and enhance the market competitiveness of companies.

[0045] In summary, the present invention solves many problems in the prior art through an efficient, stable and simple pea leaf transient transformation technology, improves the efficiency of gene function research and commercial application, and provides strong technical support for the further development of plant molecular biology and genetic engineering technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1Shown is a schematic diagram of pea leaf injection of the transformation method and application of the present invention;

[0047] (This figure shows the specific process of injecting exogenous genes into pea leaves. A disposable syringe is used to inject Agrobacterium tumefaciens liquid into the back of pea leaves)

[0048] Figure 2 Shown is a schematic diagram of the GUS staining results of the transformation method of the present invention and its application.

[0049] (The figure shows the GUS staining results of the control group and the treatment group of pea leaves observed under a stereoscope after GUS staining after injection, which is used to detect the expression of exogenous genes. A: Nothing was injected, no staining reaction occurred, and it was displayed in white; B: distilled water was injected, no staining reaction occurred, and it was displayed in white; C: empty EHA105 competent cells were injected, no staining reaction occurred, and it was displayed in white; D: pC1391-UBI-gusplus expression vector was injected, and a significant blue reaction was shown, indicating that the exogenous gene was successfully expressed). DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the embodiments.

[0051] Example 1

[0052] See also Figure 1-2 The present invention provides an embodiment: a method for transient transformation of pea leaves mediated by Agrobacterium tumefaciens injection, characterized in that the gene transformation system mediated by Agrobacterium tumefaciens is used in pea leaves to introduce exogenous genes into pea leaf cells by injection and achieve their transient expression.

[0053] An application of a method for transient transformation of pea leaves mediated by Agrobacterium tumefaciens, comprising the above-mentioned method for transient transformation of pea leaves mediated by Agrobacterium tumefaciens, and the steps are as follows:

[0054] (1) Pea seed preparation and cultivation: Select plump and healthy pea seeds and plant them in substrate soil. The cultivation conditions are: 16 hours of light (2000 Lux), temperature 22°C, humidity 70%; 8 hours of darkness, temperature 22°C. When peas grow to 4 fully expanded compound leaves, they can be used for leaf injection experiments;

[0055] (2) Preparation of infection solution: Prepare 10× Infiltration buffer and injection working solution according to Table 1 and Table 2:

[0056] Table 1 10× Infiltration buffer formula

[0057]

[0058] Table 2 Working solution formula

[0059]

[0060] 10 μL of Agrobacterium tumefaciens EHA105 competent cells containing pC1391-UBI-gusplus plasmid were inoculated into YEP liquid medium containing kanamycin (Kan) and rifampicin (Rif) resistance, and cultured at 28°C and 200 rpm with shaking until OD600 reached 0.6-0.8. After collecting the cells by centrifugation, they were resuspended with injection working solution to OD600 = 0.6-1;

[0061] (3) Leaf injection: Use a 1 mL disposable syringe to inject the prepared Agrobacterium tumefaciens infection solution into the back of pea leaves. Set up 3 replicates for each treatment group, and inject 3 leaves in each replicate. During the injection, make sure that the leaves evenly absorb the bacterial solution. After the injection, use absorbent paper to remove the excess infection solution and mark it;

[0062] (4) Transient expression: The injected pea plants were placed in a dark environment at 28°C and 50% humidity for 3 days to ensure that the exogenous gene was fully expressed;

[0063] (5) GUS staining and analysis: After 3 days, the injected pea leaves were taken for GUS staining. The GUS staining steps include:

[0064] ① Wash the leaves and immerse them in GUS staining solution;

[0065] ② Vacuum for 20 to 30 minutes to allow the dye to enter the leaf tissue;

[0066] ③ Incubate at 37°C for 24 hours;

[0067] ④ Use 75% ethanol to decolorize until the leaves of the control group are colorless.

[0068] The staining results showed that the pea leaves injected with the pC1391-UBI-gusplus expression vector showed a significant blue color, and the blue color was darker, indicating that the exogenous gene was effectively expressed.

[0069] System optimization experiment

[0070] In order to improve the efficiency of instantaneous conversion, the following system optimization experiments were conducted:

[0071] (1) Optimization of bacterial solution concentration: The effect of foreign gene introduction was optimized by adjusting the concentration of Agrobacterium tumefaciens bacterial solution (OD600 value was 0.6-1). Experiments showed that the transformation efficiency was optimal when the OD600 value was 0.8.

[0072] (2) Optimization of culture conditions: By adjusting the culture conditions (temperature, humidity, etc.), the dark culture environment of 28°C and 50% humidity was finally determined to be the optimal condition, which is conducive to the efficient expression of exogenous genes.

[0073] (3) Optimization of leaf injection depth and position: Adjust the injection depth and position to ensure that the bacterial solution is fully diffused into the leaf tissue and further improve the transformation efficiency.

[0074] As described above, the technical solution of the present invention has the following characteristics:

[0075] (1) Easy to operate, suitable for rapid screening and research in laboratories and biotechnology companies;

[0076] (2) High transformation efficiency, which can achieve effective expression of exogenous genes in a short time;

[0077] (3) The results are stable and reproducible, suitable for applications such as gene function screening and promoter verification.

[0078] Through the present invention, the transient expression of exogenous genes in pea leaves can be achieved quickly and effectively, the experimental cycle can be significantly shortened, the research efficiency can be improved, and it is suitable for scientific research and commercial development of biological companies.

Claims

1. A method for transient transformation of pea leaves mediated by Agrobacterium tumefaciens, characterized in that: In pea leaves, the Agrobacterium tumefaciens-mediated gene transformation system is used to introduce foreign genes into pea leaf cells through injection and achieve their transient expression.

2. An application of a method for transient transformation of pea leaves mediated by Agrobacterium tumefaciens, characterized in that The method comprises the method of instantaneous transformation of pea leaves mediated by Agrobacterium tumefaciens according to claim 1, wherein the steps are as follows: (1) Pea seed preparation and cultivation: Select plump and healthy pea seeds for sowing and wait until they grow to 4 compound leaves and unfold as experimental materials; (2) Preparation of infection solution: Prepare Agrobacterium tumefaciens competent cell suspension and transformation working solution through optimized formula to ensure maximum transformation efficiency; (3) Leaf injection: Use a disposable syringe to inject the resuspended Agrobacterium tumefaciens solution into the back of pea leaves to ensure that the exogenous gene can fully enter the leaf cells; (4) Transient expression: Through optimized culture conditions, the transient expression of exogenous genes in pea leaves is ensured to be stable and efficient; (5) GUS staining and analysis: The expression of exogenous genes is detected and verified through the GUS reporter gene system to quickly obtain experimental results.

Images