A method for transforming an exogenous gene into prunella vulgaris and application thereof

Abstract

A genetic transformation method for transferring exogenous genes into Prunella vulgaris and its application include the following steps: using Prunella vulgaris seedlings growing in substrate soil before flowering as infection material; introducing an expression vector containing the exogenous gene into Agrobacterium rhizogenes to prepare Agrobacterium rhizogenes infection solution; before infection, obliquely cutting and injuring the base of the Prunella vulgaris stem, removing the original root system, placing the Prunella vulgaris in the Agrobacterium rhizogenes infection solution, and then infecting using ultrasound; after infection, planting the Prunella vulgaris in the substrate, culturing in the dark, and continuing to grow under long-day conditions until hairy roots are produced. This invention achieves efficient, simplified, tissue culture-free, and visualized hairy root genetic transformation in Prunella vulgaris, with a genetic transformation rate of over 70% after 8 weeks of culture, providing a novel technical approach for the genetic improvement and functional gene research of Prunella vulgaris.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a genetic transformation method for transferring exogenous genes into Prunella vulgaris and its application. Background Technology

[0002] Prunella vulgaris ( Prunella vulgaris Prunella vulgaris is an important plant used for both food and medicine. Its dried fruit spikes can be used in medicine, and its tender leaves are edible, giving it extremely high economic value. As a traditional medicinal material, Prunella vulgaris was first recorded in the "Shennong's Classic of Materia Medica" and has significant effects such as lowering blood pressure, lowering blood sugar, antibacterial, anti-inflammatory, anti-allergic, antiviral, and enhancing immunity.

[0003] Currently, due to the interplay of issues such as explant characteristics, Agrobacterium tumefaciens compatibility, and co-culture conditions, a stable genetic transformation system for Prunella vulgaris has not yet been established, greatly limiting in-depth research on its functional genes and metabolic pathways. Regarding how to achieve stable genetic transformation of Prunella vulgaris, especially a simple and efficient method for exogenous gene transformation without relying on a strictly sterile tissue culture environment, no relevant reports have been found both domestically and internationally. Summary of the Invention

[0004] The purpose of this invention is to provide a genetic transformation method for transferring exogenous genes into Prunella vulgaris and its application, which achieves efficient, simplified, tissue culture-free, and visualized hairy root genetic transformation in Prunella vulgaris. Positive transformed roots can be observed after 3 weeks of culture after transformation, and the transformation rate is over 70% after 8 weeks of culture. This provides a brand-new technical approach for the genetic improvement and functional gene research of Prunella vulgaris.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A genetic transformation method for transferring exogenous genes into Prunella vulgaris includes the following steps: 1) Use Prunella vulgaris seedlings growing in substrate soil before flowering as infection material; 2) Introduce the expression vector containing the exogenous gene into Agrobacterium rhizogenes to prepare Agrobacterium rhizogenes infection solution; 3) Make oblique cuts at the base of the stem of Prunella vulgaris used as the infection material, remove the original root system, put the Prunella vulgaris into the Agrobacterium rhizogenes infection solution in step 2), and then use ultrasonic method to infect it. 4) Plant the infected Prunella vulgaris in the substrate, and after culturing in the dark, continue to grow under long-day conditions until hairy roots are produced.

[0006] Preferably, in step 1), the infecting material is a Prunella vulgaris seedling in its vegetative growth stage, with leaves and stem base.

[0007] Furthermore, in step 2), the expression vector is a binary recombinant plasmid carrying a Ruby visualization gene, a GFP fluorescent reporter gene, or a metabolic enzyme-related gene.

[0008] Furthermore, in step 2), the fermentation medium for Agrobacterium rhizogenes is TY medium.

[0009] Furthermore, in step 2), the method for preparing the Agrobacterium rhizogenes infection solution is as follows: Agrobacterium rhizogenes cells are resuspended in tobacco suspension, the bacterial concentration is adjusted to an OD600 value of 0.8~1.2, and acetylsuccinone (AS) is added and allowed to stand in the dark.

[0010] Furthermore, in step 3), the length of the oblique cut is 2-3 mm.

[0011] Furthermore, in step 3), when using ultrasound to infect, the ultrasound power is 140W~160W.

[0012] Preferably, in step 3), when using ultrasonic inoculation, the inoculation time is 1-5 min.

[0013] This invention provides the application of the genetic transformation method in improving the rooting rate and / or genetic transformation rate of Prunella vulgaris hairy roots.

[0014] This invention provides the application of the genetic transformation method in the study of functional genes in Prunella vulgaris.

[0015] In this invention, before infection, the base of the Prunella vulgaris stem is obliquely cut and the original root system is removed. On the one hand, this creates wounds at the root base, promoting the invasion of Agrobacterium rhizogenes; on the other hand, since the original root system is non-transgenic, only the new roots induced by Agrobacterium rhizogenes can be transgenic, thus facilitating the acquisition of transgenic root systems.

[0016] In this invention, the non-sterile genetic transformation of Prunella vulgaris is mediated by Agrobacterium rhizogenes, a Gram-negative bacterium belonging to the genus Agrobacterium of the family Rhizobium. Agrobacterium rhizogenes integrates its own Ri plasmid T-DNA into the genome of Prunella vulgaris, thereby spontaneously inducing the formation of hairy roots at the wound base of the stem of Prunella vulgaris. This provides an efficient platform for the expression and phenotypic analysis of exogenous genes in the roots of Prunella vulgaris.

[0017] The present invention has the following beneficial effects: Based on the growth characteristics of Prunella vulgaris, this invention establishes a genetic transformation system suitable for non-sterile tissue culture of Prunella vulgaris hairy roots through targeted infection methods, combined with ultrasonic power and infection time. This provides technical support for the functional gene research and pharmacodynamic component analysis of Prunella vulgaris, and is of great significance for promoting its genetic improvement and resource utilization.

[0018] Using this invention, under non-sterile conditions, Agrobacterium rhizogenes-mediated rapid genetic transformation of Prunella vulgaris hairy roots has a short cycle and high transformation efficiency. The transformation efficiency of hairy roots induced to become transgenic Prunella vulgaris can reach more than 70%, and transgenic materials can be obtained in large quantities in a short time. This invention realizes a simple, efficient, and low-cost genetic transformation of Prunella vulgaris, providing a new approach for the genetic transformation of Prunella vulgaris, and also laying the foundation for gene function research and molecular breeding of Prunella vulgaris. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the infection process of Prunella vulgaris root segments in Embodiment 1 of the present invention; Figure 2 The 35S::RUBY vector spectrum in Embodiment 1 of the present invention; Figure 3 This is a comparison diagram of wild-type and positively colored *Prunella vulgaris* plants in Example 1 of this invention; Figure 4 This is a PCR identification diagram of wild-type and positive plants of Prunella vulgaris in Example 1 of the present invention; Figure 5 The effects of different methods and different ultrasound durations on the induction of hairy roots of Prunella vulgaris in Example 1 of this invention are shown; different lowercase letters indicate significant differences (p<0.05). Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments.

[0021] Examples and Comparative Examples 1. Material preparation Using *Prunella vulgaris* planted in substrate soil as experimental material, *Prunella vulgaris* plants that had grown for about 60 days under long-day conditions and were in good condition were selected as explants. The strain was Agrobacterium rhizogenes K599; Recombinant plasmid carrying the Ruby reporter gene.

[0022] 2. Experimental Procedure 2.1 Activation and fermentation culture of Agrobacterium rhizogenes Activation culture: Take out the glycerol bacteria carrying the binary expression recombinant plasmid (K599::RUBY plasmid) of Ruby visualization gene stored at -80℃, take 30μL of bacterial culture in a clean bench, and inoculate it into TY liquid medium (10 mL). After inoculation, place the centrifuge tube in a constant temperature shaker at 30℃ and 200 rpm for 14-16 hours to activate the bacterial culture.

[0023] Expanded culture: Inoculate all the activated bacterial solution into an Erlenmeyer flask containing TY liquid culture medium (100 mL) and place it in a shaker at 30℃ and 200 rpm for 12 hours to expand the culture.

[0024] The TY liquid culture medium contains CaCl2 solution (1 mL, 1 M) and spectinomycin Spec (100 μL, 50 mg / mL).

[0025] 2.2 Preparation of Infection Solution After expansion culture, the bacterial suspension was aliquoted into 50 mL centrifuge tubes and centrifuged at 5000 rpm for 10 min at 20°C. The bacterial cells were collected, and the supernatant was discarded. Residual TY medium was washed away with tobacco suspension, and the bacterial cells were resuspended in small, repeated additions of tobacco suspension to adjust the bacterial concentration to OD600 = 0.8–1.2. Acetyleugenone AS was added to the resuspended suspension at a ratio of 1 μL per 5 mL of suspension, and the suspension was incubated in the dark for 1 hour before use.

[0026] 2.3 Explant treatment and infection Infection was carried out using different methods: Comparative example: The stem base of *Prunella vulgaris* was incised at least three times obliquely, with cuts of 2-3 mm, and the original roots were removed. Approximately 100 μL of bacterial solution was injected into the wounds at the stem base using a syringe. After injection, the plants were placed in the bacterial solution and soaked on a shaker at 50 rpm for 50 minutes.

[0027] Example: The stem base of Prunella vulgaris was obliquely incised at least three times, with incisions of 2-3 mm, and the original roots were removed. Ultrasonic treatment was then performed at a power of 150W for 1 min, 2 min, and 4 min, respectively, and labeled as 1#, 2#, and 3#. After ultrasonic treatment, the plants were placed in the bacterial solution and soaked on a shaker at 50 rpm for 50 min.

[0028] The *Prunella vulgaris* was then planted in sterilized substrate soil. The remaining bacterial solution was slowly dripped around the infected plants. The plants were incubated in darkness for 24 hours. After removing the film, they were cultured under long-day conditions (16 hours of light / 8 hours of darkness) in a normal greenhouse environment with high humidity, and watered regularly. The soil composition was vermiculite to nutrient soil in a volume ratio of 2:8. The soil was moistened with boiling water and then cooled.

[0029] 2.4 Screening and Identification of Positive Hairy Roots The *Prunella vulgaris* plants were cultured at 25-28℃ for 2-3 months for comparative observation. Preliminary screening was performed by observing the appearance of red markers indicating the RUBY gene on the surface of the hairy roots, or by detecting the fluorescent signal emitted by the fluorescent protein using a portable fluorescent flashlight. Subsequently, genomic DNA was extracted from potentially positive roots, and the transformation of the target gene was verified by PCR amplification. The verification results are shown in [link to relevant documentation]. Figure 4Wherein, Marker: DNA tag, used to indicate the size of DNA bands; Plasmid "+": positive control using 35S::RUBY plasmid as template; ddH2O "-": negative control using ddH2O as template; WT: DNA extracted from the control plant WT as template; 1#, 2#, 3#: DNA extracted from the red roots of transgenic Prunella vulgaris 1#, 2#, and 3# as templates, respectively.

[0030] Depend on Figure 4 As can be seen, the target band was amplified in Plasmid "+" and in samples 1#, 2#, and 3#, further demonstrating at the molecular level that the RUBY visualized gene transformation was successful in Prunella vulgaris samples 1#, 2#, and 3#.

[0031] For the genetic transformation rate of genes under different infection methods, please refer to [link / reference]. Figure 5 The genetic transformation rate is calculated as follows: Genetic transformation rate = (Number of positive seedlings with red roots ÷ Number of seedlings in all treatments) × 100% Depend on Figure 5 It is evident that different infection methods have varying effects on the induction of hairy roots in Prunella vulgaris. After eight weeks of culture, the genetic transformation rate of hairy roots in the injection-treated group was 17%. The group treated with ultrasound for 1 minute had a higher rate than the injection group, while the groups treated with ultrasound for 2 minutes and 4 minutes showed significantly improved induction rates of hairy roots in Prunella vulgaris. Among these, the group treated with ultrasound for 2 minutes had the highest induction rate, reaching 70% at the eighth week after treatment.

Claims

1. A method for transforming an exogenous gene into a Prunella vulgaris, comprising the following steps: 1) Using a Prunella vulgaris seedling grown in a substrate soil before flowering as an infection material; 2) Introducing an expression vector containing an exogenous gene into Agrobacterium rhizogenes to prepare an Agrobacterium rhizogenes infection solution; 3) Obliquely cutting and scratching the basal part of the stem of the Prunella vulgaris as the infection material, removing the original root system, placing the Prunella vulgaris into the Agrobacterium rhizogenes infection solution in step 2), and then using an ultrasonic method for infection; 4) Planting the infected Prunella vulgaris into a substrate, culturing in the dark, and then continuing to grow under long-day conditions until hairy roots are generated.

2. The method for genetic transformation of transferring exogenous genes into Prunella vulgaris according to claim 1, characterized in that, In step 1), the infection material is a Prunella vulgaris seedling in the vegetative growth stage, with leaves and a stem base.

3. The method of claim 1, wherein the foreign gene is introduced into the Prunella vulgaris. In step 2), the expression vector is a binary expression recombinant plasmid carrying a Ruby visual gene, a GFP fluorescent reporter gene, or a metabolic enzyme-related gene.

4. The method of claim 1, wherein the foreign gene is introduced into the Prunella vulgaris. In step 2), the fermentation medium of the Agrobacterium rhizogenes is a TY medium.

5. The method of claim 1, wherein the foreign gene is introduced into the Prunella vulgaris. In step 2), the preparation method of the Agrobacterium rhizogenes infection solution is: resuspending the Agrobacterium rhizogenes bacteria with a tobacco suspension, adjusting the concentration of the bacterial solution to an OD600 value of 0.8-1.2, and adding acetosyringone, and then placing in the dark.

6. The method of claim 1, wherein the foreign gene is introduced into the Prunella vulgaris. In step 3), the length of the obliquely cut and scratched incision is 2-3 mm.

7. The method of claim 1, wherein the foreign gene is introduced into the Prunella vulgaris. In step 3), when using the ultrasonic method for infection, the ultrasonic power is 140 W-160 W.

8. The method for genetic transformation of introducing exogenous genes into Prunella vulgaris according to any one of claims 1-7, characterized in that, In step 3), when using the ultrasonic method for infection, the infection time is 1-5 min.

9. Use of the genetic transformation method of claim 1 to improve the rooting rate and / or genetic transformation rate of hairy roots of Prunella vulgaris.

10. Use of the genetic transformation method of claim 1 in the functional gene research of Prunella vulgaris.

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