Method for establishing a genetic transformation system of salvia miltiorrhiza

Abstract

The application provides a method for establishing a genetic transformation system of Salvia miltiorrhiza, and belongs to the technical field of genetic engineering. The method is that Agrobacterium rhizogenes C58C1 containing a recombinant vector PYLTAC380H-SmCYP76AH1-GFP is used to infect root segments at rhizome joints, and then the root segments are taken out and placed in vermiculite for culture. After 2-3 weeks of budding, the Salvia miltiorrhiza seedlings can be subjected to transformation screening and detection. The application provides a genetic transformation method system of Salvia miltiorrhiza, which can realize efficient gene transformation without tissue culture under non-aseptic conditions.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a method for genetic transformation of Salvia miltiorrhiza. Background Technology

[0002] Salvia miltiorrhiza is a commonly used traditional Chinese medicine. Its small genome and few chromosomes result in a short generation cycle in transgenic regenerated plants, making it a valuable model plant for medicinal research. Genetic engineering can assist in the targeted breeding of high-yielding, highly resistant, and high-quality new varieties, or increase the content of secondary metabolites in the original medicinal material for the extraction of active ingredients, thus meeting the huge market demand for Salvia miltiorrhiza. However, the Agrobacterium-mediated transformation system using leaf disc transformation requires tissue culture, which demands high conditions, is influenced by many factors, takes a long time, and is susceptible to contamination. Furthermore, the long breeding cycle significantly limits the progress of new variety selection, failing to meet the diversified and precise needs of Salvia miltiorrhiza production. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for establishing a genetic transformation system for Salvia miltiorrhiza, which utilizes the rhizome-segment of Salvia miltiorrhiza as explants, and achieves efficient gene transformation under non-sterile conditions without the need for tissue culture.

[0004] To achieve the above objectives, the specific solution adopted by the present invention is as follows: A method for establishing a genetic transformation system for Salvia miltiorrhiza involves infecting the root segment at the junction of the rhizome and stem of Salvia miltiorrhiza with Agrobacterium rhizome containing a recombinant vector, removing the segment and placing it in vermiculite for culture, and screening and testing the Salvia miltiorrhiza seedlings 2-3 weeks after germination.

[0005] As a further optimization of the above method, the recombinant vector is PYLTAC380H-SmCYP76AH1-GFP. Specifically, using cDNA from Salvia miltiorrhiza leaves as a template, and the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers, the recombinant vector was cloned... SmCYP76AH1 The recombinant vector pYLDonarⅠ-35S-GFP was cloned and recombined in one step. After correct sequencing, the recombinant vector pYLDonarⅠ-35S-SmCYP76AH1-GFP was assembled into the expression vector pYLTAC380H to obtain the recombinant vector pYLTAC380H-SmCYP76AH1-GFP.

[0006] As a further optimization of the above method, the method specifically involves: [the following text appears to be incomplete and requires further context: "containing Danshen..."] SmCYP76AH1 The recombinant vector PYLTAC380H-SmCYP76AH1-GFP was transformed into Agrobacterium rhizogenes C58C1. Positive colonies were picked, expanded, and then the bacterial culture was resuspended. The OD of the bacterial culture was adjusted. 600The concentration of the inoculum was 0.6-0.8. Root segments of 3-5 cm from the junction of the rhizome and stem of *Salvia miltiorrhiza* were completely immersed in the inoculum and incubated on a shaker at 28℃ and 200 rpm for 20-30 min. After incubation, the roots were removed and placed in vermiculite, covered with plastic wrap, and kept in the dark and moist for 24 h. Then, the roots were placed in a 28℃ incubator for 16 h of light and 8 h of dark incubation. After sprouting, the roots were cultured for another 2-3 weeks until they had 4-6 leaves, at which point screening and testing were performed.

[0007] Furthermore, the resuspension is performed using an infiltration buffer containing 1 / 2×MS, 100 uM acetylsalicylic acid and 3% sucrose.

[0008] As a further optimization of the above method, the screening detection includes PCR screening and GFP fluorescence screening; The PCR screening refers to extracting DNA from plant leaves and then amplifying the hygromycin gene. HPT Ri plasmid gene in C58C1 rolB and SmCYP76AH1 Genes, simultaneously amplified HPT , rolB and SmCYP76AH1 The plants were initially identified as positive. The GFP fluorescence screening refers to performing fluorescence scanning on plants that have been preliminarily identified as positive plants through PCR screening. Seedlings with green fluorescence in their leaves are identified as transgenic positive plants.

[0009] Beneficial Effects: This invention proposes for the first time a genetic transformation method for *Salvia miltiorrhiza* that requires neither a sterile environment nor tissue culture. Using the rhizome-root junction as explants, *Agrobacterium rhizogenes* C58C1 containing a recombinant vector is used to infect the root segment at the rhizome junction. No sterile environment or tissue culture is required; after infection, the seedlings are directly placed in vermiculite for culture. Transgenic positive plants can be screened and detected 2-3 weeks after germination. This transformation method eliminates the need for sterile environments and contamination issues, simplifies the acquisition of *Salvia miltiorrhiza* explants, shortens the post-transformation culture time, and achieves high transformation efficiency, making it a simple and efficient genetic transformation method for *Salvia miltiorrhiza*. Attached Figure Description

[0010] Figure 1 This is a flowchart of the transformation method and positive plant screening process for Salvia miltiorrhiza; Figure 2 It is the infection and culture process of the rhizome-joint segment of Salvia miltiorrhiza; Figure 3 This is a diagram showing the amplification results of hygromycin, Ri plasmid, and CYP76AH1 gene in transformed Salvia miltiorrhiza seedlings; in the diagram, "M" represents marker2000, "-" represents the negative control of untransformed Salvia miltiorrhiza, and "+" represents the positive control; Figure 4 This is a graph showing the GFP fluorescence detection results in transformed Salvia miltiorrhiza seedlings. Detailed Implementation

[0011] In Agrobacterium-mediated transformation of Salvia miltiorrhiza, leaf disc transformation is commonly used, employing leaves as explants, or both leaves and petioles as explants. Explant pre-culture, co-culture time, Agrobacterium infection concentration, and duration are all crucial factors affecting Agrobacterium-mediated transformation efficiency. Explant culture, post-infection inhibition and subsequent induction of Agrobacterium, and tissue culture processes require stringent conditions, are influenced by numerous factors, are lengthy, and are susceptible to contamination.

[0012] To address the above problems, the technical solution of the present invention is improved as follows: Using cDNA from Salvia miltiorrhiza leaves as a template, the expression F: tcagatctcgagctcaagcttATGAGATCTATGAATCTGG TGGATGC (SEQ ID NO: 1), R: cgactgcagaattcgaagcttGTTCTGCCTATGTGCAATGTAATCG (SEQ ID NO: 2) were used as primers for cloning. SmCYP76AH1 The recombinant vector pYLDonarⅠ-35S-GFP was cloned and recombined in one step. After correct sequencing, the recombinant vector pYLDonarⅠ-35S-SmCYP76AH1-GFP was assembled into the expression vector pYLTAC380H, obtaining the expression assembly vector pYLTAC380H-SmCYP76AH1-GFP, which was then transformed into Agrobacterium rhizogenes C58C1. The Agrobacterium rhizogenes C58C1 containing the recombinant expression vector pYLTAC380H-SmCYP76AH1-GFP was used to infect root segments at the rhizome junction. These segments were then removed and placed in vermiculite culture. After budding, the seedlings were screened and tested 2-3 weeks later.

[0013] The transformation method and positive plant screening process of Salvia miltiorrhiza are as follows: Figure 1 As shown.

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0015] Infection and culture of the rhizome junction of Salvia miltiorrhiza Will contain Danshen SmCYP76AH1 The recombinant vector PYLTAC380H-SmCYP76AH1-GFP was transformed into Agrobacterium rhizogenes C58C1. Positive colonies were picked and cultured extensively. The bacterial cells were then resuspended in infusion buffer (1 / 2×MS, 100 uM acetylsuccinone, 3% sucrose). The OD of the bacterial culture was adjusted. 600A 0.6-0.8 μg solution was used as the inoculum. A 3-5 cm root segment from the rhizome junction of *Salvia miltiorrhiza* was completely immersed in the inoculum and incubated on a shaker at 28°C and 200 rpm for 20-30 minutes. After incubation, the segment was removed and placed in vermiculite, covered with plastic wrap, and kept in the dark and moist for 24 hours. It was then placed in a 28°C incubator for 16 hours of light and 8 hours of dark incubation. After approximately two weeks, buds emerged, and the segment was further cultured for 2-3 weeks until 4-6 leaves appeared, at which point screening and testing were performed. The inoculum and culture process for the *Salvia miltiorrhiza* rhizome junction segment is as follows: Figure 2 As shown.

[0016] Detection of hygromycin, Ri plasmid, and CYP76AH1 gene in transformed Salvia miltiorrhiza seedlings Because the PYLTAC380H vector carries a hygromycin selection gene. HPT Agrobacterium C58C1 contains the Ri plasmid, and the recombinant vector gene transformed is Danshen. SmCYP76AH1 After transformation, Salvia miltiorrhiza seedlings approximately 3 weeks old were selected, and DNA was extracted from the leaves of each plant. Using the DNA as a template, untransformed Salvia miltiorrhiza plants served as a negative control, and Agrobacterium rhizogenes C58C1 bacterial suspension containing the recombinant vector PYLTAC380H-SmCYP76AH1-GFP served as a positive control. Hygromycin gene assays were then performed. HPT Ri plasmid gene in C58C1 rolB and SmCYP76AH1 Gene-specific primers were used for PCR amplification screening, and the selected transformed seedlings simultaneously amplified the gene. HPT , rolB and SmCYP76AH1 The plants were initially identified as positive, and the amplification results are as follows: Figure 3 As shown.

[0017] Detection of GFP fluorescence in transformed Salvia miltiorrhiza seedlings Because the multiple cloning site of the vector PYLTAC380H contains the green fluorescent protein GFP at its C-terminus, fluorescence scanning of the initially screened positive plants was performed using a fluorescence confocal microscope to further confirm the transformation of positive plants. The results showed that all 17 *Salvia miltiorrhiza* seedlings exhibited significant green fluorescence in their leaves. Figure 4 Combining HPT , rolB and SmCYP76AH1 The amplification results of the relevant genes showed that 17 of the 20 selected transgenic plants were transgenic positive plants.

[0018] Statistics on the positive rate of transformed Salvia miltiorrhiza seedlings To test the transformation efficiency of this genetic transformation method for Salvia miltiorrhiza, three batches of transformed seedlings were screened and statistically analyzed. Twenty seedlings were selected from each batch, for a total of 60 seedlings. The positive screening results are shown in Table 1. All three batches had more than 16 positive seedlings, and the average transformation rate was approximately 82.7%.

[0019] Table 1. Number and percentage of positive plants among transformed Salvia miltiorrhiza seedlings.

[0020] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. A method for establishing a genetic transformation system for *Salvia miltiorrhiza*, characterized in that: Using cDNA from Salvia miltiorrhiza leaves as a template, and the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers, cloned SmCYP76AH1 The recombinant vector pYLDonarⅠ-35S-GFP was cloned and recombined in one step. After the sequencing was correct, the recombinant vector pYLDonarⅠ-35S-SmCYP76AH1-GFP was assembled into the expression vector pYLTAC380H to obtain the recombinant vector pYLTAC380H-SmCYP76AH1-GFP. Will contain Danshen SmCYP76AH1 The recombinant vector PYLTAC380H-SmCYP76AH1-GFP was transformed into Agrobacterium rhizogenes C58C1. Positive colonies were picked, expanded, and then the bacterial culture was resuspended. The OD of the bacterial culture was adjusted. 600 The concentration of the inoculum was 0.6-0.

8. Root segments of 3-5 cm from the junction of the rhizome and stem of *Salvia miltiorrhiza* were completely immersed in the inoculum and incubated on a shaker at 28℃ and 200 rpm for 20-30 min. After incubation, the roots were removed and placed in vermiculite, covered with plastic wrap, and kept in the dark and moist for 24 h. Then, the roots were placed in a 28℃ incubator for 16 h of light and 8 h of dark incubation. After sprouting, the roots were cultured for another 2-3 weeks until they had 4-6 leaves, at which point screening and testing were performed.

2. The method for establishing according to claim 1, characterized in that: The resuspension was performed using an infiltration buffer containing 1 / 2×MS, 100 uM acetylsalicylic acid and 3% sucrose.

3. The method for establishing according to claim 1, characterized in that: The screening tests include PCR screening and GFP fluorescence screening; The PCR screening refers to extracting DNA from plant leaves and then amplifying the hygromycin gene. HPT Ri plasmid gene in C58C1 rolB and SmCYP76AH1 Genes, simultaneously amplified HPT , rolB and SmCYP76AH1 The plants were initially identified as positive. The GFP fluorescence screening refers to performing fluorescence scanning on plants that have been preliminarily identified as positive plants through PCR screening. Seedlings with green fluorescence in their leaves are identified as transgenic positive plants.

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