Visual marker genetic transformation method for phalaenopsis leaves
By using the PeMYB23 gene-induced anthocyanins as screening marks in Phalaenopsis leaves, a transient transformation system was established, which solved the problem of low genetic transformation screening efficiency in Phalaenopsis leaves, and achieved rapid and simple transgenic screening, which was a Phalaenopsis. Technical support is provided by genetic engineering research.
Patent Information
- Application Number
- CN202411716195.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art is difficult to achieve rapid and efficient genetic transformation screening in Phalaenopsis leaves, and there is no report on using leaves as transformation receptors, limiting the study of specifically expressing genes in leaves.
By using anthocyanins induced by the Phalaenopsis PeMYB23 gene as screening markers, a transient transformation system of Phalaenopsis leaves was established, and the PeMYB23 gene was mediated by Agrobacterium to transfer the PeMYB23 gene into Phalaenopsis leaves were quickly screened using the color changes of anthocyanins.
It realizes the rapid, simple and efficient genetically modified screening of Phalaenopsis leaves, avoids complex detection and destructive problems in traditional methods, and provides important technical support for the genetic engineering breeding and molecular biology research of Phalaenopsis.
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Figure CN119464362A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of molecular biology and biotechnology, and in particular relates to a method for genetic transformation of moth orchid leaves with visualized marking. Background Art
[0002] Anthocyanin is a natural water-soluble pigment widely distributed in various parts of plants. It not only gives plants rich colors, but also enhances their ability to resist environmental stresses, such as strong light, ultraviolet rays and low temperatures. As a safe and non-toxic natural pigment, anthocyanin has a variety of health functions such as antioxidants and is widely used in food, medicine and cosmetics. The biosynthetic pathway and regulatory mechanism of anthocyanin have been deeply studied in model plants such as petunia, snapdragon and Arabidopsis. The biosynthesis of anthocyanin is regulated by three major transcription factors: MYB, bHLH and WD40. Among them, R2R3-MYB is particularly critical for the regulation of anthocyanin synthesis. It can directly regulate the expression of synthetic genes, play a core role in anthocyanin regulation, and determine the final coloration of plants.
[0003] Phalaenopsis belongs to the genus Phalaenopsis in the orchid family. It is one of the most widely cultivated and popular species in the orchid family for its bright colors, elegant flower shapes and long flowering period. It is known as the "Queen of Orchids" and plays an important role in the fields of ornamental, ecological and economics. The improvement of Phalaenopsis traits (such as flower color, leaf color, flower shape, etc.) is the main goal of genetic breeding. The traditional genetic transformation of Phalaenopsis selects the protocorms germinated from Phalaenopsis seeds as the transformation receptor, but this method has many disadvantages such as long culture cycle, easy to produce chimeras, and low transformation efficiency. Therefore, it is particularly necessary to establish a method that can quickly perform trait detection and gene function analysis in Phalaenopsis plants. The transient transformation method is mediated by Agrobacterium tumefaciens and can quickly evaluate the function and expected phenotype of exogenous genes. Compared with traditional stable transformation, transient expression does not require T-DNA to be integrated into the host genome, and has the advantages of simple operation, short cycle, and high expression efficiency. In addition, the establishment of a transient transformation system provides more possibilities for the application of technologies such as RNA interference and gene editing. At present, the Phalaenopsis transient transformation system has been successfully transformed in flower organs, but there are no reports of using leaves as transformation receptors at home and abroad, which greatly limits the research on genes specifically expressed in leaves. Therefore, it is necessary to develop a Phalaenopsis leaf transient transformation system that can effectively solve the above problems.
[0004] Traditional plant genetic transformation screening mainly relies on exogenous screening markers and reporter genes (such as GUS, GFP, LUC, etc.), but this method has the disadvantages of being invisible to the naked eye, complex detection, high destructiveness, and background interference. As a new type of reporter gene, plant endogenous anthocyanins have the advantages of being visible to the naked eye, non-destructive, easy to detect, and safe. They can be used for early screening of transformants and have been applied in tobacco, apple, rubber, mulberry and other plants. However, there are no reports on the application of anthocyanins as screening markers for the screening of instantaneous transformation systems in Phalaenopsis leaves. Summary of the invention
[0005] In order to solve the above-mentioned technical problems, the present invention provides a method for using anthocyanins induced by the Phalaenopsis PeMYB23 gene as a screening marker. This method is applied to the transient transformation screening of Phalaenopsis leaves, which is expected to greatly improve the efficiency of transgenic screening of Phalaenopsis leaves. It has the advantages of simple detection, short cycle, high expression efficiency, etc., and can provide important technical support for genetic engineering breeding and molecular biology research of Phalaenopsis.
[0006] To achieve the above object, the present invention adopts the following technical solution:
[0007] In one aspect, the present invention provides the use of the Phalaenopsis PeMYB23 gene as a transgenic marker in screening plant transgenic materials, wherein the nucleotide sequence of the Phalaenopsis PeMYB23 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.
[0008] Preferably, the anthocyanin phenotype induced by the PeMYB23 gene is used as a visual marker to identify whether the target gene has been successfully transformed in the plant.
[0009] On the other hand, the present invention also provides the use of Phalaenopsis PeMYB23 gene in regulating plant anthocyanin synthesis, wherein the nucleotide sequence of the Phalaenopsis PeMYB23 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.
[0010] Preferably, the synthesis of plant anthocyanins is promoted by overexpressing the PeMYB23 gene.
[0011] Preferably, the plant includes Phalaenopsis.
[0012] On the other hand, the present invention also provides a method for genetic transformation of Phalaenopsis leaves by visual marking, wherein the PeMYB23 gene with a nucleotide sequence such as that shown in SEQ ID NO:1 is transferred into Phalaenopsis leaves through Agrobacterium-mediated, and rapid screening is performed using the color change of anthocyanins to achieve visual and non-destructive identification of transformants.
[0013] Specifically, the target gene and the PeMYB23 gene are simultaneously constructed into a plasmid vector to obtain a recombinant plasmid; the recombinant plasmid is introduced into Agrobacterium competent cells to make them into transformation engineering bacteria, and the Agrobacterium containing the recombinant plasmid is introduced into Phalaenopsis leaves by injection.
[0014] Preferably, the Agrobacterium is Agrobacterium tumefaciens EHA105.
[0015] Preferably, the method of introducing the recombinant plasmid into Agrobacterium competent cells includes electroporation, heat shock or chemical transformation.
[0016] The present invention has the following beneficial effects:
[0017] The method of the present invention is to transfer the PeMYB23 gene into the leaves of Phalaenopsis by Agrobacterium-mediated, and to perform rapid screening by utilizing the color change of anthocyanin, so as to realize visualization of transformants and identification non-destructively. Compared with the traditional genetic transformation method, the present invention does not need to rely on exogenous reporter genes and screening markers, and has the advantages of simple operation, short cycle, high expression efficiency, and easy observation. The implementation process includes cloning and vector construction of the PeMYB23 gene, engineering bacteria transformation of the recombinant plasmid, instantaneous transformation of the leaves and detection of anthocyanin accumulation. The instantaneous transformation system finally established can realize early evaluation of the conversion efficiency of the target gene by constructing the target gene and the PeMYB23 gene into the recombinant plasmid at the same time, and realize early evaluation of the conversion efficiency of the target gene by the anthocyanin accumulation situation, so as to provide a new technical approach for the exploration of Phalaenopsis genetic engineering breeding, molecular biology research and functional genes. The technology of the present invention is not only applicable to Phalaenopsis, but also has the potential for wide application in genetic transformation and screening of other plants. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the electrophoresis diagram of the PeMYB23 gene, where M is the 2000 DNA Marker and the right lane is the PeMYB23 gene fragment.
[0019] Figure 2 This is the phenotype of the PeMYB23 gene after transient expression in the petals of 'Ama' Phalaenopsis for one week. The petals on the left are injected with empty vector, and the petals on the right are injected with bacterial solution that overexpresses the PeMYB23 gene.
[0020] Figure 3 This is a schematic diagram of the instantaneous transformation injection of Phalaenopsis leaves.
[0021] Figure 4 This is the phenotype of the golden edge cranberry Phalaenopsis leaves one week after injection. The left side is the leaf injected with no vector, and the right side is the leaf injected with the overexpressed PeMYB23 bacterial solution. The arrow indicates the injection site.
[0022] Figure 5This is an enlarged picture of the phenotype of the Golden Edge Cranberry Phalaenopsis leaves one week after injection. The scale is 1mm.
[0023] Figure 6 This is a picture of anthocyanin extraction from leaves one week after injection of empty vector and overexpressed PeMYB23 bacterial solution.
[0024] Figure 7 The anthocyanin content in leaves of Phalaenopsis orchids injected with empty vector and overexpressed PeMYB23 gene.
[0025] Figure 8 The expression abundance of PeMYB23 and anthocyanin structural genes in Phalaenopsis leaves with no vector and transient overexpression of PeMYB23 gene. DETAILED DESCRIPTION
[0026] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.
[0027] The plant materials in the embodiment include Phalaenopsis Chia E Yenlin Variegata and Phalaenopsis aphrodite, both of which are planted in the resource nursery of South China Botanical Garden, Chinese Academy of Sciences. The nursery has temperature control facilities, with a temperature range of 15-32° C. and a humidity range of 70-85%.
[0028] Example 1: Cloning of Phalaenopsis anthocyanin-regulated transcription factors
[0029] Through relative expression pattern and homology analysis in the transcriptome database, the transcription factor that positively regulates anthocyanin biosynthesis in Phalaenopsis virginiana was discovered. After online comparison (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), it was found that the amino acid sequence of this gene was 100% similar to PeMYB23 and 95.38% similar to PeMYB2, so it was named PeMYB23. Using the leaves of Phalaenopsis virginiana as the material, total RNA was extracted, 1 μg of high-quality total RNA was taken for reverse transcription, and PCR was performed using specific primers PeMYB23-F: 5'-ATGGGAAGGAATCCGAGCTG-3' and PeMYB23-R: 5'-TTAGAACTGTTTCAAAAGCT-3'. Using the obtained cDNA of the golden-edged cranberry leaves as a template, the PCR reaction was carried out using PeMYB23-F and PeMYB23-R primers. Selection FastPfu Fly PCR SuperMix (Cat. No. AS231) was used for amplification. The reaction procedure was as follows: 1 cycle of 98°C / 10s, 30 cycles of 98°C / 10s, 56°C / 5s, 72°C / 10s, and 1 cycle of 72°C / 1min. The amplified PeMYB2 gene fragment was Figure 1 As shown, the PeMYB2 gene fragment was recovered using the Meiji gel DNA recovery kit (Cat. No.: D2111) and then connected to the Qingke cloning vector pClone007 Versatile Simple Vector Kit (Cat. No.: TSV-007VS) for gene sequencing. The sequencing results were compared with the transcriptome data, and the results showed that the nucleotide sequence of the golden edge cranberry Phalaenopsis PeMYB23 gene was successfully cloned, as shown in SEQ ID NO: 1:
[0030] ATGGGAAGGAATCCGAGCTGTTCGAAGGAAGGGCTCAACAAGGGAGCATGGACTGCCG
[0031] TCGAAGACAAGCTCTTGATTAAATACGTCAATGCTCATGGCGAAGGCAAATGGACGACG
[0032] GTGCCCTATATAGCCGGGCTGAAAAGGTCGGGCAAGAGCTGTCGTCTTCGATGGTTAAA
[0033] CTACCTGAGGCCAAACGTCAAACGTGGGAACTTTTCTGAGGAGGAAATCGACCTCATAA
[0034] TCAGGCTGCATAAACTGCTCGGCAATAGATGGTCTCTGATCGCTGGGAGGATACCAGGC
[0035] AGAACTGACAACGAAATAAAGAACTACTGGAACACAACCTTGGCGAAGAAGACAACCT
[0036] CACAGCATCCATCACAAAAGCCACCAGCTTCAAATCAAGCACTGCCATCCCCATCATCTT
[0037] CTCTATCACCTCAATCCACAAATAACAAAACTTTGAATAGACCCAAGGCCCTGAGGTGC
[0038] AGCCAATTGGGTTTTCTAGTAGGAGAACCAACTGCAGCAGTGGCTTCTGTGTTGCCTAA
[0039] TCAGAACACCAAAGCCAGCACCATTGCTGAAGAAGAACTGCTTCAGGAGGAGGAGGA
[0040] GAATGTAGTCTGGAACTGCAGCTTTGATGAATATAATGCAGTGTTTGCTGATGAGGATTT
[0041] GATGGAGTTCAACGGAATGCAGGATTTTGAGAGCTGGATGCTGAATGATGATGGTTGTT
[0042] ACCTTCCTGATGATGAGAACATAAATTGGCTAACTTCTTTGTTTGATATGGAAGGGGAGCTTTTGAAACAGTTCTAA。
[0043] The corresponding amino acid sequence is shown in SEQ ID NO:2:
[0044] MGRNPSCSKEGLNKGAWTAVEDKLLIKYVNAHGEGKWTTVPYIAGLKRSGKSCRLRWLN
[0045] YLRPNVKRGNFSEEEIDLIIRLHKLLGNRWSLIAGRIPGRTDNEIKNYWNTTLAKKTTSQHPS
[0046] QKPPASNQALPSPSSSLSPQSTNNKTLNRPKALRCSQLGFLVGEPTAAVASVLPNQNTKASTI
[0047] AEEELLQEEEENVVWNCSFDEYNAVFADEDLMEFNGMQDFESWMLNDDGCYLPDDENINWLTSLFDMEGELLKQF。
[0048] Example 2: Construction of a Visualization Label Vector
[0049] The PeMYB23 gene cloning vector sequenced correctly in Example 1 was used as a template, and the homologous recombination primers were designed using the Takara online website (https: / / www.takarabio.com / learning-centers / cloning / primer-design-and-other-tools). The primer sequences were as follows: PeMYB23-C17F: 5'-ACATTTAAATACTAGATGGGAAGGAATCCGAGCTG-3'; PeMYB23-C17R: 5'-TACCGGATCCACTAGTGAACTGTTTCAAAAGCT-3'. The pGreen 35S-GFP(C17) vector (Li, J., Wu, K., Li, L. et al. Identification of HpMYB1 inducing anthocyanin accumulation in Hippeastrum Hybridum tepals by RNA-seq. BMC Plant Biol 23, 594 (2023)) was double-digested with Thermo Fisher EcoRⅠ (Cat. No.: FD0274) and XbaⅠ (Cat. No.: FD0684) restriction enzymes. The PeMYB23 gene fragment was connected to the pGreen 35S-GFP(C17) vector using the full-strand gold homologous recombinase (Cat. No.: CU201). The homologous recombinant vector was transformed into Escherichia coli DH5α (Cat. No.: TSC-C01), and positive clones were selected for sequencing. The sample culture solution with correct sequencing was propagated and the recombinant plasmid was extracted using the Meiji Plasmid Rapid Extraction Kit (Cat. No.: P1001).
[0050] pGreen 35S-GFP(C17)-PeMYB23 was used for downstream experiments.
[0051] Example 3: Transformation of engineered bacteria with a visual marker vector
[0052] Take out two tubes of cryopreserved Agrobacterium competent cells EHA105 (Cat. No.: AC1012) from -80℃, place them at room temperature or in the palm of your hand to warm them slightly, and add 1μg of empty vector plasmid pGreen 35S-GFP (C17) and recombinant plasmid pGreen 35S-GFP (C17)-PeMYB23 respectively after they melt, and gently shake the bottom of the tube by hand to mix them evenly. Then, place the bacterial solution on ice for 5 minutes, treat it in liquid nitrogen for 5 minutes, and place it in a 37℃ water bath for 5 minutes, and finally place it in an ice bath for 5 minutes. Add 700μL of LB liquid culture medium without antibiotics and culture it at 28℃, 200rpm for 2 hours. After the culture was completed, centrifuge at 6000 rpm for 1 minute, collect the cells and keep about 100 μL of the supernatant, resuspend the cells and evenly spread them on LB solid plates containing Kan (50 mg / L) and rifampicin (20 mg / L) antibiotics, and invert them in a 28°C incubator for 3 days. Pick the positive clones, verify the results by colony PCR and sequence them.
[0053] Example 4: Transient overexpression in leaves of Phalaenopsis vulgaris
[0054] Take 100 μL of the Agrobacterium bacterial solution obtained in Example 3, add 10 mL of LB liquid medium containing Kan (50 mg / L) and rifampicin (20 mg / L) antibiotics, culture at 28°C, 180 rpm shaker for about 24 hours until the bacterial solution OD reaches 0.8-1.0, centrifuge at 5000×g for 5 minutes at room temperature, remove the supernatant, resuspend the bacterial block with 10 mL of sterile water, centrifuge at 5000×g for 5 minutes at room temperature, remove the supernatant, add the resuspension (containing 2.02 g MgCl2, 0.97 g MES and 200 μM acetosyringone per liter, the solvent is water, and the pH value is adjusted to 5.8) to adjust the OD to 0.8-1.0 to prepare the final infection solution. In order to preliminarily verify the gene function of PeMYB23, select the newly opened 'Ama' Phalaenopsis flowers, use a 1 mL syringe without a needle, and inject about 500 μL of bacterial solution from the back of the petals. After the injection, the Phalaenopsis plants were placed in an artificial climate chamber for dark culture overnight and then transferred to normal culture. The culture environment was 26℃, 10000Lux, 12 hours of light / 24℃, 0Lux, 12 hours of dark period. After 5 days of culture, it was observed that the petals injected with the overexpressed PeMYB23 gene (right side) had obvious anthocyanin accumulation, while the petals injected with the empty vector (left side) had no anthocyanin accumulation ( Figure 2 ), the anthocyanin coloring phenomenon is still maintained when the Phalaenopsis flowers wither. The results show that the PeMYB23 gene has the function of inducing the formation of anthocyanins and can be used in the development of a genetic transformation system with visual markers.
[0055] At the same time, the final infection solution prepared above was injected into the yellow area of the golden edge cranberry orchid leaves. Because its color is lighter, it can eliminate the interference of the background color on the anthocyanin observation, making it easier to observe the production of anthocyanins. Figure 3 As shown, a 1mL syringe was selected to obliquely insert the needle into the center of the Phalaenopsis leaf, and about 200μL of bacterial solution was injected until a water-stained area appeared on the leaf. The injected Phalaenopsis plant was placed in an artificial climate chamber for dark culture overnight and then transferred to normal culture. The culture environment was 26℃, 10000Lux, 12 hours of light / 24℃, 0Lux, 12 hours of dark period. After 7 days of culture, it was observed that the control leaves injected with no load did not accumulate anthocyanins, while the leaves injected with the overexpressed PeMYB23 gene showed obvious anthocyanin accumulation ( Figure 4 , Figure 5 , Figure 6 ). The results showed that the transient transformation system in Phalaenopsis leaves has been successfully established, and the anthocyanin phenotype induced by the PeMYB23 gene can be used as a visual marker to directly identify whether the transformation is successful by the naked eye.
[0056] Example 5: Anthocyanin content in Phalaenopsis leaves and identification of transgenic
[0057] The pH differential method was used to detect the content of anthocyanins in Phalaenopsis leaves with no load and overexpression of PeMYB23 gene. Take 0.1g of fresh Phalaenopsis leaf material from the injection area, freeze it in liquid nitrogen, grind the sample into powder using a ball mill, then transfer all the samples to a 5mL centrifuge tube, add anthocyanin extract (1% hydrochloric acid methanol, v / v), shake and mix, place in a 4℃ refrigerator away from light for 24h, and centrifuge to remove impurities to obtain anthocyanin extract. The extract and two buffers (KCl buffer with pH 1 and NaAc buffer with pH 4.5) were mixed in a volume ratio of 1:4, and the solvent was used as a blank control. It was placed at room temperature away from light for 20-30min. Add it to a 500μL cuvette with a light path of 1cm, and measure the absorbance values at 520nm and 700nm using a UV spectrophotometer. Anthocyanin content (mg / g) = ΔD × V × F × M × 1000 / (ε×m), where ΔD = (A520nm-A700 nm) pH 1.0-(A520 nm-A700 nm) pH 4.5; V: dilution volume (unit L); F: dilution factor; M: the relative molecular mass of cyanidin-3-glucoside is 449.2 g / mol; ε: the molar extinction coefficient of cyanidin-3-glucoside is 26900 L / (mol×cm); m: sample mass (unit g).
[0058] The results showed that the anthocyanin content in leaves overexpressing PcMYB23 gene was 83.6 μg / g, which was 6.8 times that of the unexpressed leaves. Figure 7). This indicates that PcMYB23 successfully induced anthocyanins in the leaves of Phalaenopsis virginiana, thereby changing the leaf color.
[0059] Real-time quantitative PCR was used to detect the expression levels of anthocyanin-related genes in the empty vector and PcMYB23 gene injection areas. The specific primer sequences are shown in Table 1. It was found that anthocyanin-related synthesis genes were significantly upregulated in the PcMYB23 gene injection area ( Figure 8 ). The surface can promote the expression of key genes in the anthocyanin synthesis pathway in Phalaenopsis leaves.
[0060] Table 1
[0061]
[0062] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field should fall within the protection scope determined by the claims of the present invention.
Claims
1. Application of Phalaenopsis PeMYB23 gene as a transgenic marker in screening transgenic plant materials, the nucleotide sequence of Phalaenopsis PeMYB23 gene is shown in SEQ ID NO:
1.
2. The use according to claim 1, characterized in that: The amino acid sequence of the Phalaenopsis PeMYB23 gene is shown in SEQ ID NO:
2.
3. The use according to claim 1, characterized in that: The anthocyanin phenotype induced by the PeMYB23 gene is used as a visual marker to identify whether the target gene has been successfully transformed in the plant.
4. Application of Phalaenopsis PeMYB23 gene in regulating plant anthocyanin synthesis, the nucleotide sequence of the Phalaenopsis PeMYB23 gene is shown in SEQ ID NO:
1.
5. The use according to claim 4, characterized in that: The amino acid sequence of the Phalaenopsis PeMYB23 gene is shown in SEQ ID NO:
2.
6. The use according to claim 4, characterized in that: By overexpressing the PeMYB23 gene, plant anthocyanin synthesis is promoted.
7. The use according to claim 1 or 4, characterized in that: The plants include Phalaenopsis.
8. A method for genetic transformation of Phalaenopsis leaves by visualizing markers, characterized in that: The PeMYB23 gene with a nucleotide sequence as shown in SEQ ID NO: 1 is transferred into Phalaenopsis leaves through Agrobacterium-mediated transformation, and rapid screening is performed using the color change of anthocyanins to achieve visual and non-destructive identification of transformants.
9. The method according to claim 8, characterized in that The method comprises the following steps: constructing the target gene and the PeMYB23 gene into a plasmid vector at the same time to obtain a recombinant plasmid; introducing the recombinant plasmid into Agrobacterium competent cells to make them into transformation engineering bacteria, and introducing the Agrobacterium containing the recombinant plasmid into Phalaenopsis leaves by injection.
10. The method according to claim 8, characterized in that The Agrobacterium is Agrobacterium tumefaciens EHA105.