Anoectochilus formosanus PHR transcription factor ArPHR2, coding gene and application

Abstract

The invention discloses an anoectochilus formosanus PHR transcription factor ArPHR2, a coding gene and application. The amino acid sequence of the transcription factor ArPHR2 is as shown in SEQ ID NO.2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.1; the invention also provides application of the transcription factor ArPHR2 and the coding gene thereof in relieving water loss of anoectochilus formosanus and increasing the content of kinsenoside. The transcription factor ArPHR2 is overexpressed in the anoectochilus formosanus by using a genetic engineering technology, and the main component kinsenoside of the anoectochilus formosanus is detected by using a high performance liquid chromatography. After the ArPHR2 gene is instantaneously over-expressed in the anoectochilus formosanus, the content of kinsenoside is remarkably increased, water loss of the anoectochilus formosanus is relieved, and an important theoretical basis and gene resources are provided for cultivation of a high-quality new variety of the anoectochilus formosanus.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and metabolic regulation technology, specifically involving a PHR transcription factor ArPHR2 of Anoectochilus roxburghii, its encoding gene, and its application. Background Technology

[0002] Golden Thread Lotus ( Anoectochilus roxburghii *Wall.) Lindl.* is a perennial herb belonging to the genus *Anoectochilus* in the family Orchidaceae. Also known as Golden Thread Orchid, Variegated Golden Thread Orchid, and Golden Thread Orchid, it is a rare and precious traditional Chinese medicine, possessing both medicinal and edible properties. Modern research indicates that *Anoectochilus roxburghii* glycosides are the main active medicinal components, distributed throughout the entire plant, with the highest content in the leaves. These glycosides exhibit pharmacological activities such as anti-liver damage and antioxidant effects, and their efficacy is superior to silymarin. Research on the regulatory mechanism of *Anoectochilus roxburghii* glycoside accumulation will provide theoretical support for improving the quality of *Anoectochilus roxburghii* and stabilizing the quality of the medicinal material.

[0003] The PHR (PHOSPHATE STARVATION RESPONSE) transcription factor family is a key factor regulating phosphorus homeostasis and low-phosphorus response in plants. Especially under phosphorus-deficient conditions, it plays a core regulatory role and synergistically resists related abiotic stresses such as drought, hormone stress, and salt stress. It primarily regulates plant growth, development, and secondary metabolism through phosphorus-responsive stress mechanisms. PHR belongs to the MYB superfamily and contains a conserved MYB-CC domain composed of 50-53 amino acids in a helix-random coil-helix configuration. Studies have shown that PHR family genes are widely involved in plant root differentiation, phosphorus signal transduction, and other growth and development processes. PHR genes can interact with signaling pathways such as strigolactones (SLs), cytokinins, and sugar metabolism, synergistically regulating phosphorus starvation response and growth. For example, under low-phosphorus conditions, OsPHR2 promotes strigolactone synthesis and enhances phosphorus uptake by directly activating the expression of NSP1 and NSP2. Simultaneously, it can respond to abiotic stresses and interact with environmental microorganisms. In Pinellia ternata, PtPHR1 can respond to phosphorus starvation, inducing benzoic acid production and realizing the biosynthesis of phenylpropanoid alkaloids. The PHR gene has also been reported to participate in the symbiosis between plants and arbuscular mycorrhizal fungi (AMF), promoting symbiotic colonization and enhancing symbiotic phosphorus uptake under low phosphorus conditions. Therefore, rationally utilizing the regulatory function of the PHR gene, through direct or indirect regulation of downstream target gene networks, can improve plant secondary metabolism. Currently, whether the PHR transcription factor participates in phosphorus metabolism and regulates the biosynthesis of active substances such as roximate in Anoectochilus roxburghii remains unclear. Therefore, by mining functional genes, technical support can be provided for increasing roximate content and achieving genetic engineering breeding. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a transcription factor ArPHR2 from *Anoectochilus roxburghii*, its encoding gene, and its application.

[0005] The amino acid sequence of ArPHR2, the PHR transcription factor of Anoectochilus roxburghii described in this invention, is shown in SEQ ID NO.2.

[0006] The present invention also provides the application of the above-mentioned Anoectochilus roxburghii PHR transcription factor ArPHR2 in alleviating water loss and increasing the content of Anoectochilus roxburghii glycosides.

[0007] The present invention also provides a coding gene encoding the above-mentioned Anoectochilus roxburghii PHR transcription factor ArPHR2, the coding gene having the nucleotide sequence shown in SEQ ID NO.1.

[0008] The aforementioned coding gene contains a MYB-CC domain.

[0009] The aforementioned encoded genes were obtained through cloning in Anoectochilus roxburghii and / or artificial synthesis.

[0010] The present invention also provides a plant transient overexpression vector containing the encoding gene of the above-mentioned Anoectochilus roxburghii PHR transcription factor ArPHR2.

[0011] The transient overexpression vector for the above-mentioned plants was pHB-ArPHR2-YFP.

[0012] The present invention also provides the application of the gene encoding the above-mentioned PHR transcription factor ArPHR2 of Anoectochilus roxburghii in alleviating water loss and increasing the content of Anoectochilus roxburghii glycosides.

[0013] Beneficial effects: This invention clones a PHR transcription factor ArPHR2 from *Anoectochilus roxburghii*. ArPHR2 The gene is highly expressed in leaves and flowers and is located in the cell nucleus. Transient overexpression of the gene in Anoectochilus roxburghii leaves can increase the content of Anoectochilus roxburghii glycosides and alleviate water loss in Anoectochilus roxburghii, which is of great significance for realizing genetic engineering breeding. Attached Figure Description

[0014] Figure 1 for ArPHR2 Image of PCR amplification results of the gene; Figure 2 This is a diagram showing the amino acid sequence alignment analysis of homologous proteins of transcription factor ArPHR2; Figure 3 for ArPHR2 A diagram illustrating gene expression patterns in different tissues; Figure 4 Subcellular localization map of transcription factor ArPHR2 in tobacco epidermal cells; Figure 5 for ArPHR2Response diagram of genes in transient overexpression of Anoectochilus roxburghii; Figure 6 For overexpression ArPHR2 Figure showing the effect of water loss phenotype on detached leaves of Anoectochilus roxburghii; Figure 7 For overexpression ArPHR2 The effect of detached leaves of Anoectochilus roxburghii on water loss rate; Figure 8 for ArPHR2 A graph showing the changes in roximate glycosides during transient overexpression of the gene in *Anoectochilus roximate*. Detailed Implementation

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but the present invention is not limited to the embodiments.

[0016] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (4th Edition), or as recommended in the reagent instructions.

[0017] Example 1: ArPHR2 Gene cloning and sequence analysis Plant material preparation: *Anoectochilus roxburghii* tissue culture seedlings (variety "ArHX187635"), seedling height 18-20 cm. The cultivation medium is a 1:1 mixture of Pinsbury peat moss and vermiculite. The cultivation containers are black square pots with dimensions of 6 cm:8.5 cm:8 cm (length:width:height). After washing the *Anoectochilus roxburghii* tissue culture seedlings, they are evenly spread on a tray lined with double-layered filter paper. After the plants have dried, they are transplanted into the black square pots filled with the mixed medium. The cultivation is carried out in a greenhouse (25℃, 60% humidity, light intensity approximately 50 μmol / m²). -2 s -1 The leaves were cultured in a light-dark cycle of 16 h and 8 h. After 2 weeks of culture, 0.2 g of the leaves were taken, immediately wrapped in aluminum foil, frozen in liquid nitrogen for 15 minutes, and then stored in an ultra-low temperature freezer at -80°C.

[0018] Total RNA extraction was performed using the SteadyPure Universal RNA Extraction Kit (Accurate Biology, AG21019, China). cDNA was synthesized via reverse transcription using the Evo M-MLV Reverse Transcription Premixed Kit (Accurate Biology, AG21019, China).

[0019] Primer design and synthesis: using *Anoectochilus roxburghii* (a type of orchid) as a primer. Anoectochilus roxburghii Among the genes with high homology to this gene PHRBased on the gene, primers for this gene sequence were designed and synthesized at Zhejiang Youkang Biotechnology Co., Ltd. Using cDNA from the leaves of *Anoectochilus roxburghii* (variety "ArHX187635") as a template, the upstream and downstream primer sequences were designed as follows: F- ArPHR2 :5'-ATGAGCAATTCAGATTTGAAGA -3',R- ArPHR2 : 5'-TCACTCCGATGGCGTATT -3', PCR amplification was performed according to the following reaction system and conditions: 25 μL system containing 12.5 μL PrimeSTAR Max Premix DNAPolymerase (purchased from TaKaRa), 10 μmol / L primer F- ArPHR2 and primer R- ArPHR2 Add 1 μL of each of the following: cDNA 2 μL, and ddH2O to a final volume of 25 μL. Reaction conditions: pre-denaturation 98℃ for 3 min; 98℃ for 10 s; 58℃ for 15 s; 72℃ for 1 min; 34 cycles; extension at 72℃ for 5 min. ArPHR2 PCR amplification results of gene sequences as follows Figure 1 As shown. After gel electrophoresis of the amplified fragments, the products were recovered and ligated into the cloning vector pMD18-T (purchased from TaKaRa) using Accurate Taq Master Mix DNA Polymerase (purchased from Hunan Aike Rui Biotechnology Co., Ltd.). The ligation was then transformed into E. coli DH5α (purchased from Shanghai Weidi Biotechnology Co., Ltd.). After successful transformation, the samples were sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. ArPHR2 The full-length open reading frame sequence of the gene is 1500 bp, and the detailed sequence results are shown in SEQ ID NO.1.

[0020] Example 2: Amino acid sequence alignment analysis of homologous proteins of transcription factor ArPHR2 according to ArPHR2 The open reading frame sequence of the gene deduced that transcription factor ArPHR2 is a protein composed of 499 amino acid residues, the detailed sequence of which is shown in SEQ ID NO.2. Using DNAMAN software, the amino acid sequences of transcription factor ArPHR2 were compared with homologous proteins in Dendrobium officinale (DcPHR2) and rice (OsPHR2), and the results are as follows. Figure 2 As shown, the transcription factor ArPHR2 was found to contain conserved MYB-DNA-binding and MYB-CC domains.

[0021] Example 3: ArPHR2 Analysis of relative gene expression levels in different tissues The preparation process of plant materials is the same as in Example 1. Take 0.2g each of the root, stem, leaf and flower of Anoectochilus roxburghii, freeze in liquid nitrogen for 15 minutes and then store in an ultra-low temperature freezer at -80℃.

[0022] The methods for extracting total RNA from different tissues and synthesizing cDNA were the same as in Example 1. ArPHR2 Design specific primers qRTF- for the gene's cDNA sequence ArPHR2 :5'-TGGGTAGCGATGATTGGA-3', qRTR- ArPHR2 :5'-CGAAGGTAACTGTTGATGGG-3', Golden Thread Lotus ArAction-α This is an internal reference gene. The upstream and downstream primer sequences are: F- ArActin-α :5'-GCTAGTGGCCGTACAACTGG-3',R- ArActin-α The PCR reaction mixture was 5'-GCCAGCAAGGTCCAATCGAA-3'. Quantitative detection was performed using a Bio-Rad real-time quantitative PCR instrument. The PCR reaction system consisted of: 10 μL 2×SYBR qPCR SuperMix (purchased from TransGen), primer F- ArActin - α and primer R- ArActin - α 0.4 μL of each of the following: cDNA 2 μL, water to a total volume of 20 μL. The reaction program was 95℃ for 30 s; 95℃ for 5 s; 58℃ for 30 s; 39 cycles; 95℃ for 5 s; 65℃ for 5 s. Each treatment was repeated 3 times biologically and 3 times technically. Two [reaction methods were employed]. −ΔΔCT The data was analyzed using SPSS for statistical analysis and GraphPad Prism for plotting. ArPHR2 Gene expression patterns, such as Figure 3 As shown , It can be seen that, ArPHR2 The expression level was highest in the leaves of Anoectochilus roxburghii, followed by the flowers.

[0023] Example 4: Subcellular localization of transcription factor ArPHR2 in tobacco epidermal cells.

[0024] Construction of plant expression vectors: using ArPHR2 Using the -pMD18-T plasmid as a template, a design with... BamH I, SpePrimers with specific restriction enzyme sites were used for PCR amplification and ligated into the pMD18-T vector. The double enzyme digestion products were recovered from the gel and linked to the linearized pHB-YFP vector with the same restriction enzyme sites to obtain the plant expression vector pHB-ArPHR2-YFP (abbreviated as ArPHR2-YFP). This vector was transformed into Escherichia coli, and after verification by PCR in the bacterial culture, the plasmid was extracted and transformed into Agrobacterium competent cells GV3101.

[0025] Transient conversion of tobacco leaves: Agrobacterium monoclonal strains containing ArPHR2-YFP and pHB-YFP (empty vector) were selected and added to a solution containing 50 mg L... -1 Kan and 25 mg L -1 In Rif LB liquid medium, incubate at 28℃ on a shaker at 200 rpm until the OD600 reaches approximately 1.2. Centrifuge at 5000 rpm for 15 min, collect the bacteria, resuspend in MS liquid medium, adjust the OD600 to 0.6, and simultaneously add acetylsyleugenone (AS) and MES buffer (final concentrations of 0.2 mM and 10 mM, respectively), mix gently, and incubate in the dark at room temperature for at least 3 h. Inject the bacterial suspension into the underside of tobacco leaves using a 1 mL disposable syringe, incubate in the dark for 48 h, and cut 1 cm sections. 2 The injected tobacco leaves were observed under a laser confocal microscope, and the results are as follows: Figure 4 As shown, the transcription factor ArPHR2 is located in the cell nucleus.

[0026] Example 5: ArPHR2 Gene response in transient overexpression of Anoectochilus roxburghii The vector construction process is the same as in Example 4. In this example, the plant transient overexpression vector is pHB-YFP.

[0027] Transformation of *Anoectochilus roxburghii*: Recombinant vectors containing ArPHR2-YFP and Agrobacterium strain GV3101 containing pHB-YFP (empty vector) were added to LB liquid medium containing antibiotics Kan50 and Rif100, respectively, and cultured on a shaker at 28℃ and 200 rpm until the OD600 reached 0.6. The bacterial solution was then applied to prepared *Anoectochilus roxburghii* leaves, marked, and incubated in the dark for 3 days before sampling. ArPHR2 0.2 g each of leaves with transient gene overexpression and unloaded leaves were frozen in liquid nitrogen and then stored at -80°C. The extraction of total RNA and the synthesis of cDNA for different treatments were performed using the same methods as in Example 1. Quantitative detection was performed using a Bio-Rad real-time quantitative PCR instrument. The primer sequence and relative expression level detection procedures were the same as in Example 3. The results are as follows: Figure 5 As shown, compared to no load, ArPHR2 The transcriptional level of the gene was higher in the leaves of Anoectochilus roxburghii with transient overexpression, proving that the gene was successfully expressed in Anoectochilus roxburghii.

[0028] Example 6: ArPHR2 The role of genes in drought stress during transient overexpression in Anoectochilus roxburghii The carrier construction process is the same as in Example 4.

[0029] Transformation of *Anoectochilus roxburghii*: *Agrobacterium* strain GV3101 containing the recombinant vector ArPHR2-YFP and the empty vector pHB-YFP (EV) was added to LB liquid medium containing antibiotics Kan50 and Rif100, respectively, and cultured on a shaker at 28℃ and 200 rpm until the OD600 reached 0.6. The prepared *Anoectochilus roxburghii* leaves were infected with the bacterial solution, marked, and treated in the dark for 3 days. Samples were taken and weighed at 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, and 48 h. The water loss rate was calculated using the formula (original weight - weight at different time points) / original weight * 100%, and a curve was plotted using Graphpad Prism. Figure 7 As shown. Using a hole punch, holes were drilled from different... ArPHR2 Three identical discs were taken from leaves with transient gene overexpression and three from unloaded leaves for water loss phenotype observation at 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, and 48 h. The results are as follows: Figure 6 As shown. By Figure 6 , Figure 7 It can be seen that overexpression ArPHR2 It reduced the water loss rate of Anoectochilus roxburghii, thus alleviating drought stress.

[0030] Example 7: ArPHR2 Extraction and detection of roximate from transient gene overexpression Prepare normal phosphorus and low phosphorus Hoglandian nutrient solutions. Purchase Hoglandian nutrient solution powder (phosphorus-free and calcium nitrate-free) from Haibo Biotechnology. Dissolve 1.215g of Hoglandian nutrient solution powder (phosphorus-free and calcium nitrate-free) in 1000mL of distilled water by heating and autoclaving at 115℃ for 20 minutes. For normal phosphorus Hoglandian nutrient solution, add 0.136g / L of KH2PO4. For low phosphorus Hoglandian nutrient solution, add 0.00136g / L of KH2PO4 and 0.07376g / L of KCl. Adjust the pH of the prepared nutrient solution to 5.80. The method for transforming *Anoectochilus roxburghii* is the same as in Example 5. After infecting the *Anoectochilus roxburghii* leaves, mark them. After dark treatment and cultivation under normal phosphorus and low phosphorus conditions for 3 days, collect fresh *Anoectochilus roxburghii* (dried at 60℃ until moisture content ≤13.0%) or dried products for later use. Accurately weigh 0.2000 g of *Anoectochilus roxburghii* sample into a stoppered Erlenmeyer flask, add 50.0 mL of 70% ethanol solution, seal tightly, weigh, shake well, and let stand at room temperature for 15 min. Then, extract by sonication (500 W, 40 kHz) for 10 min. After cooling, weigh again, replenish the lost weight with 70% ethanol solution, shake well, and filter through a 0.45 μm filter membrane to obtain the test solution. Blank sample solution: 70% ethanol solution. The extracted test solution and blank sample solution were used to determine the content of *Anoectochilus roxburghii* glycosides using a Waters 2695 series high-performance liquid chromatography system from Shimadzu Instruments (Suzhou) Co., Ltd. The reference operating conditions for liquid chromatography are as follows: Column: Agilent Zorbax NH2 column (4.6 mm × 250 mm, 5 μm); Mobile phase: acetonitrile:water = 85:15 (volume ratio); Flow rate: 1.0 mL / min; Column temperature: 25℃; Nebulizer temperature: 70℃; Injection volume: 10 µL; N2 flow rate: 1.5 L / min; Gain: 4; Detection wavelength: 215 nm, with real-time peak intensity monitoring. After the determination, a standard working curve was plotted based on the peak area. The standard working solutions of *Anoectochilus roxburghii* glycosides were injected into the HPLC system for determination, and the peak areas were recorded. The standard working curve was plotted with the logarithm of concentration (logC) on the x-axis and the logarithm of peak area (logA) on the y-axis.

[0031] The results are as follows Figure 8 As shown, compared to no-load, in ArPHR2 Transient overexpression of the gene resulted in higher levels of anoectochilin, and the levels were higher under low phosphorus conditions than under normal phosphorus conditions.

[0032] Unless otherwise specified, all technologies mentioned above refer to existing technologies.

[0033] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A transcription factor ArPHR2 from *Anoectochilus roxburghii* PHR, characterized in that, The amino acid sequence of the transcription factor ArPHR2 is shown in SEQ ID NO.

2.

2. The application of the ArPHR2 transcription factor of Anoectochilus roxburghii as described in claim 1 in alleviating water loss and increasing the content of Anoectochilus roxburghii glycosides.

3. A gene encoding the ArPHR2 transcription factor of *Anoectochilus roxburghii* as described in claim 1, characterized in that, The encoding gene is a nucleotide sequence as shown in SEQ ID NO.

1.

4. The gene encoding the ArPHR2 transcription factor of *Anoectochilus roxburghii* PHR according to claim 3, characterized in that, The coding gene contains a MYB-CC domain.

5. A plant transient overexpression vector, characterized in that, It includes the gene encoding the ArPHR2 transcription factor of Anoectochilus roxburghii as described in claim 3.

6. The plant transient overexpression vector according to claim 5, characterized in that, The transient overexpression vector for the plant was pHB-ArPHR2-YFP.

7. The application of the gene encoding ArPHR2, the PHR transcription factor of Anoectochilus roxburghii as described in claim 3, in alleviating water loss and increasing the content of Anoectochilus roxburghii glycosides.

Images