Anoectochilus roxburghii WOX transcription factor ArWOX8, coding gene and application thereof

Abstract

The invention discloses an anoectochilus formosanus WOX transcription factor ArWOX8 as well as a coding gene and application thereof. The amino acid sequence of the transcription factor ArWOX8 is shown as SEQ ID NO.2, and the nucleotide sequence of the transcription factor ArWOX8 is shown as SEQ ID NO.1; the invention also provides application of the WOX transcription factor protein and the coding gene thereof in increasing the content of kinsenoside. ArWOX8 is overexpressed in anoectochilus formosanus by using a genetic engineering technology, and the main component of anoectochilus formosanus is detected to be kinsenoside by high performance liquid chromatography; after the ArWOX8 is instantaneously over-expressed in the anoectochilus formosanus, the content of kinsenoside is remarkably increased, and an important theoretical basis and a gene resource are provided for cultivation of a high-quality new variety of the anoectochilus formosanus.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering and metabolic substance regulation, and specifically relates to a roxburghii WOX transcription factor ArWOX8, a coding gene and an application thereof in increasing the roxburghii glycoside content. Background Art

[0002] Golden thread lotus ( Anoectochilus roxburghii (Wall.) Lindl. is a perennial herbaceous plant of the genus Anoectochilus in the Orchidaceae family. The entire plant can be used as medicine, with a mild, sweet flavor. Anoectochilus roxburghii is a rare and precious Chinese medicinal herb, known as the "King of Medicine." Modern pharmacological research indicates that its main component, anoectochilin, possesses hepatoprotective and other benefits. Research on the regulatory mechanisms of anoectochilin accumulation has important theoretical and practical significance for the selection and breeding of superior Anoectochilus roxburghii varieties and the improvement of herbal quality.

[0003] The WUSCHEL-related homeobox (WOX) transcription factor family is a plant-specific transcription factor that plays an important role in maintaining stem cell stability, establishing embryonic development patterns, building leaf polarity, and developing ovules and anthers. It is also a transcription factor necessary for regulating biological processes such as plant environmental stress responses. It belongs to the Homeobox (HOX) superfamily and contains a conserved helix-loop-helix-turn-helix domain consisting of 60 to 65 amino acids. Studies have shown that WOX family genes are widely involved in the growth and development of plant roots, stems, leaves, flowers, fruits, seeds, embryonic development, and other processes. For example, the DoWOX4 protein of Dendrobium officinale can directly bind to and activate key genes in the polysaccharide synthesis pathway. DoGMP1 However, in Anoectochilus roxburghii, it remains unclear whether WOX transcription factors regulate the biosynthesis of active ingredients. Therefore, it is necessary to increase the content of anoectochilin through genetic engineering to provide technical support for genetic engineering breeding. Summary of the Invention

[0004] In order to solve the problem that there is currently no method for increasing the content of roxburghii glycosides through genetic modification, the present invention provides a roxburghii WOX transcription factor ArWOX8, an encoding gene and an application thereof.

[0005] The present invention provides a roxburghii WOX transcription factor ArWOX8. The amino acid sequence of the transcription factor ArWOX8 is shown in SEQ ID NO.2.

[0006] The present invention provides a gene encoding the above-mentioned Anoectochilus roxburghii WOX transcription factor ArWOX8, and the above-mentioned gene encoding is a nucleic acid sequence shown as SEQ ID NO.1.

[0007] Preferably, the above encoding gene contains a WUS domain.

[0008] Preferably, the above encoding gene is obtained by cloning and / or artificial synthesis in Anoectochilus roxburghii.

[0009] The present invention provides a plant transient overexpression vector comprising the coding gene of the above-mentioned Anoectochilus roxburghii WOX transcription factor ArWOX8.

[0010] The present invention provides an application of the above-mentioned roxburghii WOX transcription factor ArWOX8 or the encoding gene of the above-mentioned roxburghii WOX transcription factor ArWOX8 in increasing the content of anoectochiloside.

[0011] Beneficial effects: 1) The present invention cloned a WOX transcription factor ArWOX8 from Anoectochilus roxburghii. ArWOX8 It is highly expressed in roots and leaves and localized in the cell nucleus. Its transient overexpression in roxburghii leaves can significantly increase the content of roxburghii glycoside; 2) The present invention provides a basis for its effective application, which is of great significance for increasing the content of roxburghii glycoside, especially for realizing genetic engineering breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 for ArWOX8 PCR amplification results of the gene; Figure 2 This is the amino acid sequence alignment analysis of ArWOX8 homologous proteins; Figure 3 for ArWOX8 Diagram of gene expression patterns in different tissues; Figure 4 This is the subcellular localization map of ArWOX8 in tobacco epidermal cells; Figure 5 for ArWOX8 Response diagram in transient overexpression of Anoectochilus roxburghii; Figure 6 for ArWOX8 Figure 3. Changes in anoectochiloside content during transient overexpression of Anoectochilus roxburghii. DETAILED DESCRIPTION

[0013] The technical solutions of the present invention are described in detail below through examples, but the protection scope of the present invention is not limited to the examples.

[0014] For experimental methods in the following examples where specific conditions are not specified, they were generally used according to conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (fourth edition), or the conditions recommended in the reagent instructions.

[0015] Example 1: ArWOX8 Gene cloning and sequence analysis Plant material preparation: Anoectochilus roxburghii (cultivar "ArHX187635"), 15-20 cm tall. Wash the Anoectochilus roxburghii seedlings, spread them evenly on a tray lined with double-layer filter paper, air-dry, and transplant them into peat pots. In a greenhouse (25°C, 60% humidity, and a light intensity of approximately 50 μmol m -2 s -1 After 2 weeks of culture, 0.2 g of leaves were collected, immediately wrapped in tin foil, frozen in liquid nitrogen for 15 minutes, and then stored in a -80°C ultra-low temperature freezer.

[0016] Total RNA was extracted using the SteadyPure Universal RNA Extraction Kit (Accurate Biology, AG21019, China), and cDNA was synthesized by reverse transcription using the Evo M-MLV Mix Kit with gDNA Clean for qPCR (Accurate Biology, AG21019, China).

[0017] Primer design and synthesis: Anoectochilus roxburghii ) with higher homology to this gene WOX Based on the gene, primers for the gene sequence were designed and synthesized at Youkang Biotechnology Co., Ltd. Using cDNA from the leaves of Anoectochilus roxburghii (cultivar "ArHX187635") as the template, the upstream and downstream primer sequences were: F-primer: 5'-ATGGAGTGGAAGAAGGATGA -3', and R-primer: 5'-CTAAAGCTCCCAGGAGTTATCC -3'. PCR amplification was performed using the following reaction system and conditions: 25 μL system containing 12.5 μL LA Taq DNA Polymerase (purchased from TaKaRa), 1 μL each of 10 μmol / L primers F and R, 2 μL cDNA, and ddH2O to 25 μL. Reaction conditions: initial denaturation at 98°C for 3 min; 34 cycles of 98°C for 10 s, 60°C for 15 s, and 72°C for 1 min; and extension at 72°C for 5 min. ArWOX8 The PCR amplification results of the gene sequence are as follows Figure 1 As shown, the band size is approximately 700 bp, which is in line with expectations. The amplified fragment was recovered and ligated into the cloning vector pMD18-T (purchased from TaKaRa), transformed into Escherichia coli DH5α (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and sent to Youkang Bio for sequencing after identification. ArWOX8 The full length of the gene open reading frame sequence is 762 bp, and the detailed sequence results are shown in SEQ ID NO.1.

[0018] Example 2: Amino acid sequence alignment analysis of ArWOX8 homologous proteins Based on the open reading frame sequence of ArWOX8, it was deduced that ArWOX8 is a protein composed of 253 amino acid residues. The detailed sequence is shown in SEQ ID NO. 2. The amino acid sequence of ArWOX8 and its homologous proteins in Dendrobium officinale and Phalaenopsis were aligned using DNAMAN software. The results are as follows: Figure 2 As shown. Figure 2 It can be found that ArWOX8 contains a conserved Homologous domain, an Acid domain and a WUS domain, which are typical WOX family domains.

[0019] Example 3: ArWOX8 Relative expression analysis in different tissues The preparation process of plant materials was the same as in Example 1. 0.2 g each of the root, stem, leaf, and flower of Anoectochilus roxburghii were taken, frozen in liquid nitrogen for 15 minutes, and then stored in a -80°C ultra-low temperature freezer.

[0020] The extraction of total RNA from different tissues and the synthesis of cDNA were the same as in Example 1. ArWOX8 Design specific primers F-qRT- ArWOX8 :5'-TGGAAGAAGGATGACGGAG-3',R-qRT- ArWOX8 :5'-ATCTGACGACGGAGAACCT-3'; ArActin-α is the internal reference gene, and the upstream and downstream primer sequences are: F- ArActin-α :5'-GCTAGTGGCCGTACAACTGG-3',R- ArActin-α : 5'-GCCAGCAAGGTCCAATCGAA-3'. Quantitative detection was performed on a Bio-Rad real-time quantitative PCR instrument. The PCR reaction system consisted of 10 μL 2×SYBR qPCR SuperMix (purchased from Quanshijin), 0.4 μL each of the upstream and downstream primers, 2 μL of cDNA, and water to a total volume of 20 μL. The reaction program was 95°C for 30 s; 95°C for 5 s, 58°C for 30 s, 39 cycles, 95°C for 5 s, and 65°C for 5 s. Each treatment was replicated three times biologically and three times technically. Two −ΔΔCT The data were analyzed using SPSS, and the graphs were drawn using Graphpad Prism. ArWOX8 Expression patterns such as Figure 3 As shown in the figure, the expression level is highest in roots and leaves, followed by stems, and lowest in flowers. This expression pattern is basically consistent with the distribution of anoectochiloside content.

[0021] Example 4: Subcellular localization of ArWOX8 in tobacco epidermal cells.

[0022] Plant expression vector construction: ArWOX8 -pMD18-T plasmid was used as a template, and primers with BamHI and SpeI were designed for PCR amplification and ligated to the pMD18-T vector. After the double enzyme digestion product was recovered on gel, it was linked to the linearized pHB-YFP vector with the same restriction enzyme cutting sites to obtain pHB- ArWOX8 -YFP recombinant vector was transformed into Escherichia coli, and the plasmid was extracted after bacterial liquid PCR verification and transformed into Agrobacterium competent GV3101.

[0023] Transient transformation of tobacco leaves: Pick out the leaves containing pHB- ArWOX8 -YFP and pHB-YFP (empty) Agrobacterium monoclonal clones were added to LB liquid medium containing Kan50 and Rif100, cultured on a shaker at 28°C at 200 rpm until OD600 was about 1.2, centrifuged at 5000 rpm for 15 min, and resuspended in MS liquid medium after collection, adjusted to OD600 of 0.6, and AS and MES (final concentrations of 0.2 mM and 10 mM, respectively) were added, gently mixed, and placed in the dark at room temperature for more than 3 hours. Use a 1 mL disposable syringe to inject the bacterial solution into the back of tobacco leaves, culture in the dark for 48 hours, and cut 1 cm 2 The injected tobacco leaves were observed under a laser confocal microscope. Figure 4 As shown, ArWOX8 is localized in the cell nucleus, which is consistent with the characteristics of a transcription factor.

[0024] Example 5: ArWOX8 Response in transient overexpression of Anoectochilus roxburghii The vector construction process is the same as that in Example 4. In this example, the plant transient overexpression vector is pHB-YFP.

[0025] Transformation of Anoectochilus roxburghii: Transform the ArWOX8- The recombinant vector of YFP and the Agrobacterium strain GV3101 of pHB-YFP (empty vector) were added to LB liquid culture medium containing antibiotics Kan50 and Rif100, respectively, and cultured on a shaker at 28°C at 200 rpm until OD600 reached 0.6. The prepared leaves of golden thread lotus were infected with the bacterial liquid, marked, and cultured in the dark for 3 days before sampling. 0.2 g of ArWOX8 transient overexpression and empty vector were frozen with liquid nitrogen and stored in an ultra-low temperature refrigerator at -80°C. The extraction of total RNA from different treatments and the synthesis of cDNA were the same as in Example 1. Quantitative detection was performed on a Bio-Rad real-time quantitative PCR instrument. The primer sequence and relative expression detection process were the same as in Example 3. The results are shown in FIG. Figure 5 As shown, compared with no-load, ArWOX8 The transcription level was significantly increased in the leaves of transiently overexpressed Anoectochilus roxburghii.

[0026] Example 6: ArWOX8 Extraction and Detection of Anoectochiloside in Transient Overexpression The method for transforming Anoectochilus roxburghii is the same as in Example 5. After infection, mark the leaves of Anoectochilus roxburghii, incubate in the dark for 3 days, and then collect fresh Anoectochilus roxburghii (dried at 60°C to a moisture content of ≤ 13.0%) or dried samples, pulverize, and pass through a 60-mesh sieve for later use. Accurately weigh 0.2 g (accurate to 0.001 g) of Anoectochilus roxburghii sample into a stoppered Erlenmeyer flask. Add 50.0 mL of 70% ethanol solution, stopper tightly, and weigh the sample. Shake thoroughly, incubate at room temperature for 15 minutes, and then extract using ultrasound (500 W, 40 kHz) for 10 minutes. After cooling, weigh the sample, adjust the weight, and add 70% ethanol solution to compensate for the loss. Shake thoroughly, filter through a 0.45 μm filter to obtain the test solution. Blank sample solution: 70% ethanol solution. The extracted test solution and blank sample solution were analyzed for anoectochilin content using a Waters 2695 series HPLC system from Shimadzu Instruments (Suzhou) Co., Ltd. The reference working conditions for liquid chromatography are as follows: chromatographic 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°C; nebulizer temperature: 70°C; injection volume: 10 μL; N2 flow rate: 1.5 L / min; gain value: 4; detection wavelength: 215 nm, and real-time monitoring of peak intensity. After the determination was completed, a standard working curve was drawn based on the peak area. The standard series of working solutions of anoectin were injected into the high-performance liquid chromatograph for determination, and the chromatographic peak areas were recorded. The standard working curve was drawn with the logarithm of the concentration (logC) as the horizontal axis and the logarithm of the peak area (logA) as the vertical axis. The results are shown in Figure 2. Figure 6 As shown, compared with no load, ArWOX 8 The content of anoectochiloside in transiently overexpressed gene was significantly increased.

[0027] The technologies not specifically mentioned above are all referenced to the existing technologies.

[0028] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A WOX transcription factor ArWOX8 of Anoectochilus roxburghii, characterized in that The amino acid sequence of the transcription factor ArWOX8 is shown in SEQ ID NO.

2.

2. A gene encoding the Anoectochilus roxburghii WOX transcription factor ArWOX8 according to claim 1, characterized in that: The coding gene is the nucleic acid sequence shown in SEQ ID NO.

1.

3. The coding gene of the Anoectochilus roxburghii WOX transcription factor ArWOX8 according to claim 2, characterized in that The encoding gene contains a WUS domain.

4. The coding gene of the Anoectochilus roxburghii WOX transcription factor ArWOX8 according to claim 2, characterized in that The coding gene is obtained by cloning and / or artificial synthesis in Anoectochilus roxburghii.

5. A plant transient overexpression vector, characterized in that: A gene encoding the roxburghii WOX transcription factor ArWOX8 according to any one of claims 2 to 4.

6. Use of the roxburghii WOX transcription factor ArWOX8 according to claim 1 or the gene encoding the roxburghii WOX transcription factor ArWOX8 according to any one of claims 2 to 4 in increasing the content of anoectochiloside.

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