Schizonepeta transcription factor gene S19891 as well as encoded product and application thereof

Abstract

The invention relates to a genetic engineering body, and particularly discloses a schizonepeta transcription factor gene St19891 as well as a product coded by the schizonepeta transcription factor gene St19891 and application of the The nucleotide sequence of the gene is as shown in SEQ ID NO.1, the amino acid sequence of a coded product is as shown in SEQ ID NO.2, a schizonepeta plant in which a schizonepeta transcription factor St19891 is inhibited is obtained by cloning and amplifying the schizonepeta transcription factor gene St19891, constructing a gene silencing vector and inhibiting in a schizonepeta body, and the schizonepeta transcription factor gene St19891 has the advantages that the schizonepeta transcription factor gene St19891 has the inhibition effect on the schizonepeta plant; the invention solves the problem that the transcription factors related to monoterpene synthesis regulation in the schizonepeta are not separated and identified in the existing research, and can provide a basis for genetic engineering for improving the schizonepeta and increasing the content of effective components such as pulegone in the schizonepeta.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a schizonepeta transcription factor gene St19891 and a product encoded by the gene and application thereof. Background Art

[0002] Schizonepeta tenuifolia is a plant of the Lamiaceae family and the genus Nepeta, an annual herb. It was first called "false Su" and recorded in "Shennong's Herbal Classic", and the name "Schizonepeta" was first recorded in "Wu Pu's Herbal Classic". It has the effects of dispelling wind, clearing rashes, and eliminating sores. It is commonly used for colds, headaches, measles, rubella, and the initial stage of sores. Schizonepeta volatile oil has antiviral, anti-inflammatory, analgesic, anti-tumor, immunomodulatory, antibacterial, and hemostatic pharmacological effects. It is mainly composed of monoterpenes and sesquiterpenoids, such as pulegone, menthone, caryophyllene, etc. The "Chinese Pharmacopoeia" (Volume 1 of the 2020 edition) stipulates that the content of volatile oil in Schizonepeta medicinal materials shall not be less than 0.60%, and the content of pulegone in the volatile oil of Schizonepeta medicinal materials shall not be less than 0.020%. The monoterpene compound pulegone is a key component of the Schizonepeta medicinal material index, with anti-inflammatory, insecticidal, antipyretic and analgesic effects. Exploring the biosynthesis and regulatory mechanism of pulegone in Schizonepeta tenuifolia is of great significance for revealing the quality changes of Schizonepeta tenuifolia medicinal materials and the screening of high-quality production areas and germplasm seedlings.

[0003] Using geranyl pyrophosphate (GPP) synthesized by the MEP / MVP pathway of terpenoid synthesis as a substrate, limonene is produced under the catalysis of limonene synthase. Limonene is further hydroxylated and dehydrogenated to give isomenthenone, which is further reduced to isopulegone, and pulegone is obtained under the action of isomerase. The biosynthetic pathway of pulegone in Nepeta tenuifolia was further verified based on the VIGS gene silencing system. However, the transcription factors involved in regulating the synthesis of pulegone, a menthane monoterpene, in Nepeta tenuifolia have not yet been isolated and identified. Summary of the invention

[0004] The purpose of the present invention is to solve the problem that the transcription factors regulating the monoterpene synthesis pathway in Nepeta have not been separated and identified in the existing research, and provide Nepeta transcription factor gene St19891 and the products encoded by it and applications.

[0005] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses the nucleotide sequence of the Nepeta tenuifolia transcription factor gene St19891 as shown in SEQ ID NO.1.

[0006] The present invention also discloses a product encoded by the above-mentioned schizonepeta transcription factor gene St19891, and the amino acid sequence of the encoded product is shown in SEQ ID NO. 2. The product is RNA, polypeptide or protein.

[0007] The invention also discloses the above-mentioned schizonepeta transcription factor gene St19891 specific primers, including an upstream primer: 19891-F: 5'-ACAATAATCATCATCACGGACAGC-3'; and a downstream primer: 19891-R: 5'-CGCCTCATCTTCCCGCTT-3'.

[0008] The invention also discloses a gene silencing vector comprising the schizonepeta transcription factor gene St19891, wherein the expression vector is pTRV2.

[0009] The present invention also discloses the use of the above-mentioned nepeta transcription factor gene St19891 in regulating monoterpenoid compounds, including transferring the nepeta transcription factor gene St19891 into cells, inhibiting the expression of the nepeta transcription factor gene St19891 in the host, and the nepeta transcription factor gene St19891 inhibiting the synthesis of monoterpenoid compounds in nepeta. The monoterpenoid compounds include pulegone and limonene.

[0010] Beneficial effects: Compared with the prior art, the schizonepeta transcription factor St19891 gene provided by the present invention is cloned and prepared from schizonepeta plants for the first time. The schizonepeta transcription factor St19891 is a key regulatory gene in the schizonepeta monoterpene synthesis pathway, which can be used to regulate the pulegone content of schizonepeta, and the protein can be used to regulate the biosynthesis of schizonepeta monoterpene compounds. The gene provided by the present invention can be used to increase the content of pulegone, a medicinal ingredient in schizonepeta, through genetic engineering technology, providing a research direction for schizonepeta molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is an agarose gel image of the gene silencing fragment of the Nepeta tenuifolia transcription factor gene St19891;

[0012] Figure 2 is the gene silencing efficiency of the Nepeta tenuifolia transcription factor gene St19891;

[0013] Figure 3 The changes of limonene content in Nepeta after silencing of Nepeta transcription factor gene St19891;

[0014] Figure 4 The changes of pulegone content in Nepeta after silencing of Nepeta transcription factor gene St19891;

[0015] Figure 5 The expression changes of genes related to the pulegone biosynthesis pathway after silencing of the Nepeta tenuifolia transcription factor gene St19891; DETAILED DESCRIPTION

[0016] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0017] The technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products. TM Nucleic acid dyes were purchased from Shanghai Biosai Biotechnology Co., Ltd.; GeneJET Gel Extraction Kit was purchased from Thermo Fisher Scientific; 2×RapidTaq Master Mix (P222); FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics–rich) (RC401), HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R312-01 / 02), and DH5α chemically competent cells (C502) were purchased from Novazonic Biotechnology Co., Ltd.; GV3101 competent cells (AC1001) were purchased from Shanghai Weidi Biotechnology Co., Ltd.; restriction endonucleases such as EcoRⅠ (1040A) and BamHⅠ (1010S) were purchased from TaKaRa; sequence primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; other reagents were imported or domestic analytical grade reagents.

[0018] 1. Cloning of the Silence Fragment of the Nepeta Transcription Factor Gene St19891

[0019] The cloning of the St19891 silent fragment used forward primers: upstream primer (sequence such as SEQ ID NO.3): 19891-F: 5'-ACAATAATCATCATCACGGACAGC-3'; downstream primer (sequence such as SEQ ID NO.4): 19891-R: 5'-CGCCTCATCTTCCCGCTT-3'. The full-length sequence of the gene encoding the transcription factor gene St19891 of Nepeta tenuifolia was used as a template for PCR amplification. The amplification system is as follows: 5μL of 2×Rapid Taq Master Mix enzyme, 0.2μL of primers primer-F and primer-R, 1μL of template, and the rest is supplemented with sterile double distilled water. Reaction conditions: 95℃ pre-denaturation for 2min, 95℃ denaturation for 20s, 60℃ annealing for 20s, 72℃ extension for 1min30s, 72℃ extension for 5min after 35 cycles, and 4℃ storage. So far, the cloning of the silent fragment of the Nepeta tenuifolia transcription factor gene St19891 has been obtained.

[0020] 2. Construction of gene silencing vector for the Nepeta tenuifolia transcription factor gene St19891

[0021] The pTRV2 vector was digested with BamHI and EcoRI restriction enzymes. The digestion reaction system was as follows: 40μL pTRV2 vector, 2.5μL BamHI and EcoRI restriction enzymes, and 5μL 10×K buffer. Reaction conditions: digestion at 37℃ for 3 hours, and the linearized pTRV2 vector was obtained. The St191891 gene silencing fragment containing the homology arm sequence of the vector was obtained by synthesizing upstream and downstream primers and amplifying them. The gene silencing vector of the Nepeta transcription factor gene St19891 was constructed by homologous recombination. Reaction system: 4μL St19891 gene silencing fragment containing homology arms, 3μL linearized pTRV2 vector, 2μL 5×CE Buffer, and 1μL Exnase II. Reaction conditions: 37℃ for 30min. The gene silencing vector of Nepeta St19891 was obtained. The gene silencing vector of Nepeta St19891 was transformed into Agrobacterium GV3101 competent cells. The transformation method was as follows: after the competent cells were melted on ice, 5 μL of the gene silencing vector of Nepeta St19891 was added thereto, followed by standing on ice for 5 min, reacting the standing cells in liquid nitrogen for 5 min, immediately transferred to a 37°C water bath for 5 min, standing on ice for 2 min, adding 700 μL of antibiotic-free LB liquid culture medium thereto, and resuscitated at 28°C, 200 rpm for 3 h. The resuscitated cells were spread on LB plates containing 50 mg / L kanamycin and 20 mg / L rifampicin, and cultured at 28°C for 60 h.

[0022] 3. Gene silencing of the transcription factor gene St19891 in Nepeta tenuifolia

[0023] The GV3101 strain transformed with the St19891 gene silencing vector was cultured in 20 mL of LB liquid medium containing 50 mg / L kanamycin at 28 ° C with shaking until the OD600 was about 0.6. The bacterial solution was centrifuged at 5000 rpm for 10 min at room temperature, the culture medium was discarded to collect the bacteria, the bacteria were resuspended in a buffer containing 10 mM MgCl2 and 10 mM MES and the OD600 was adjusted to 0.4, and acetosyringone was added to it to a final concentration of 20 μM. It was left to stand for activation at 25 ° C in the dark for 3 hours. The activated bacterial solution was mixed with the GV3101 bacterial solution containing pTRV1 activated under the same conditions at a ratio of 1:1. The GV3101 bacterial solution transformed with the pTRV2 silencing fragment of the St19891 transcription factor of Nepeta was used as a negative control for further comparative analysis.

[0024] Ten-day-old Nepeta plants were used as infection materials, and the Agrobacterium solution was injected into the Nepeta plants from the back of the leaves using a 1mL syringe. The injected Nepeta was left to stand at 25°C for 24 hours in the dark, and then cultured normally in an incubator for 20 days. The silenced plant leaves were then collected for expression verification and component content determination.

[0025] IV. Verification of silencing efficiency of transcription factor gene St19891 in Nepeta tenuifolia

[0026] The leaves of the silencing plants were quickly frozen in liquid nitrogen and used for RNA extraction. Total RNA was extracted according to the instructions of the plant polysaccharide and polyphenol RNA extraction kit, and the first-strand cDNA library was obtained by reverse transcription using total RNA as a template. QRT-PCR was used to verify the silencing efficiency of the transcription factor St19891 of Nepeta and the changes in the expression of genes related to the biosynthesis pathway of pulegone. The PCR amplification system was: 2×ChamQ Blue Universal SYBR qPCRMaster Mix 5μL, cDNA template 1μL, upstream and downstream primers 0.2μL each, and sterile water was used to make up 10μL. The reaction conditions were: 95℃ pre-denaturation for 30s, 95℃ denaturation for 10s, 60℃ extension for 30s, and 40 cycles; the melting curve was the default program of the instrument. β-actin in Nepeta was used as the internal reference gene, and the relative expression of the gene was calculated using the 2^(-△△CT) method.

[0027] like Figure 2 , Figure 5 As shown, the expression of St19891 gene in plants infected with Nepeta decreased by about 60%, indicating that the St19891 gene in Nepeta was successfully silenced, further indicating that the silenced plants were successfully constructed. In Nepeta plants where Nepeta St19891 was successfully silenced, the gene expression levels of Nepeta menthane monoterpenoid biosynthesis-related genes L3OH (limonene-3-hydroxylase), IPD (isomenthenol dehydrogenase), and IPR (isomenthenone reductase) increased significantly, indicating that the expression level of St19891 gene was negatively correlated with the expression level of genes related to the biosynthesis of menthane monoterpenoids, among which the expression level of L3OH decreased the most, indicating that the St19891 gene may further negatively regulate the biosynthesis of Nepeta monoterpenoid compounds by regulating the expression level of L3OH gene.

[0028] 5. Changes in volatile oil content in plants with silencing of the transcription factor gene St19891 in Nepeta tenuifolia

[0029] Collect the leaves of Nepeta plants after silencing the Nepeta transcription factor St19891. Accurately weigh 1g into a centrifuge tube, add 2 5mm diameter steel balls and n-hexane containing 30ng / mL camphor internal standard. In a tissue grinder, grind at 60HZ for 60s, add an appropriate amount of anhydrous sodium sulfate to the ground sample to remove moisture, and centrifuge at 12000rpm for 5min at room temperature, aspirate the supernatant of the centrifuged sample and filter through a 0.22μm filter membrane. The filtered sample is analyzed by GC. The analysis conditions were as follows: sample injection volume was 1 μL, no splitting, carrier gas was high-purity nitrogen, injection port temperature was 220°C, FID detector temperature was 250°C, chromatographic column model was Agilent 19091S-433-HP-5, 30m×250μm×0.25μm, and program temperature conditions were as follows: 0°C for 3 min, heating to 90°C at a rate of 3°C / min, then heating to 150°C at a rate of 5°C / min, and finally heating to 220°C at a rate of 10°C / min and maintaining at 5°C. The peak area of ​​camphor was used as the internal standard to correct the peak area of ​​limonene and pulegone in the volatile oil, and the effect of silencing the transcription factor gene St19891 on the content of limonene and pulegone in the volatile oil was further calculated.

[0030] like Figure 3 , Figure 4 As shown, the contents of limonene and pulegone in the plants with St19891 gene silenced showed an increasing trend, further indicating that the St19891 gene negatively regulates the biosynthesis of monoterpenoid compounds in Nepeta.

[0031] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. Nepeta transcription factor gene St19891, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The product encoded by the Nepeta tenuifolia transcription factor gene St19891 according to claim 1, characterized in that The amino acid sequence of the product is shown in SEQ ID NO.

2.

3. The product according to claim 2, characterized in that The product is RNA, polypeptide or protein.

4. The specific primer of the Nepeta tenuifolia transcription factor gene St19891 as claimed in claim 1, characterized in that: include Upstream primer: 19891-F: 5′-cgtgagctcggtaccggatccACAATAATCATCATCACGGACAGC-3′; and downstream primer: 19891-R: 5′-gtgagtaaggttaccgaattcCGCCTCATCTTCCCGCTT-3′.

5. A virus-induced gene silencing vector comprising the Nepeta tenuifolia transcription factor gene St19891 according to claim 1.

6. The gene silencing vector according to claim 5, characterized in that The expression vector is pTRV2.

7. Use of the Nepeta tenuifolia transcription factor gene St19891 according to claim 1 in regulating monoterpenoid compounds.

8. The use according to claim 7, characterized in that The method comprises the following steps: transferring the Nepeta tenuifolia transcription factor gene St19891 into cells, expressing the Nepeta tenuifolia transcription factor gene St19891 in host cells, and regulating the synthesis of monoterpenoid compounds by the Nepeta tenuifolia transcription factor St19891.

9. The construction of the gene silencing vector according to claim 5 or 6, characterized in that: The following steps are involved: (1) Cloning of the silencing fragment of the Nepeta tenuifolia transcription factor gene St19891 The cloning of the St19891 silent fragment used forward primers: upstream primer: 19891-F: 5'-ACAATAATCATCATCACGGACAGC-3'; downstream primer: 19891-R: 5'-CGCCTCATCTTCCCGCTT-3'; PCR amplification was performed using the full-length sequence of the gene encoding the Nepeta transcription factor gene St19891 as a template; the cloning of the Nepeta transcription factor gene St19891 silent fragment was obtained; (2) Construction of gene silencing vector for the Nepeta tenuifolia transcription factor gene St19891 The pTRV2 vector was digested with BamHI and EcoRI restriction enzymes to obtain a linearized pTRV2 vector, and a gene silencing vector of the Nepeta tenuifolia transcription factor gene St19891 was constructed by homologous recombination.

10. The construction of the gene silencing vector according to claim 9, characterized in that: The following steps are involved: (1) Cloning of the silencing fragment of the Nepeta tenuifolia transcription factor gene St19891 The cloning of the St19891 silent fragment used forward primers: upstream primer: 19891-F: 5'-ACAATAATCATCATCACGGACAGC-3'; downstream primer: 19891-R: 5'-CGCCTCATCTTCCCGCTT-3'; PCR amplification was performed using the full-length sequence of the gene encoding the Nepeta transcription factor gene St19891 as a template; the amplification system was as follows: 5μL of 2×RapidTaq Master Mix enzyme, 0.2μL each of primers primer-F and primer-R, 1μL of template, and the rest was supplemented with sterile double distilled water; reaction conditions: pre-denaturation at 95℃ for 2min, denaturation at 95℃ for 20s, annealing at 60℃ for 20s, extension at 72℃ for 1min30s, extension at 72℃ for 5min after 35 cycles, and storage at 4℃; the clone of the Nepeta transcription factor gene St19891 silent fragment was obtained. (2) Construction of gene silencing vector for the Nepeta tenuifolia transcription factor gene St19891 The pTRV2 vector was digested with BamHⅠ and EcoRⅠ restriction endonucleases, and the digestion reaction system was as follows: 40 μL of pTRV2 vector, 2.5 μL each of BamHⅠ and EcoRⅠ restriction endonucleases, and 5 μL of 10×K buffer; reaction conditions: digestion at 37°C for 3 hours, and a linearized pTRV2 vector was obtained; a gene silencing vector of the Nepeta tenuifolia transcription factor gene St19891 was constructed by homologous recombination; reaction system: 4 μL of St19891 gene silencing fragment, 3 μL of linearized pTRV2 vector, 2 μL of 5×CE Buffer, and 1 μL of Exnase II; reaction conditions: reaction at 37°C for 30 minutes, and a gene silencing vector of Nepeta tenuifolia St19891 was obtained.

Images