Schizonepeta transcripion factor gene s19891 and the product encoded thereby and use thereof

Abstract

The present application relates to genetic engineering body, specifically disclose the schizonepeta transcription factor gene St19891 and the product and application coded thereof, the nucleotide sequence of this gene is as shown in SEQ ID NO.1, the amino acid sequence of the coded product is as shown in SEQ ID NO.2, through the cloning, amplification, construction gene silencing vector of schizonepeta transcription factor gene St19891, inhibition is carried out in schizonepeta in vivo, and the schizonepeta transcription factor St19891 inhibited schizonepeta plant is obtained, this schizonepeta transcription factor gene St19891, solve the problem that the monoterpenes synthesis regulation related transcription factor in the existing research about schizonepeta has not been separated and identified, the present application can provide the basis for the genetic engineering of improving schizonepeta and improving the content of effective components such as schizonepeta pulegone.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a Schizonepeta tenuifolia transcription factor gene St19891, a product encoded by the gene and application thereof. BACKGROUND

[0002] Schizonepeta tenuifolia is an annual herb of the Labiatae family and the Schizonepeta genus. It was first recorded as “Jian Su” in the Shennong Bencao Jing, and the name “Schizonepeta” was first recorded in the Wu Pu Bencao. It has the effects of relieving superficies and dispelling wind, promoting rash and resolving sore. It is commonly used in modern times for cold, headache, measles, wind rash and sore at the initial stage. The volatile oil of Schizonepeta tenuifolia has pharmacological effects such as antiviral, anti-inflammatory, analgesic, antitumor, immunoregulatory, antibacterial and hemostatic effects. It is mainly composed of monoterpene and sesquiterpene compounds such as p-menthane-3,8-diol, menthone, caryophyllene, etc. The Chinese Pharmacopoeia (2020 edition, first volume) stipulates that the volatile oil content of Schizonepeta tenuifolia medicinal materials shall not be less than 0.60%, and the p-menthane-3,8-diol content in the volatile oil of Schizonepeta tenuifolia medicinal materials shall not be less than 0.020%. Monoterpene p-menthane-3,8-diol is the key component of Schizonepeta tenuifolia medicinal materials, and has the effects of anti-inflammatory, insecticidal, antipyretic and analgesic. Exploring the biosynthesis and regulation mechanism of p-menthane-3,8-diol in Schizonepeta tenuifolia has very important significance for revealing the quality change of Schizonepeta tenuifolia medicinal materials and screening high-quality production areas and germplasm seedlings.

[0003] The geranyl pyrophosphate (GPP) synthesized by the terpenoid synthesis pathway MEP / MVP pathway is used as a substrate to generate limonene under the catalysis of limonene synthase. Limonene is further hydroxylated and dehydrogenated to obtain isomenthone, and isomenthone is further reduced to obtain isomenthone, which is isomerized by isomerase to obtain menthone. The biosynthesis pathway of menthone in Schizonepeta tenuifolia is further verified based on the VIGS gene silencing system. However, the transcription factor involved in the regulation of the synthesis of menthone, a monoterpene of the menthane class, in Schizonepeta tenuifolia has not been isolated and identified. SUMMARY

[0004] The present application aims to solve the problem that the transcription factor involved in the regulation of the monoterpene synthesis pathway in Schizonepeta tenuifolia has not been isolated and identified in existing research, and provides a Schizonepeta tenuifolia transcription factor gene St19891, a product encoded by the gene and application thereof.

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

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

[0007] The application also discloses the specific primer of the above-mentioned Schizonepeta Tran-scription Factor Gene St19891, which comprises an upstream primer 19891-F: 5'-ACAATAATCATCATCACGGACAGC-3' and a downstream primer 19891-R: 5'-CGCCTCATCTTCCCGCTT-3'.

[0008] The application also discloses a gene silencing vector containing the above-mentioned Schizonepeta Tran-scription Factor Gene St19891, wherein the expression vector is pTRV2.

[0009] The application also discloses the application of the above-mentioned Schizonepeta Tran-scription Factor Gene St19891 in regulating monoterpenes, which comprises the following steps: introducing the Schizonepeta Tran-scription Factor Gene St19891 into cells, inhibiting the expression of the Schizonepeta Tran-scription Factor Gene St19891 in a host, and inhibiting the synthesis of monoterpenes in Schizonepeta by the Schizonepeta Tran-scription Factor Gene St19891. The monoterpenes comprise menthone and limonene.

[0010] Advantages: Compared with the prior art, the Schizonepeta Tran-scription Factor St19891 gene provided by the application is first cloned and prepared from Schizonepeta plants. The Schizonepeta Tran-scription Factor St19891 is a key regulatory gene of the monoterpenes synthesis pathway of Schizonepeta, and can be used for regulating the menthone content of Schizonepeta. The protein can be applied to regulating the biosynthesis of monoterpenes of Schizonepeta. The gene provided by the application can be used to improve the content of the medicinal component menthone in Schizonepeta by genetic engineering technology, and provides a research direction for the molecular breeding of Schizonepeta. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 It is an agarose gel diagram of the Schizonepeta Tran-scription Factor Gene St19891 gene silencing fragment;

[0012] Figure 2 It is the Schizonepeta Tran-scription Factor Gene St19891 gene silencing efficiency;

[0013] Figure 3 It is the change of the limonene content in Schizonepeta after the Schizonepeta Tran-scription Factor Gene St19891 gene silencing;

[0014] Figure 4 It is the change of the menthone content in Schizonepeta after the Schizonepeta Tran-scription Factor Gene St19891 gene silencing;

[0015] Figure 5 It is the change of the expression amount of the menthone biosynthesis pathway related gene after the Schizonepeta Tran-scription Factor Gene St19891 gene silencing; DETAILED DESCRIPTION

[0016] The above and other technical features and advantages of the present application will be more apparent from the following description taken in connection with the accompanying drawings.

[0017] The technical means used in the examples are conventional means known to those skilled in the art, and the raw materials used are commercially available. Non-toxic Super Gel Blue TM The nucleic acid dye was purchased from Shanghai Bayscience Biotech Co., Ltd.; the gel recovery kit GeneJET Gel Extraction Kit was purchased from Thermo Fisher Scientific; 2x Rapid Taq Master Mix (P222); FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics-rich) (RC401), reverse transcription kit HiScript III 1stStrand cDNA Synthesis Kit (+gDNA wiper) (R312-01 / 02), DH5a chemically competent cells (C502) were purchased from Novogene Bioinformatics Co., Ltd.; GV3101 competent cells (AC1001) were purchased from Shanghai Uptide Biotech Co., Ltd.; restriction endonucleases such as EcoR I (1040A) and BamH I (1010S) were purchased from TaKaRa; sequence primers were synthesized by Shanghai Shengong Bioengineering Co., Ltd.; other reagents were imported or domestic analytical pure reagents.

[0018] I. Cloning of Schizonepeta trancription factor gene St19891 silencing fragment

[0019] The cloning of St19891 silencing fragment used forward primer: upstream primer (sequence as SEQ ID NO. 3): 19891-F: 5'-ACAATAATCATCATCACGGACAGC-3'; downstream primer (sequence as SEQ ID NO. 4): 19891-R: 5'-CGCCTCATCTTCCCGCTT-3'. The full-length sequence of Schizonepeta trancription factor gene St19891 encoding gene was used as a template for PCR amplification. The amplification system was as follows: 2x Rapid Taq Master Mix enzyme 5 μL, primer primer-F and primer-R each 0.2 μL, template 1 μL, and the rest was supplemented with sterile double distilled water. The reaction conditions were as follows: 95°C pre-denaturation for 2 min, 95°C denaturation for 20 s, 60°C annealing for 20 s, 72°C extension for 1 min 30 s, 35 cycles of 72°C extension for 5 min, and 4°C storage. Thus, the cloning of Schizonepeta trancription factor gene St19891 silencing fragment was obtained.

[0020] II. Construction of Schizonepeta trancription factor gene St19891 gene silencing vector

[0021] The pTRV2 vector was selected by BamH I and EcoR I restriction enzymes, and the enzyme reaction system was as follows: pTRV2 vector 40 μL, BamH I and EcoR I restriction enzymes 2.5 μL each, 10×K buffer 5 μL. The reaction condition was that the enzyme cutting was carried out at 37 °C for 3 hours, and thus the linearized pTRV2 vector was obtained. The St19891 gene silencing fragment containing the homologous arm was obtained by synthesizing the upstream and downstream primers containing the vector homologous arm sequence and amplifying. The St19891 gene silencing vector of Schizonepeta was constructed by using the method of homologous recombination. The reaction system was as follows: St19891 gene silencing fragment containing the homologous arm 4 μL, linearized pTRV2 vector 3 μL, 5×CE Buffer 2 μL, Exnase II 1 μL. The reaction condition was that the reaction was carried out at 37 °C for 30 min. Thus the St19891 gene silencing vector of Schizonepeta was obtained. The St19891 gene silencing vector of Schizonepeta was transformed into the Agrobacterium GV3101 competent cell, and the transformation method was as follows: the competent cell was thawed on ice, and then 5 μL of the St19891 gene silencing vector of Schizonepeta was added, followed by standing on ice for 5 min, and then the cell was reacted in liquid nitrogen for 5 min, and then it was immediately transferred to a 37 °C water bath for 5 min, and then it was stood on ice for 2 min, and then 700 μL of the LB liquid medium without antibiotics was added, and then it was recovered at 28 °C and 200 rpm for 3 h. The recovered cell was plated on the LB plate containing 50 mg / L of kanamycin and 20 mg / L of rifampicin, and then it was cultured at 28 °C for 60 h.

[0022] III. Gene silencing of the transcription factor gene St19891 in Schizonepeta

[0023] The GV3101 strain transformed with the St19891 gene silencing vector was cultured in 20 mL of the LB liquid medium containing 50 mg / L of kanamycin at 28 °C and under shaking until the OD600 was about 0.6. The bacterial liquid was centrifuged at 5000 rpm for 10 min at room temperature, and then the culture medium was discarded and the bacterial body was collected, and then the bacterial body was resuspended with the buffer containing 10 mM MgCl2 and 10 mM MES and the OD600 was adjusted to 0.4, and then acetyl eugenol was added to a final concentration of 20 μM. The activated bacterial liquid was mixed with the GV3101 bacterial liquid containing pTRV1 under the same conditions at a ratio of 1:1. The GV3101 bacterial liquid transformed with the pTRV2 not containing the St19891 silencing fragment of Schizonepeta was used as a negative control for further comparative analysis.

[0024] Using catnip plants sown for ten days as infection material, Agrobacterium tumefaciens solution was injected into the catnip plants from the back of the leaves using a 1 mL syringe. After injection, the catnip plants were left to stand in the dark at 25°C for 24 hours. After standing, the plants were cultured normally in an incubator for 20 days. The leaves of the silenced plants were then collected for expression level verification and component content determination.

[0025] IV. Verification of the silencing efficiency of the transcription factor gene St19891 in Catnipae lanceolata

[0026] Silenced leaves of *Nepeta cataria* plants were flash-frozen in liquid nitrogen for RNA extraction. Total RNA was extracted according to the instructions of the plant polysaccharide and polyphenol RNA extraction kit. First-strand cDNA libraries were obtained by reverse transcription using the total RNA as a template. Quantitative RT-PCR was used to verify the silencing efficiency of the *Nepeta cataria* transcription factor St19891 and the changes in the expression levels of genes related to the menthol biosynthesis pathway. The PCR amplification system consisted of: 5 μL of 2×ChamQ Blue Universal SYBR qPCRMaster Mix, 1 μL of cDNA template, 0.2 μL each of forward and reverse primers, and 10 μL of sterile water. The reaction conditions were: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 60℃ extension for 30 s, for 40 cycles; the melting curve was set to the instrument's default program. β-actin in *Nepeta cataria* was used as an internal reference gene, and the relative expression level of the gene was calculated using the 2^(-ΔΔCT) method.

[0027] like Figure 2 , Figure 5 As shown, the expression level of the St19891 gene in infected *Nepeta cataria* plants decreased by approximately 60%, indicating successful silencing of the St19891 gene and further demonstrating the successful construction of silenced plants. In *Nepeta cataria* plants with successfully silenced St19891, the gene expression levels of genes related to the biosynthesis of menthol monoterpenes—L3OH (limonene-3-hydroxylase), IPD (isomenthenol dehydrogenase), and IPR (isomenthenone reductase)—significantly increased. This indicates a negative correlation between the expression level of the St19891 gene and the expression levels of genes related to the biosynthesis of menthol monoterpenes. The largest decrease in L3OH expression suggests that the St19891 gene may further negatively regulate the biosynthesis of monoterpenes in *Nepeta cataria* by regulating the expression level of the L3OH gene.

[0028] V. Changes in volatile oil content in plants with the St19891 transcription factor gene silenced in Catnip

[0029] The leaves of the Artemisia argyi plant after silencing of the transcription factor St19891 are collected. 1 g is weighed into a centrifuge tube, 2 steel balls with a diameter of 5 mm and n-hexane containing 30 ng / mL camphor internal standard are added. In a tissue grinder, 60 Hz grinding for 60 s, an appropriate amount of anhydrous sodium sulfate is added to the ground sample to remove moisture, and centrifuged at 12000 rpm for 5 min at room temperature. The supernatant of the sample after centrifugation is aspirated and filtered through a 0.22 μm filter. The filtered sample is analyzed by GC. The analysis conditions are as follows: sample injection amount is 1 μL, no split, carrier gas is high-purity nitrogen, injection port temperature is 220°C, FID detector temperature is 250°C, chromatographic column type is Agilent 19091S-433-HP-5, 30 m x 250 μm x 0.25 μm, and the programmed temperature conditions are as follows: 0°C for 3 min, then increased to 90°C at a rate of 3°C / min, then increased to 150°C at a rate of 5°C / min, and finally increased to 220°C at a rate of 10°C / min and maintained at 5°C. The peak area of camphor is used as an internal standard to correct the peak area of limonene and menthone in the volatile oil, and the effect of silencing of the transcription factor gene St19891 on the content of limonene and menthone in the volatile oil is further calculated.

[0030] As shown in Figure 3 , Figure 4 The contents of limonene and menthone in the Artemisia argyi plant after silencing of the St19891 gene both show an upward trend, further indicating that the St19891 gene negatively regulates the biosynthesis of monoterpenes in Artemisia argyi.

[0031] The above only describes the preferred embodiments of the present application, which are only illustrative but not limiting. Those skilled in the art understand that many changes, modifications and even equivalents can be made to the present application within the spirit and scope defined by the claims of the present application, but all will fall within the protection scope of the present application.

Claims

1. Nepeta cataria transcription factor gene St 19891, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The catnip transcription factor gene according to claim 1 St The product coded 19891 is characterized by, The amino acid sequence of the product is shown in SEQ ID NO.

2.

3. The application of the virus-induced gene silencing vector of the catnip transcription factor gene St19891 as described in claim 1 in increasing the content of limonene and menthol in catnip.

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