Schizonepeta myb transcription factor gene smyb8774 and virus-induced gene silencing vector and application thereof

Abstract

This invention relates to genetically engineered organisms, specifically disclosing the Nepeta cataria MYB transcription factor gene. St MYB8774 and its encoded product, and a virus-induced gene silencing vector; the nucleotide sequence of this gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded product is shown in SEQ ID NO.2. This was achieved by silencing the MYB transcription factor gene in *Nepeta cataria*. St Cloning, amplification, and inhibition of MYB8774 in Nepeta cataria resulted in Nepeta cataria plants with inhibited MYB transcription factor StMYB8774, thereby increasing the content of terpenoids such as menthol and limonene. This invention provides... St Virus-induced gene silencing vectors of the MYB8774 gene can increase the content of terpenoids such as menthone and limonene, which are the active medicinal components of Catnip, through genetic engineering technology, providing a research direction for molecular breeding of Catnip.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the Nepeta cataria MYB transcription factor gene. St MYB8774 and its encoded products, its virus-induced gene silencing vectors, and their applications. Background Technology

[0002] Catnip ( Schizonepeta tenuifolia *Nepeta cataria* (also known as *Nepeta cataria*) is an annual herb belonging to the Lamiaceae family and the *Nepeta* genus. It was first recorded as "Jia Su" in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), while the name "Jing Jie" (Nepeta cataria) first appeared in the *Wu Pu Bencao* (Wu Pu's Materia Medica). It is used to relieve exterior syndromes, promote rash eruption, and eliminate sores. In modern times, it is commonly used for colds, headaches, measles, rubella, and early-stage sores. The volatile oil of *Nepeta cataria* has pharmacological effects such as antiviral, anti-inflammatory, analgesic, antitumor, immunomodulatory, antibacterial, and hemostatic properties. It is mainly composed of monoterpenes and sesquiterpenes, such as menthone, menthol, and caryophyllene. The *Chinese Pharmacopoeia* (2020 edition, Part I) stipulates that the volatile oil content in *Nepeta cataria* should not be less than 0.60%, and the menthone content in the volatile oil should not be less than 0.020%. Monoterpenes such as menthone are key components in the indicators of *Nepeta cataria*, possessing anti-inflammatory, insecticidal, antipyretic, and analgesic effects. Investigating the biosynthesis and regulatory mechanisms of terpenoids such as menthone in Catnip is of great significance for revealing the quality changes of Catnip medicinal materials and for screening high-quality production areas, germplasm and seedlings.

[0003] With the increasing maturity of genome sequencing technology, the genome sequencing of *Nepeta cataria* has been completed. Based on genomic and transcriptomic data, the biosynthetic pathway of menthol in *Nepeta cataria* has been elucidated. Geranyl pyrophosphate (GPP), synthesized via the MEP / MVP pathway, is used as a substrate to generate limonene under the catalysis of limonene synthase. Limonene is further hydroxylated and dehydrogenated to give isomenthol. The limonene-3-hydroxylase StL3OH involved in this step has been isolated and identified, and its role in the hydroxylation and dehydrogenation of limonene and the biosynthesis of menthol in *Nepeta cataria* has been discovered using the virus-induced gene silencing system VIGS. However, the transcription factor that regulates the synthesis of menthane monoterpenes menthol in *Nepeta cataria* by modulating limonene-3-hydroxylase StL3OH has not yet been isolated and identified. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to address the problem in existing research that transcription factors regulating the monoterpene synthesis pathway in Nepeta cataria have not yet been isolated and identified, and to provide the MYB transcription factor gene. St MYB8774 and its encoded products and applications. Another object of this invention is to provide the MYB transcription factor gene. St Virus-induced gene silencing vector and construction method of MYB8774.

[0005] Technical solution: To achieve the above objectives, this invention discloses the MYB transcription factor gene. St The nucleotide sequence of the MYB8774 gene is shown in SEQ ID NO. 1.

[0006] This invention also discloses the above-mentioned Nepeta cataria MYB transcription factor gene. St The product encoded by MYB8774 has an amino acid sequence as shown in SEQ ID NO. 2.

[0007] This invention also discloses the above-mentioned Nepeta cataria MYB transcription factor gene. St MYB8774-specific primers include: upstream primer: MYB8774-F: 5'-cgtgagctcggtaccggatccTTACATGAAATAATCTAATTCTAAACTCTCCG-3'; downstream primer: MYB8774-R: 5'-gtgagtaaggttaccgaattcCTTACAGATGCTTCACCTCAAAATCA-3'.

[0008] This invention also discloses a gene containing the above-mentioned Nepeta cataria MYB transcription factor gene. St The gene silencing vector for MYB8774, wherein the expression vector is pTRV2.

[0009] This invention also discloses the verification of the interaction between the above-mentioned catnip MYB transcription factor gene StMYB71 and the catnip limonene-3-hydroxylase StL3OH promoter, including the transformation of the catnip MYB transcription factor gene and the catnip limonene-3-hydroxylase StL3OH promoter into yeast cells, and the point-to-point verification of the interaction between the two.

[0010] The monoterpenoids are menthone and limonene.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: The catnip MYB transcription factor provided by the present invention St The MYB8774 gene was cloned and prepared for the first time from the catnip plant. (Catnip MYB transcription factor) St MYB8774 is a key regulatory gene in the monoterpene synthesis pathway of Nepeta cataria, and can be used to regulate the content of terpenoids such as menthol and limonene in Nepeta cataria. St The virus-induced gene silencing vector MYB8774 can be applied to regulate the menthane monoterpene biosynthesis pathway in Nepeta cataria, which is based on the MEP / MVA pathway. This invention provides... StVirus-induced gene silencing vectors of the MYB8774 gene can increase the content of terpenoids such as menthone and limonene, which are the active medicinal components of Catnip, through genetic engineering technology, providing a research direction for molecular breeding of Catnip. Attached Figure Description

[0012] Figure 1 Agarose gel electrophoresis image of the silenced fragment of the StMYB8774 gene of the MYB transcription factor gene in Nepeta cataria. Figure 2 The gene silencing efficiency of the MYB transcription factor gene StMYB8774 in Nepeta cataria; Figure 3 Changes in limonene content after silencing the StMYB8774 transcription factor gene in Nepeta cataria; Figure 4 Changes in menthone content after silencing the StMYB8774 transcription factor gene of Nepeta cataria; Figure 5 The changes in the expression levels of genes related to the biosynthetic pathway of menthane monoterpenes after silencing the MYB transcription factor gene StMYB8774 in Nepeta cataria. Figure 6 This study used yeast one-hybrids to demonstrate the relationship between the MYB transcription factor StMYB8774 from *Nepeta cataria* and the limonene-3-hydroxylase promoter StL3OHpro from *Nepeta cataria*. Detailed Implementation

[0013] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0014] The technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0015] Non-toxic Super GelBlue TMNucleic acid dyes were purchased from Shanghai Bioscient Biotechnology Co., Ltd.; the GeneJET Gel Extraction Kit was purchased from Thermo Fisher Scientific; 2×Rapid Taq MasterMix (P222); the FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics–rich) (RC401), the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R312-01 / 02), and DH5α chemicompetent cells (C502) were purchased from Novizan Biotechnology Co., Ltd.; GV3101 competent cells (AC1001) were purchased from Shanghai Weidi Biotechnology Co., Ltd. Eco RⅠ(1040A), Bam Restriction endonucleases such as HⅠ(1010S) were purchased from TaKaRa; sequence primers were synthesized by Shanghai Sangon Biotech Co., Ltd.; other reagents were either imported or domestically produced analytical grade reagents.

[0016] I. Nepeta cataria MYB transcription factor gene St Cloning of the silent fragment of MYB8774 St Cloning of the MYB8774 silenced fragment utilized forward primers: upstream primer: MYB8774-F: 5'-cgtgagctcggtaccggatccTTACATGAAATAATCTAATTCTAAACTCTCCG-3'; downstream primer: MYB8774-R: 5'-gtgagtaaggttaccgaattcCTTACAGATGCTTCACCTCAAAATCA-3'. The MYB transcription factor gene from *Nepeta cataria* was used. St The full-length sequence encoding the MYB8774 gene was used as a template for PCR amplification. The amplification system was as follows: 5 μL of 2×Rapid Taq Master Mix enzyme, 0.2 μL each of primers primer-F and primer-R, 1 μL of template, and the remainder was made up with sterile double-distilled water. Reaction conditions: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles, followed by a final extension at 72℃ for 5 min, and storage at 4℃. The *Nepeta cataria* MYB transcription factor gene was thus obtained. St The MYB8774 silencing fragment was cloned, and its nucleotide sequence is shown in SEQ ID NO. 1.

[0017] Construction of a gene silencing vector for the MYB transcription factor gene StMYB8774 from Nepeta cataria Select pTRV2 vector Bam HⅠ and Eco The pTRV2 vector was digested with RⅠ restriction endonuclease in the following reaction system: 40 μL of pTRV2 vector. Bam HⅠ and Eco 2.5 μL each of RⅠ restriction endonucleases and 5 μL of 10×K buffer were added. Reaction conditions: 37℃ for 3 hours. This yielded the linearized pTRV2 vector. Then, the Nepeta cataria MYB transcription factor gene was constructed using homologous recombination. St Gene silencing vector for MYB8774. Reaction system: St 4 μL of the MYB8774 gene silencing fragment, 3 μL of linearized pTRV2 vector, 2 μL of 5×CE Buffer, and 1 μL of Exnase II were added. Reaction conditions: 37 ℃ for 30 min. This yielded *Nepeta cataria*. St Gene silencing vector for MYB8774.

[0018] The above-mentioned catnip St The gene silencing vector of MYB8774 was transformed into Agrobacterium GV3101 competent cells. The transformation method was as follows: competent cells were thawed on ice and then catnip was added to them. St 5 μL of the MYB8774 gene silencing vector was added, followed by incubation on ice for 5 min. The cells were then incubated in liquid nitrogen for 5 min, immediately transferred to a 37°C water bath for 5 min, and incubated on ice for 2 min. 700 μL of antibiotic-free LB broth was added, and the cells were incubated at 28°C and 200 rpm for 3 h. The revived cells were then plated on LB agar plates containing 50 mg / L kanaciline and 20 mg / L rifampin and cultured at 28°C for 60 h.

[0019] Gene silencing of the MYB transcription factor gene StMYB8774 in Catnipae tinctoria GV3101 strain transformed with the StMYB8774 gene silencing vector was cultured in 20 mL of LB broth containing 50 mg / L kanamycin at 28°C with shaking until the OD600 reached approximately 0.6. The bacterial culture was centrifuged at 5000 rpm for 10 min at room temperature, the culture medium was discarded, and the bacterial cells were collected. The cells were resuspended in a buffer containing 10 mM MgCl2 and 10 mM MES, and the OD600 was adjusted to 0.4. Acetyleugenol was added to a final concentration of 20 μM. The culture was then activated at 25°C in the dark for 3 h. The activated bacterial culture was then mixed 1:1 with GV3101 culture containing pTRV1 activated under the same conditions. A negative control, GV3101 culture transformed without the StMYB8774 silencing fragment pTRV2, was used for further comparative analysis.

[0020] 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.

[0021] Verification of the silencing efficiency of the MYB transcription factor gene StMYB8774 in Catnipae tinctoria 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* MYB transcription factor StMYB8774 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 qPCR Master Mix, 1 μL of cDNA template, 0.2 μL each of upstream primer AGATGTGGGAAGAGTTGC and downstream primer CTGAGGCGAGCCGAGTA, 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.

[0022] like Figure 2 , Figure 5As shown, the expression level of the StMYB8774 gene decreased by approximately 70% after infection with *Nepeta cataria* plants, indicating successful silencing of the *Nepeta cataria* StMYB8774 gene. When the StMYB71 gene in *Nepeta cataria* was silenced, the expression levels of genes related to the biosynthesis of menthol monoterpenes, including LS, L3OH, ISPD, and ISPR, all showed a significant increase, indicating a negative correlation between the expression level of the StMYB8774 gene and the expression levels of genes related to the biosynthesis of menthol monoterpenes. This further suggests that the StMYB8774 gene may negatively regulate the biosynthesis of menthol monoterpenes in *Nepeta cataria*. Therefore, it can be achieved through... St The MYB8774 gene silencing vector was used to increase the bioavailability of menthol monoterpenes.

[0023] Changes in volatile oil content in plants with silenced MYB transcription factor gene StMYB8774 in Catnip Leaves of *Nepeta cataria* plants were collected after silencing the MYB transcription factor StMYB8774. 1 g of the sample was accurately weighed into a centrifuge tube, and two 5 mm diameter steel balls and n-hexane containing 30 ng / mL camphor internal standard were added. The sample was ground in a tissue homogenizer at 60 Hz for 60 s. An appropriate amount of anhydrous sodium sulfate was added to the ground sample to remove moisture, and the sample was centrifuged at 12000 rpm for 5 min at room temperature. The supernatant was collected and filtered through a 0.22 μm filter. The filtered sample was analyzed by GC. The analytical conditions were as follows: sample injection volume of 1 μL, splitless, carrier gas of high-purity nitrogen, injection port temperature of 220℃, FID detector temperature of 250℃, column type: Agilent 19091S-433-HP-5, 30 m × 250 μm × 0.25 μm, and temperature program: 0℃ for 3 min, ramped to 90℃ at a rate of 3℃ / min, ramped to 150℃ at a rate of 5℃ / min, and finally ramped to 220℃ at a rate of 10℃ / min and held at 5℃. The peak areas of limonene and menthone in the volatile oil were corrected using the peak area of ​​camphor as an internal standard. Further calculations of the MYB transcription factor gene were performed. St The effect of MYB8774 silencing on the content of limonene and menthone in volatile oil.

[0024] like Figure 3 , Figure 4 As shown, when the StMYB8774 gene in catnip is suppressed, the contents of menthol and limonene in catnip both increase significantly, further proving that the MYB transcription factor StMYB8774 gene in catnip negatively regulates the biosynthesis of menthol monoterpenes in catnip.

[0025] The *Nepeta cataria* MYB transcription factor gene StMYB8774 and the *Nepeta cataria* limonene-3-hydroxylase promoter StL3OHpro yeast one-hybrid The StMYB8774 cloning utilized the following primers: Upstream primer: MYB8774-AD-F: 5'-gccatggaggccagtgaattcATGGGAAGAACACCGTGCTG -3'; Downstream primer: MYB8774-AD-R: 5'-cagctcgagctcgatggatccTTACATGAAATAATCTAATTCTAAACTCTCCG-3'. The StMYB transcription factor gene was used as a precursor. St The full-length sequence encoding the MYB8774 gene was used as a template for PCR amplification. The amplification system was as follows: 5 μL of 2×Rapid Taq Master Mix enzyme, 0.2 μL each of primers primer-F and primer-R, 1 μL of template, and the remainder was made up with sterile double-distilled water. Reaction conditions: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 20 s, 60℃ annealing for 20 s, 72℃ extension for 1 min 30 s, 35 cycles, followed by a 5 min extension at 72℃, and storage at 4℃. The *Nepeta cataria* MYB transcription factor gene was thus obtained. St MYB8774.

[0026] Select pGADT7 vector Bam HⅠ and Eco The enzyme was digested with RⅠ restriction endonuclease. The digestion reaction system was as follows: 40 μL of pGADT7 vector. Bam HⅠ and Eco 2.5 μL each of RⅠ restriction endonuclease and 5 μL of 10×K buffer. Reaction conditions: digestion at 37℃ for 3 hours, thus obtaining the linearized pGADT7 vector.

[0027] Constructing the Nepeta cataria MYB transcription factor gene using homologous recombination St MYB8774 gene silencing vector, reaction system: St 4 μL of the MYB8774 gene fragment, 3 μL of linearized pGADT7 vector, 2 μL of 5×CE Buffer, and 1 μL of Exnase II were added. Reaction conditions: 37 ℃ for 30 min. This yielded *Nepeta cataria*. St Yeast one-hybrid vector MYB8774.

[0028] The cloning of the *Nepeta cataria* limonene-3-hydroxylase promoter StL3OHpro was performed using the following primers: upstream primer primer-F: 5'-agtggtctctgtccagtcctTTTCAAACGGATCTGCAGGTG-3'; downstream primer primer-R: 5'-ggtctcagcagaccacaagtCTCAATTAATCCTCCTAATTAATCAACTAA-3'; PCR amplification was performed using *Nepeta cataria* genomic DNA as a template. The amplification system was as follows: 5 μL of 2×Rapid Taq Master Mix enzyme, 0.2 μL each of primer primer-F and primer-R, 1 μL of template, and the remainder was made up with sterile double-distilled water. Reaction conditions: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 20 s, 60℃ annealing for 20 s, 72℃ extension for 1 min 30 s, 35 cycles, followed by a 5 min extension at 72℃, and storage at 4℃. The *Nepeta cataria* limonene-3-hydroxylase promoter StL3OHpro gene fragment was thus obtained.

[0029] The pAbAi vector was digested with BamHI and EcoRI restriction endonucleases. The digestion reaction system was as follows: 40 μL pAbAi vector, 2.5 μL each of BamHI and EcoRI restriction endonucleases, and 5 μL 10×K buffer. The reaction conditions were: digestion at 37℃ for 3 hours, thus obtaining the linearized pAbAi vector.

[0030] Then, a yeast one-hybrid vector for *Nepeta cataria* StL3OHpro was constructed using homologous recombination. The reaction mixture consisted of 4 μL of the *Nepeta cataria* limonene-3-hydroxylase promoter StL3OHpro gene fragment, 3 μL of linearized pAbAi vector, 2 μL of 5×CE Buffer, and 1 μL of Exnase II. The reaction conditions were 37 ℃ for 30 min. This yielded the yeast one-hybrid vector for *Nepeta cataria* StL3OHpro.

[0031] The yeast one-hybrid vector of *Nepeta cataria* StL3OHpro was obtained by digestion with BstBI at 37℃ for 1 h to obtain a linearized vector. The linearized vector was then transformed into Y1HGold competent yeast cells according to the manufacturer's instructions. The transformed competent cells were plated on SD-Ura medium and incubated at 30℃. Single colonies were picked and transferred to YPDA medium. After incubation, the bacterial culture was centrifuged to collect the cells, and then diluted 10-fold, 100-fold, and 100-fold with sterile water, respectively. These diluted cells were then spotted onto SD-Ura medium containing different concentrations of AbA for self-activation detection and AbA concentration screening.

[0032] Yeast cells containing the StL3OHpro yeast one-hybrid vector were prepared as competent cells and then transformed with catnip. StThe yeast one-hybrid vector MYB8774 was used, and the transformed yeast cells were plated on SD-Ura / Leu medium and cultured at 30°C. Single colonies were picked and cultured in YPDA medium with shaking. After incubation, the bacterial culture was centrifuged to collect the cells, and diluted 10-fold, 100-fold, and 100-fold with sterile water, respectively, and spotted onto SD-Ura / Leu medium containing AbA and cultured at 30°C.

[0033] like Figure 6 As shown, *Nepeta cataria* StL3OHpro exhibits self-activation. The self-activation of *Nepeta cataria* StL3OHpro was inhibited at a concentration of 600 mg / L, which was selected as the inhibitory concentration for abamin (AbA). When two yeast one-hybrid vectors were co-transformed into yeast cells, culture revealed that the yeast cells grew normally on the culture medium compared to the control group, indicating that *Nepeta cataria* transcription factors... St MYB8774 and StL3OHpro have a direct interaction.

[0034] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. Nepeta cataria MYB transcription factor gene St MYB8774 is characterized by, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The Nepeta cataria MYB transcription factor gene as described in claim 1 St The product encoded with MYB8774 is characterized by, 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 can be RNA, polypeptide, or protein.

4. The Nepeta cataria MYB transcription factor gene as described in claim 1 St The specific primers for MYB8774 are characterized by, Including upstream primer: MYB8774-F: 5'-cgtgagctcggtaccggatccTTACATGAAATAATCTAATTCTAAACTCCG-3'; Downstream primer: MYB8774-R 5'-gtgagtaaggttaccgaattcCTTACAGATGCTTCACCTCAAAATCA-3'.

5. Contains the Nepeta cataria MYB transcription factor gene as described in claim 1 St The virus-induced gene silencing vector of MYB8774.

6. Nepeta cataria MYB transcription factor gene St Application of MYB8774 and its virus-induced gene silencing vector in regulating sesquiterpenoid compounds.

7. The application as described in claim 6, characterized in that, This includes introducing the Nepeta cataria MYB transcription factor gene into cells. St MYB8774 or a virus-induced gene silencing vector expresses the Nepeta cataria MYB transcription factor gene in host cells. St MYB8774 or inhibition of the MYB transcription factor gene in Nepeta cataria St MYB71 expression regulates the synthesis of monoterpenoids.

8. The method for constructing the gene silencing vector according to claim 5, characterized in that, Includes the following steps: (1) Nepeta cataria MYB transcription factor gene St Cloning of the silent fragment of MYB8774 St Cloning of the MYB8774 silenced fragment utilized forward primers: upstream primer: MYB8774-F: 5'-cgtgagctcggtaccggatccTTACATGAAATAATCTAATTCTAAACTCTCCG-3'; downstream primer: MYB8774-R: 5'-gtgagtaaggttaccgaattcCTTACAGATGCTTCACCTCAAAATCA-3', targeting the MYB transcription factor gene from *Nepeta cataria*. St The full-length sequence encoding the MYB8774 gene was used as a template for PCR amplification; the MYB transcription factor gene of *Nepeta cataria* was obtained. St Cloning of the MYB8774 silencing fragment, nucleotide sequence as shown in SEQ ID NO. 1; (2) Construction of a gene silencing vector for the MYB transcription factor gene StMYB8774 from Nepeta cataria Select pTRV2 vector Bam HⅠ and Eco The gene was digested with RⅠ restriction endonuclease, and then the Nepeta cataria MYB transcription factor gene was constructed using homologous recombination. St Gene silencing vector for MYB8774.

9. The method for constructing a gene silencing vector according to claim 8, characterized in that, Includes the following steps: (1) Nepeta cataria MYB transcription factor gene St Cloning of the silent fragment of MYB8774 St Cloning of the MYB8774 silenced fragment utilized forward primers: upstream primer: MYB8774-F: 5'-cgtgagctcggtaccggatccTTACATGAAATAATCTAATTCTAAACTCTCCG-3'; downstream primer: MYB8774-R: 5'-gtgagtaaggttaccgaattcCTTACAGATGCTTCACCTCAAAATCA-3', targeting the MYB transcription factor gene from *Nepeta cataria*. St The full-length sequence encoding the MYB8774 gene was used as a template for PCR amplification. The amplification system was as follows: 5 μL of 2×Rapid Taq Master Mix enzyme, 0.2 μL each of primers primer-F and primer-R, 1 μL of template, and the remainder was made up with sterile double-distilled water. The reaction conditions were: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles, followed by 72℃ extension for 5 min, and storage at 4℃ to obtain the Nepeta cataria MYB transcription factor gene. St Cloning of the MYB8774 silencing fragment, nucleotide sequence as shown in SEQ ID NO. 1; (2) Construction of a gene silencing vector for the MYB transcription factor gene StMYB8774 from Nepeta cataria Select pTRV2 vector Bam HⅠ and Eco The pTRV2 vector was digested with RⅠ restriction endonuclease in the following reaction system: 40 μL of pTRV2 vector. Bam HⅠ and Eco 2.5 μL each of RⅠ restriction endonucleases and 5 μL of 10×K buffer; reaction conditions: digestion at 37℃ for 3 hours, thus obtaining the linearized pTRV2 vector; the Nepeta cataria MYB transcription factor gene was constructed using homologous recombination. St MYB8774 gene silencing vector; reaction system: St 4 μL of the MYB8774 gene silencing fragment, 3 μL of linearized pTRV2 vector, 2 μL of 5×CE Buffer, and 1 μL of Exnase II were added. The reaction conditions were: 37 ℃ for 30 min. This yielded *Nepeta cataria* (a type of herb). St Gene silencing vector for MYB8774.

10. The application of the nepeta MYB transcription factor gene StMYB8774 in regulating limonene biosynthesis in nepeta through its interaction with nepeta limonene-3-hydroxylase StL3OH.

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