Aspidium adiantum aspiMYB5 gene and application in regulating aspidium adiantum phenol biosynthesis

Abstract

This invention discloses tree fern. AspiMYB5 The application of genes in regulating the biosynthesis of tree fern phenols belongs to the field of genetic engineering technology, which involves regulating the biosynthesis of tree fern phenols within plants. AspiMYB5 The expression level of the gene, thereby regulating the key enzyme gene of tree fern phenol. AspiPKS6 The expression of [a specific enzyme] and the synthesis of tree fern phenol. This invention uses a yeast library screening method to screen genes affecting key enzymes. AspiPKS6 Expressed transcription factors AspiMYB5 The results were verified using yeast single-hybrid technology (Y1H) and electrophoretic mobility variation analysis (EMSA). AspiMYB5 and AspiPKS6 The promoter exhibits direct physical binding in vitro. Dual-luciferase reporter gene assays further demonstrate that... AspiMYB5 and AspiPKS6 When the promoter is co-expressed, the fluorescence signal intensity is significantly reduced, further confirming... AspiMYB5 It can inhibit AspiPKS6 AspiMYB5 Promoter activity. This invention not only improves the theoretical research on the biosynthetic pathway of tree fern phenols, but also provides key target genes and technical support for regulating the content of secondary metabolites in rare plants through genetic improvement.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to tree ferns. AspiMYB5 Genes and their application in regulating the biosynthesis of tree fern phenols. Background Technology

[0002] tree fern ( Alsophila spinulosa It belongs to the genus *Cyathea* of the family Cyatheaceae. Alsophila Tree ferns, also known as tree ferns, are a rare type of fern. As the only extant woody fern, they are called a "living fossil" for studying paleontology and the evolution of the Earth. Tree ferns have significant medicinal value and produce numerous secondary metabolites, from which approximately 99 organic compounds have been isolated. Most of these are from the stems of the tree fern, and the main types of compounds are flavonoids, steroids and triterpenoids, and phenylpropanoids.

[0003] Aalsophilin is a novel compound isolated and identified from the traditional medicinal tree tree *Cyathea spinulosa*. It is unique to *Cyathea spinulosa* and possesses strong antioxidant activity. Its biosynthetic pathway is as follows: styrylpyrone synthase (SPS) and stilbene synthase (STS) condense 2 and 3 molecules of malonyl-CoA, respectively, onto a hydroxycinnamoyl-CoA group, generating hispidin and piceatannol, respectively. Then, the 7',8'-trans-dihydrofuran ring in alsophilin is biocatalyzed by an oxidase. In vitro enzyme activity assays were conducted, selecting eight genes highly expressed in the xylem from the 103 PKS genes previously identified in *Cyathea spinulosa* to generate recombinant proteins. These proteins were then used as substrates for in vitro enzyme activity assays with p-coumaroyl-CoA and caffeoyl-CoA, and detected by LC-MS. The results showed that... AspiPKS6 It has high SPS activity and can catalyze the conversion of p-coumaroyl-CoA to hispidin precursor bis-Noryangonin, as well as the conversion of caffeoyl-CoA to hispidin.

[0004] Within plants, a series of signal transduction pathways activate transcription factors. These factors bind to their corresponding cis-acting elements, activating the RNA polymerase II transcription complex and initiating the transcriptional expression of specific genes. Ultimately, the gene products regulate internal and external signals. Transcription factors play a crucial pivotal role in regulating the synthesis and accumulation of secondary metabolites. Some transcription factor protein products can precisely activate or inhibit the transcription of multiple rate-limiting enzymes along the entire secondary metabolic pathway, thereby determining the type, content, and spatiotemporal distribution of the final products.

[0005] However, current research lacks a clear understanding of the key transcription factors in the tree fern phenol synthesis pathway. Screening transcription factors that affect key enzymes is crucial for improving the tree fern phenol synthesis pathway.

[0006] Based on this, the present invention designs tree fern. AspiMYB5 To address the aforementioned issues, we need to explore the role of genes in regulating the biosynthesis of tree fern phenols. Summary of the Invention

[0007] To address the aforementioned shortcomings of existing technologies, this invention provides tree ferns. AspiMYB5 Genes and their application in regulating the biosynthesis of tree fern phenols.

[0008] To achieve the above objectives, the present invention provides the following technical solution: Tree ferns used to regulate the biosynthesis of tree ferns. AspiMYB5 Genes, the ones mentioned AspiMYB5 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0009] To better achieve the objectives of this invention, the present invention also provides tree fern. AspiMYB5 Genes and their application in regulating the biosynthesis of tree fern phenols, through regulating the body's internal structure of plants. AspiMYB5 The expression level of the gene, thereby regulating the key enzyme gene of tree fern phenol. AspiPKS6 The expression of and the synthesis of tree fern phenol.

[0010] Furthermore, the aforementioned AspiMYB5 Genes used to negatively regulate key enzyme genes of tree fern phenol AspiPKS6 The expression of this substance reduces the content of milkweed alkaloid, the precursor of tree fern alkaloids, thereby inhibiting the synthesis of tree fern alkaloids.

[0011] Furthermore, utilizing AspiMYB5 The method for regulating the biosynthesis of tree fern phenols by gene is as follows: constructing a system containing... AspiMYB5 A gene recombinant vector is used to transfer the recombinant vector into a plant to induce gene recombination. AspiMYB5 Overexpression inhibits the synthesis of tree fernol.

[0012] To better achieve the objectives of this invention, the present invention also provides a recombinant vector, the recombinant vector comprising the aforementioned... AspiMYB5 Gene.

[0013] Furthermore, the recombinant vector is selected from pGADT7-Rec2, pET-28a(+) or pBI121.

[0014] To better achieve the objectives of this invention, the present invention also provides methods for verification. AspiMYB5 and AspiPKS6 The engineered bacteria with an interactive relationship contain the recombinant vector described above.

[0015] Furthermore, the engineered bacteria are selected from Escherichia coli, yeast, or Agrobacterium.

[0016] To better achieve the objectives of this invention, the present invention also provides AspiMYB5 Application of genes in screening or identifying substances that regulate the biosynthesis pathway of tree fern phenols.

[0017] Compared with the prior art, the beneficial effects of this invention are as follows: This invention uses a yeast library screening method to screen genes that affect key enzymes. AspiPKS6 Expressed transcription factors AspiMYB5 The results were verified using yeast single-hybrid technology (Y1H) and electrophoretic mobility variation analysis (EMSA). AspiMYB5 and AspiPKS6 The promoter exhibits direct physical binding in vitro. This was demonstrated through the creation of transgenic Arabidopsis plants and dual-luciferase experiments. AspiMYB5 right AspiPKS6 It has a significant negative regulatory effect, and overexpression AspiMYB5 This will lead to AspiPKS6 The expression level decreased. This invention not only improves the theoretical research on the biosynthetic pathway of tree fern phenols, but also provides key target genes and technical support for regulating the content of secondary metabolites in rare plants through genetic improvement. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 This invention serves as a yeast one-hybrid experiment verification. AspiMYB5 Transcription factors and AspiPKS6 A diagram showing the results of one-to-one interaction between promoters.

[0020] Figure 2 This invention provides experimental verification of electrophoretic mobility variation analysis (EMSA). AspiMYB5 Protein and AspiPKS6 The result of the promoter's in vitro physical binding.

[0021] Figure 3 For the present invention AspiPKS6 Overexpression of plant transcription levels and AspiPKS6 / AspiMYB5 Figure showing the results of transcriptional level identification in dual overexpression transgenic plants.

[0022] Figure 4 This is a fluorescence imaging image of transient expression in *Nicotiana benthamiana* leaves in the dual-luciferase reporter experiment of this invention, showing... AspiMYB5 and AspiPKS6 Changes in fluorescence signal during promoter co-expression.

[0023] Figure 5 This is a graph showing the quantitative analysis results of relative luciferase activity in the dual-luciferase reporter assay of this invention. AspiMYB5 right AspiPKS6 Inhibition of promoter activity. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, enzymes, competent cells, plasmids, etc. used are all commercially available.

[0026] Example 1: Tree fern AspiMYB5 Transcription factors and AspiPKS6 One-to-one verification of yeast single-hybrid genes (1.1) AspiMYB5 and AspiPKS6 Cloning of gene sequences The pith, xylem, phloem, and sclerenchyma of the tree fern stem were separated. RNA was extracted from these four parts using the Plant RNA Extraction Kit (AG21019) from Aikerui Biotechnology Co., Ltd., and cDNA was obtained by reverse transcription using the Evo M-MLV Premixed Reverse Transcription Kit from Aikerui Biotechnology Co., Ltd. Based on the tree fern genome sequence information, gene-specific primers were designed at both ends of the target gene sequence and synthesized by Beijing Qingke Biotechnology Co., Ltd., to obtain the necessary primers for... AspiMYB5 and AspiPKS6 The upstream and downstream primer sequences for gene cloning are shown in Table 1.

[0027] Table 1 is used for AspiMYB5 and AspiPKS6 Upstream and downstream primer sequences for gene cloning

[0028] The target sequence was obtained by polymerase chain reaction (PCR) using the primers described above. The PCR product was then separated into target bands by agarose gel electrophoresis. The target fragment was recovered from the gel using a gel recovery kit from Kangwei Century Biotechnology Co., Ltd.

[0029] (1.2) AspiMYB5 and AspiPKS6 Construction of gene vectors 1.2.1 The plant expression vectors pGADT7-Rec2 and pHis-Rec2 were double-digested. The specific reaction system and reaction conditions are as follows: a. pGADT7-Rec2 enzyme digestion system:

[0030] b. pHis-Rec2 enzyme digestion system:

[0031] c. Enzyme digestion conditions: digestion temperature 37 ℃, digestion time 15 min, hot cap temperature 60 ℃.

[0032] 1.2.2 After enzyme digestion, the target band was separated by electrophoresis, and the target band was recovered using a gel extraction kit produced by Beijing Qingke Biotechnology Co., Ltd. The target band was then ligated using Yugong recombinant ligase. AspiMYB5 It was loaded into the plant expression vector pGADT7-Rec2; AspiPKS6 The contents were loaded into the plant expression vector pHis-rec2, and the specific reaction system and conditions are as follows: a. Connecting the reaction system:

[0033] b. Connection conditions: Temperature 50 ℃, processing time 15 min.

[0034] 1.2.3. Thaw DH5α competent cells on ice. Add all the ligation products to the competent cells and mix thoroughly. Incubate on ice for 30 min, then heat shock at 42 ℃ for 90 s, and then incubate on ice for 2 min. Add 750 μL of antibiotic-free LB medium and activate at 37 ℃ and 220 rpm for one hour. Collect the cells at a low rotation speed of 4000 rpm and... ​ The bacterial cells were spread on solid LB medium (containing 50 mg / L ampicillin); ​ The clones were plated on solid LB medium (containing 50 mg / L kanamycin) and incubated overnight at 37 °C. The next day, the obtained single clones were identified by PCR using vector primers. The primer sequences are shown in Table 2.

[0035] Table 2 Primer Sequences

[0036] 1.2.4. Electrophoretic analysis of PCR products was performed to identify positive clones. Positive single clones were picked and cultured overnight at 220 rpm and 37 ℃ in 10 mL LB medium (kanamycin, 50 mg / L; ampicillin, 50 mg / L). The next day, the bacterial strain was preserved and the cells were collected by centrifugation for plasmid extraction. The specific procedure was performed according to the instructions of the Qingke Bioparticle Small Plasmid Extraction Kit.

[0037] 1.2.5. The coding sequence of the mouse tumor suppressor protein p53 was cloned into the pGADT7-Rec2 vector and pHis-Rec2, and co-transformed into yeast as a positive control for yeast monohybridization. Undigested pGADT7-Rec2 and... ​ The vector was co-transformed into yeast as a negative control for yeast monohybridization.

[0038] (1.3) ​ and ​ Cotransfer dual-deficient culture medium 1.3.1 Take 4 tubes of 100µL Y187 yeast competent cells thawed on ice, and add 5µL of pre-cooled plasmid to each tube. ​ carrier, ​ The vector or p53 positive control, pGADT7-Rec2 empty vector negative control, etc.), 10µL of pretreated carrier DNA (heated at 95-100 ℃ for 5 min, then rapidly placed on ice, and the above procedure was repeated once), and 500µl of PEG / LiAc, were pipetted and mixed several times, and then incubated in a 30 ℃ water bath for 30 min (inverted 6-8 times to mix after 15 min of incubation).

[0039] 1.3.2 Place the tube in a 42 ℃ water bath for 15 min (invert 6-8 times during the 7.5 min water bath to mix thoroughly).

[0040] 1.3.3 After centrifugation at 5000 rpm for 40 s, the supernatant was discarded, the precipitate was resuspended in 400 µL ddH2O, and centrifuged again for 30 s before discarding the supernatant.

[0041] 1.3.4. The precipitate was resuspended in 50 µL ddH2O, then spread on yeast double-deficient medium and incubated at 29 °C for 48-96 h.

[0042] (1.4) ​ and ​ Cotransfer of three-defect culture medium Single clones from the double-deficient medium were transferred to 500 µl of double-deficient liquid medium and incubated overnight at 29 °C. Electrophoretic analysis of the cultured yeast culture was performed to identify positive clones. The yeast culture was diluted with 0.9% physiological saline to an OD value of [missing value]. 600 5 µl of yeast liquid was spotted sequentially onto the three-deficient medium at concentrations of 0.2, 0.02, and 0.002. The order was: experimental group, negative control, positive control. The three-deficient medium required the addition of a certain amount of 3-amino-1,2,4-triazole (3AT) at concentrations of 10, 20, 50, 70, and 75 mM. At a concentration of 75 mM, it was confirmed that the negative self-activation control did not produce yeast, while the experimental group and positive control showed normal yeast growth. ​ and ​ Genes can interact.

[0043] The ​ The gene sequence is SEQ ID No. 1:

[0044] The ​ The gene sequence is SEQ ID No. 2:

[0045] ​ This invention serves as a yeast one-hybrid experiment verification. ​ Transcription factors and ​ A diagram showing the results of one-to-one interaction between promoters. ​ A represents the core interaction fragment of the key enzyme gene in yeast single-hybrid screening. The fragments were split into 3, 4, and 3 segments respectively, with some overlap between each pair. The final selected interaction fragment is the end of the whole fragment. ​ Results B show that in the first segmentation, the interacting fragment was located in segment 4; in the second segmentation, the interacting fragment was located in segment 3; and in the third segmentation, the interacting fragment was located in segment 3. These three validation results indicate that yeast in the dual-deficient culture medium can grow normally, while only the experimental group and the positive control group yeast can grow normally in the triple-deficient selection culture medium containing different concentrations of 3AT.

[0046] Example 2: Tree fern ​ Transcription factors and ​ Electrophoretic gel migration assay of genes (2.1) Construction of prokaryotic expression vectors 2.1.1 The prokaryotic expression vector pET-28a(+) was subjected to double enzyme digestion. The specific reaction system and reaction conditions are as follows: a. pET-28a(+) enzyme digestion system:

[0047] b. Enzyme digestion conditions: Enzyme digestion temperature 37 ℃, enzyme digestion time 15 min, hot cap temperature 60 ℃.

[0048] 2.1.2 After enzyme digestion, the target band was separated by electrophoresis and recovered using the Qingke Biogel Recovery Kit. The target band was then ligated using Okclone ligase. ​ The contents were inserted into the plant expression vector pET-28a(+), and the specific reaction system and conditions are as follows: pET-28a(+) enzyme ligation system:

[0049] Connection conditions: temperature 50 ℃, processing time 15 min.

[0050] 2.1.3. Transform the plasmid into DH5α competent cells, following step 1.2.3. Spread the plasmid in LB medium containing kanamycin resistance and incubate overnight at 37 °C. Pick single clones from the medium for PCR electrophoresis to verify positive clones.

[0051] (2.2) Protein purification 2.2.1 Prepare two 500 mL bottles of LB culture medium. Take 2 × 100 µL of... ​ The carrier bacterial culture was added to 2×10mL LB medium (kanamycin, 50 mg / L) and cultured overnight.

[0052] 2.2.2 Add the overnight cultured bacterial suspension to 500 mL of culture medium and incubate at 37 ℃ and 220 rpm until OD... 600 When the concentration was 0.8, the sample was retained. Then, 0.3 mM IPTG was added, and the mixture was incubated overnight at 16 ℃ and 120 rpm to induce expression. After induction, the sample was retained again.

[0053] 2.2.3. After induction, aliquot the culture medium into 50 mL centrifuge tubes and centrifuge at high speed and low temperature. Centrifugation conditions: 10000 rpm, 4 ℃, centrifuge for 10 min, and discard the supernatant.

[0054] 2.2.4. Resuspend the precipitate in 30 mL of washing buffer and place on ice for later use. Add 1 mL of lysozyme and 300 μL of PMSF sequentially.

[0055] 2.2.5. Place on ice and sonicate for 30 min (45% ultrasonic power). The ultrasonic program is: 5 s operation, 15 s interval, 3-5 intermittent cycles, with each sonication session lasting 1-2 min. After disruption, centrifuge at high speed and low temperature under the following conditions: 10000 rpm, 4 ℃, for 40 min. Retain samples of the supernatant and precipitate separately.

[0056] 2.2.6 Wash 1.5 mL of beads 3-5 times with 1×PBS, and centrifuge at 1000 rpm and 4 ℃ for 1 min. Add 1.5 mL of Ni-NTA beads suspension to every 30 mL of supernatant and incubate overnight at 4 ℃ by rotation.

[0057] 2.2.7. Transfer the liquid to a 50 mL centrifuge tube and centrifuge at 1000 rpm and 4 ℃ for 5 min to remove most of the supernatant. Then aliquot the liquid into 2 mL centrifuge tubes and centrifuge again at 1000 rpm and 4 ℃ for 5 min.

[0058] 2.2.8 Wash the precipitate 3-5 times with washing buffer, centrifuging at 1000 rpm, 4 ℃, for 1 min. Retain the sample and discard the supernatant. Add elution buffer to the precipitate and incubate at 4 ℃ for 2 h. After incubation, centrifuge at high speed and low temperature at 1000 rpm, 4 ℃, for 5 min. Collect the supernatant and retain the precipitate as a sample.

[0059] 2.2.9. Determine the concentration of the supernatant. Centrifuge at 5000 rpm and 4 ℃ for 10 min to concentrate the concentration.

[0060] The reagent specifications used in step (2.2) are as follows:

[0061] (2.3) Probe preparation and EMSA experiment 2.3.1 ​ Promoter probe design: based on ​ Probes were designed based on the MYB binding element in the gene promoter region, and the binding region was analyzed using yeast single-hybrid assays. ​ The speed was gradually reduced to 50 bp.

[0062] 2.3.2 Electrophoretic Mobility Variation Analysis (EMSA) Experiment. The probe sequences used for the EMSA experiment are shown in Table 3.

[0063] Table 3 Probe sequences used for EMSA experiments

[0064] The reaction system and conditions are as follows: Reaction system:

[0065] Reaction conditions: Incubate at 37 °C for 30 min; then add 0.5 μL of 0.2M EDTA to terminate the reaction.

[0066] ​ This invention provides experimental verification of electrophoretic mobility variation analysis (EMSA). ​ Protein and ​ The diagram shows the results of the promoter's in vitro physical binding. Lane 1 shows the protein migrating downwards without probe binding; lane 2 shows the protein binding to the tag probe, resulting in stagnation; lanes 3, 4, and 5 have competitive probes added, leading to decreased tag probe binding to the protein; lane 6 shows the protein binding to the mutant probe, with no binding observed. This indicates that the screened... ​ core fragments and ​ In vitro interaction was performed.

[0067] Example 3: Tree Fern ​ , ​ Obtaining transgenic plants and the regulatory mechanisms of transcription factors (3.1) ​ Creation of transgenic Arabidopsis thaliana through overexpression 3.1.1 Building Pro ​ pBI121- ​ carrier Amplification using the Golden Gate cloning method ​ The gene CDS sequence was obtained, and the enzyme used was the Type IIS restriction enzyme (BsaI). Primer sequences are shown in Table 4.

[0068] Table 4 Primer sequences

[0069] Note: In the primer sequences, lowercase letters indicate BsaI restriction endonuclease recognition sites. Uppercase letters indicate gene-specific binding regions. The tetra-base sticky ends (CTCG and ACCG) shown are designed for targeted cloning of fragments into recipient vectors.

[0070] a. Amplification ​ Reaction system:

[0071] b. Carrier enzymatic digestion reaction system:

[0072] c. Enzyme digestion-ligation reaction:

[0073] Reaction conditions: First, treat at 37 °C for 5 min; then treat at 20 °C for 5 min; repeat the above process 15 times.

[0074] d. Transform the ligation product into *E. coli* DH5α, following steps 1, 2, and 3. The following day, perform PCR identification of the obtained single clones using the vector primers. The primers used are shown in Table 5. Table 5 Primer Sequences

[0075] e. Perform electrophoretic analysis on the PCR products to identify positive clones. Pick a single positive clone and incubate it overnight at 220 rpm and 37 ℃ in 10 mL LB medium (kanamycin, 50 mg / L). The next day, preserve the bacterial strain and collect the bacterial cells by centrifugation for plasmid extraction. Refer to the instructions of the Qingke Bioplasma Small Plasmid Extraction Kit for specific procedures. Sequencing primers RB-R / Lac-F are used for sequencing. If the sequencing result matches the target fragment sequence, it is considered Pro. ​ pBI121- ​ The vector sequence has been constructed.

[0076] 3.1.2. The built Pro ​ pBI121- ​ Vector transferred into Agrobacterium GV3101 Thaw 100 μL of GV3101 competent cells on ice; add 10 μL of the target plasmid, gently aspirate and mix, incubate on ice for 5 min; flash freeze in liquid nitrogen for 5 min, incubate at 37 ℃ for 5 min, then immediately incubate on ice for 5 min; add to 700 μL of LB medium and incubate at 28 ℃ and 220 rpm for 2-3 h with shaking; then plate on LB acclimation plates containing Rif+Kana. Pick single clones for testing and submit for sequencing. Successful sequencing indicates GV3101-35s. ​ Vector construction successful. GV3101-35s was retrieved. ​ Agrobacterium-mediated bacterial suspension was cultured in 30 mL LB liquid medium (Kana and Rif resistant) with shaking for 2-3 days until the suspension turned orange. The supernatant was discarded by centrifugation, and the suspension was resuspended in 1 / 2 MS osmotic medium until the OD600 of the resuspended suspension was approximately 0.6.

[0077] 3.1.3 Obtain transgenic Arabidopsis thaliana. Remove the siliques that have already formed. Immerse the flower buds completely in the resuspension for 30 seconds, then remove them. After the first infection, repeat the infection once a week, for two to three times. Each time, place the infected plants horizontally, seal them with plastic wrap to retain moisture, and incubate them in the dark for 24 hours before transferring them to an incubator. Infect them again after 7 days. After the Arabidopsis thaliana siliques turn yellow and dry, harvest the seeds, and then collect the T0 generation seeds.

[0078] 3.1.4 In order to obtain homozygotes, after a growth cycle of about 30 days, T0 generation seeds are sown and T1 generation seeds are harvested by dividing the plants according to the same cycle. T1 generation seeds are then sown to obtain T2 generation seedlings. T2 generation transgenic Arabidopsis seeds are harvested. The same method is used to sow and screen to T3 generation. Homozygous T3 generation seedlings and seeds are harvested for later use.

[0079] (3.2) ​ Creation of transgenic Arabidopsis thaliana through overexpression Construct pBI121- ​ The vector and primer sequences are shown in Table 6.

[0080] Table 6 Primer Sequences

[0081] The amplification, vector digestion, and vector ligation system were performed in the same manner as in step 3.1.1. The ligation product was transformed into *E. coli* DH5α. The next day, the obtained single clones were identified by PCR using the vector primers. The primer sequences are shown in Table 7.

[0082] Table 7 Primer Sequences

[0083] Positive clones were identified, the bacterial strain was preserved, and plasmids were extracted. Sequencing was performed using RB-R / Lac-F primers. The sequence aligned with the target fragment sequence, indicating it was pBI121-. ​ Vector sequence construction is complete. The constructed pBI121- ​ The vector was transferred into Agrobacterium GV3101, following step 3.1.2. This was done directly into the pre-created... ​ The inflorescences were infected onto Arabidopsis thaliana plants. ​ The flower buds of the transgenic material were completely immersed in pBI121- ​ Prepare Agrobacterium resuspension, following step 3.1.3. Collect homozygous T3 generation seedlings and seeds for later use.

[0084] (3.3) ​ Research on the regulatory mechanism 3.3.1 Each transgenic plantlet was cultivated into 4 lines, with 3-4 plants per line. Stems 5-7 cm below the flower bud were selected from T3 generation Arabidopsis seedlings and placed into enzyme-free 1.5 mL centrifuge tubes. One tube was prepared for each line, for a total of 8 tubes. These tubes were named... ​ -1~ ​ -4、 ​ -1~ ​ -4°C, place in a cryogenic grinder, and grind into powder. RNA was extracted at low temperature using the Qingke Bio RNA Extraction Kit, and reverse transcribed into cDNA using the Evo M-MLV Reverse Transcription Premixed Kit from AG Biotechnology, then stored at -20°C. Real-time quantitative polymerase chain reaction (qPCR) experiments were performed using the SYBR Green Pro Taq HS Premixed qPCR Kit from AG Biotechnology; primer sequences are shown in Table 8. Arabidopsis thaliana Actin2 was used as an internal control gene to eliminate loading errors. Finally, the data were analyzed by ΔC. t and Normalization processing. Results show that the transition... ​ In the subsequent genetically modified materials, ​ The content of [something] decreased significantly.

[0085] Table 8 Primer Sequences

[0086] ​ This invention relates to the transcriptional level of plants overexpressing AspiPKS6 and... ​ / ​ Figure showing the transcriptional level identification results of dual overexpression transgenic plants. There are four groups in total; the first group consists of wild-type WT Arabidopsis thaliana. ​ The content, the remaining three groups are three different strains, and the left column in each group is ​Overexpressing plants, right side ​ / ​ Double overexpression transgenic plants. It can be seen that... ​ It was successfully transferred into wild-type Arabidopsis thaliana that does not express this gene, and in ​ Under the influence of ​ Genes can be suppressed in plants, reducing their expression levels.

[0087] 3.3.2 Constructing pGreenII 0800-LUC- ​ pro, pGreenII 62-SK- ​ carrier a. The plant expression vectors pGreenII 0800-LUC and pGreenII 62-SK were double-digested with enzymes. The specific reaction system and reaction conditions are as follows: pGreenII 0800-LUC and pGreenII 62-SK digestion system:

[0088] Enzyme digestion conditions: digestion temperature 37 ℃, digestion time 15 min, hot capping temperature 60 ℃.

[0089] b. After enzyme digestion, the target band was separated by electrophoresis and recovered using the Qingke Biogel Recovery Kit. The target band was then ligated using Yugong Recombinant Ligase. ​ It was loaded into the plant expression vector pGADT7-Rec2; ​ The contents were loaded into the plant expression vector pHis-rec2, and the specific reaction system and conditions are as follows:

[0090] Connection conditions: temperature 50 ℃, processing time 15 min.

[0091] c. Transform into competent DH5α cells, following steps 1, 2, and 3. The following day, perform PCR identification of the obtained monoclonal antibodies using the vector primers, as shown in Table 9.

[0092] Table 9 Primer Sequences

[0093] d. Perform electrophoretic analysis on the PCR products to identify positive clones. Incubate the positive clones overnight to preserve the bacterial strain and extract plasmids.

[0094] 3.3.3 The successfully constructed pGreenII 0800-LUC- ​ pro plasmid and pGreenII 62-SK- ​The plasmid was co-transformed into Agrobacterium tumefaciens GV3101, and the recombinant plasmid was combined with the reporter vector LUC-pro using an Agrobacterium-mediated transient expression system for tobacco. ​ The drugs were injected into leaves of 4-6 week old Nicotiana benthamiana. Fluorescence was detected 48 hours post-injection using an in vivo imaging system.

[0095] ​ This is a transient fluorescence imaging image of the dual-luciferase reporter experiment in *Nicotiana benthamiana* leaves. The left side shows the empty vector and AspiPKS6 co-transformed into *Agrobacterium* followed by injection into tobacco leaves; the right side shows... AspiMYB5 Effect plasmids and AspiPKS6 The report describes how plasmids were co-transformed into Agrobacterium and injected into tobacco leaves. It can be seen that the fluorescence intensity on the right side is significantly lower than that on the left. The results show that when... AspiMYB5 and AspiPKS6 When expressing together, AspiPKS6 The promoter-driven fluorescence signal was significantly weakened, indicating that AspiMYB5 Repressed promoter-driven transcription ， Reduced AspiPKS6 Gene expression. Confirmation of transcription factors. AspiMYB5 They form a complex through protein-protein interactions, jointly participating in the regulation of key genes involved in the production of milkweed alkaloids, a precursor to tree fern phenols. AspiPKS6 Transcription. Milk alkaloids are a key precursor in the synthesis of tree fern phenols, therefore... AspiMYB5 against AspiPKS6 Negative regulation was implemented, which affected the synthesis of tree fern phenol.

[0096] Figure 5 This is a graph showing the quantitative analysis results of relative luciferase activity in the dual-luciferase reporter experiment of this invention. Quantitative analysis of the relative fluorescence intensity on the left and right sides of the tobacco leaf shows that... AspiMYB5 Under the influence of AspiPKS6 The expression decreased significantly, confirming AspiMYB5 transcription factors AspiPKS6 Key enzyme genes play a negative regulatory role.

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Tree ferns used to regulate the biosynthesis of tree ferns. AspiMYB5 Genes, characterized by, The AspiMYB5 The nucleotide sequence of the gene is shown in SEQ ID No.

2.

2. The tree fern according to claim 1 AspiMYB5 The application of genes in regulating the biosynthesis of tree fern phenols is characterized by, By regulating the plant's internal structure AspiMYB5 The expression level of the gene, thereby regulating the key enzyme gene of tree fern phenol. AspiPKS6 The expression of and the synthesis of tree fern phenol.

3. The application according to claim 2, characterized in that, The AspiMYB5 Genes used to negatively regulate key enzyme genes of tree fern phenol AspiPKS6 The expression of this substance reduces the content of milkweed alkaloid, the precursor of tree fern alkaloids, thereby inhibiting the synthesis of tree fern alkaloids.

4. The application according to claim 2, characterized in that, use AspiMYB5 The method for regulating the biosynthesis of tree fern phenols by gene is as follows: constructing a system containing... AspiMYB5 A gene recombinant vector is used to transfer the recombinant vector into a plant to induce gene recombinant expression. AspiMYB5 Overexpression inhibits the synthesis of tree fernol.

5. A recombinant vector, characterized in that, The recombinant vector comprises the one described in claim 1. AspiMYB5 Gene.

6. The recombinant vector according to claim 5, characterized in that, The recombinant vector is selected from pGADT7-Rec2, pET-28a(+) or pBI121.

7. Used for verification AspiMYB5 and AspiPKS6 Engineered bacteria with interactive relationships are characterized by, The engineered bacteria contain the recombinant vector as described in claim 5 or 6.

8. The engineered bacteria according to claim 7, characterized in that, The engineered bacteria are selected from Escherichia coli, yeast, or Agrobacterium.

9. The method according to claim 1 AspiMYB5 Application of genes in screening or identifying substances that regulate the biosynthesis pathway of tree fern phenols.

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