Regulatory gene loc108210899 for synthesis of carrot terpenoids and application thereof

Abstract

The application discloses a kind of carrot terpenoid compound synthesis's regulation gene LOC108210899 And application.The present application belongs to the field of biotechnology, the regulatory protein LOC108210899 activity or content or the expression of the coding gene of the regulatory protein can be applied in any one of the following: 1) application in the regulation of plant terpenoid compound synthesis;2) application in the preparation of the product for regulating plant terpenoid compound synthesis;3) application in the cultivation of plant with changed terpenoid compound synthesis capacity;4) application in the preparation of the product for cultivating plant with changed terpenoid compound synthesis capacity.Overexpression vector is used to make LOC108210899 Gene overexpression, or gene editing vector is used to make LOC108210899 LOC100288978 LOC100288978 LOC100288978 LOC100 Gene no expression, plant variety breeding is carried out, and carrot variety with controllable cis-ubiquinol content is obtained.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a regulatory gene for the synthesis of carotene terpenoids. LOC108210899 and its applications. Background Technology

[0002] Carrots are an important global economic crop, rich in nutrients. Their fleshy roots are rich in carotenoids, vitamins, and dietary fiber, among other nutrients, and possess certain nutritional and medicinal value, earning them the nickname "little ginseng." Terpenoids are typically the main components of plant essential oils, resins, and floral aromas, and are highly correlated with the sensory variables of carrots (terpene flavor, green flavor, earthy flavor, bitterness, and aftertaste).

[0003] Cis-Abienol is a bicyclic oxygen-containing diterpene alcohol, the same substance or isomer as cis-abienol 4 reported in balsam fir. Cis-Abienol, along with other plant-derived oxygen-containing diterpenes such as sclareol and manool, is a key precursor in the synthesis of Ambrox®, a substitute for ambergris, in the fragrance industry, and has significant economic value. The biosynthesis of cis-panthenol begins with isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are converted to farnesyl diphosphate (FPP) by farnesyl pyrophosphate synthase (ispA / Erg20). Subsequently, geraniylgeraniyl pyrophosphate synthase (CrtE) catalyzes the formation of geraniylgeraniyl diphosphate (GGPP). GGPP is then cyclized by diterpene synthase type II (diTPS) to generate the intermediate lap-13-enol diphosphate (LDPP), which is finally catalyzed by cis-panthenol synthase (Cas) to form cis-panthenol.

[0004] Therefore, regulating the content of terpenoids such as cis-panthenol in carrots through genetic engineering is an effective measure to improve the sensory quality of carrots, and discovering new transcription factors that regulate the synthesis of terpenoids can help regulate the content of terpenoids. Summary of the Invention

[0005] The main problem this invention aims to solve is how to regulate the synthesis of terpenoids in carrots.

[0006] To address the aforementioned problems, the present invention provides the use of a substance that regulates the activity or content of protein LOC108210899 or regulates the expression of the gene encoding said protein in any of the following ways: 1) Application in regulating the synthesis of plant terpenoids; 2) Applications in the preparation of products that regulate the synthesis of plant terpenoids; 3) Applications in cultivating plants with altered terpene compound synthesis capabilities; 4) Applications in the preparation of products from plants whose ability to synthesize terpenoids has been altered; The protein may be any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No:1; a2) A protein with the same function as the amino acid sequence shown in a1) by substitution and / or deletion and / or addition of one or more amino acid residues; a3) Proteins that share more than 80% identity with the amino acid sequence defined by a1) or a2) and have the same function; a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3).

[0007] The amino acid sequence of SEQ ID No: 1 is: MEVAKVVTCAGETVVVMLLVALVNAHLSALLLRDFFQGFGEYPIHNEDDYISSATKVLDAEGVEDDDNDDDEDGDFDEGEEDPSSEDYGGKSNNKENSKNGSGKGDKQGNGDEEDEEEDDEDGDEDGDDQNNDDDEEEDDDDDEGDKTEEVEDKDEAEEEEEEEDEEIQPPKKRKK.

[0008] To facilitate the purification or detection of protein LOC108210899 in a1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No:1 in the sequence listing.

[0009] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0010] The protein LOC108210899 mentioned above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0011] In this article, the regulation can be upregulation, enhancement, or increase. The regulation can also be inhibition, reduction, or downregulation.

[0012] In this article, the upregulation, enhancement, or increase of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase, or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0013] In this article, regulating the expression of the gene encoding the protein can also be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0014] Furthermore, the substance may be any of the following: c1) The nucleic acid molecule encoding the protein LOC108210899 described above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1); c5) Transgenic plant cell lines containing the nucleic acid molecules described in c1); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1); c7) Transgenic plant organs containing the nucleic acid molecules described in c1).

[0015] Furthermore, the nucleic acid molecule described in c1) can be any of the following DNA molecules: d1) The coding region sequence is the DNA molecule shown in SEQ ID No:2; d2) The nucleotide sequence is the DNA molecule shown in SEQ ID No:3; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein described above; d4) A DNA molecule that hybridizes under strict conditions with a nucleotide sequence defined by d1) or d2) and encodes the protein described above.

[0016] The present invention also provides a method for breeding plants with reduced terpene compound synthesis capacity, comprising enhancing the activity and / or content of the aforementioned protein LOC108210899 in the target plant, or / and increasing the expression level of the gene encoding the aforementioned protein LOC108210899, to obtain plants with reduced terpene compound synthesis capacity, wherein the terpene compound synthesis capacity of the plants with reduced terpene compound synthesis capacity is weaker than that of the recipient plant.

[0017] Furthermore, the method includes the following steps: (1) Construct a recombinant expression vector for the gene encoding the protein LOC108210899 described above; (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant whose ability to synthesize terpenoids is weaker than that of the recipient plant.

[0018] The present invention also provides a method for reducing the ability of plants to synthesize terpenoids, wherein the method involves enhancing the activity and / or content of the protein LOC108210899 mentioned above in the target plant, or / and increasing the expression level of the gene encoding the protein mentioned above, thereby reducing the ability of plants to synthesize terpenoids.

[0019] The present invention also provides a method for improving the ability of plants to synthesize terpenoids, the method comprising, in order to inhibit the activity and / or content of the protein LOC108210899 mentioned above in the target plant, or / and, reducing the expression level of the gene encoding the protein LOC108210899 mentioned above, thereby improving the ability of plants to synthesize terpenoids.

[0020] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0021] In this article, the plant may be carrot or Arabidopsis thaliana.

[0022] In this article, the terpene compound is cis-panthenol.

[0023] This invention clones the cis-panthenol regulatory gene in "Japanese Three Reds". LOC108210899 Recombinant expression vectors and overexpression vectors were constructed; the results were obtained through the flower-dipping method. LOC108210899 The content of cis-panthenol was reduced in transgenic Arabidopsis thaliana positive plants. Therefore, overexpression vectors can be used to... LOC108210899 Gene overexpression, or the use of gene editing vectors to... LOC108210899 The gene was not expressed, so plant breeding was carried out to obtain carrot varieties with controllable cis-panthenol content. Attached Figure Description

[0024] Figure 1 The expression levels of the LOC108210899 gene and its neighboring genes.

[0025] Figure 2The expression levels of the LOC108210899 gene in different growth and development stages and tissues of "Japanese Three Red" were analyzed.

[0026] Figure 3 The localization of LOC108210899 protein in tobacco epidermal cells.

[0027] Figure 4 In situ RNA hybridization of the LOC108210899 gene in 120-day-old fleshy roots. Wherein Ep: epidermis; Ph: phloem; Xy: xylem (scale bar is 2000 μm).

[0028] Figure 5 The relative luciferase activity of the LOC108210899 gene is shown. The BD group was co-transfected with pGreenII 0800-35S-5×GAL4-LUC and pGreenII 62-SK-BD; the BD-VP16 group was co-transfected with pGreenII 0800-35S-5×GAL4-LUC and pGreenII 62-SK-BD-VP16; the BD-0899 group was co-transfected with pGreenII 0800-35S-5×GAL4-LUC and pGreenII 62-SK-BD-0899; and the VP16-0899 group was co-transfected with pGreenII 0800-35S-5×GAL4-LUC and pGreenII 62-SK-BD-VP16-0899.

[0029] Figure 6 Construction of an overexpression vector for the LOC108210899 gene.

[0030] Figure 7 The expression level of LOC108210899 in transgenic Arabidopsis thaliana is shown. The WT group consists of wild-type Arabidopsis thaliana, while the other groups are T1 generation overexpressing Arabidopsis thaliana plants.

[0031] Figure 8 The content of cis-panthenol in transgenic Arabidopsis thaliana LOC108210899 was determined. The WT group consisted of wild-type Arabidopsis thaliana, while the LOC108210899 group consisted of T1 generation overexpressing Arabidopsis thaliana plants. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0035] The *Benjamina* oleracea var. *capitata* with the mCherry nuclear localization marker required for transient expression in the following embodiments was provided by Researcher Shuhua Huang of the Shaanxi Hybrid Rapeseed Research Center, and is described in: Shen Z, Huang S, Guo M, Zhang Y, Xu Z. The BoMIEL1-BoMYB96 Module Responds to ABA Signals to Enhance the Drought Tolerance of Cabbage (Brassica oleracea var. capitata). Plant Cell Environ. 2026 Jul 1. doi: 10.1111 / pce.70721. Epub ahead of print. PMID: 42387905. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0036] The expression vector pCAMBIA1302 in the following examples was preserved by the Henan Provincial Key Laboratory of Ion Beam Bioengineering and has been documented in: Sun Hao. Functional Study of Silicon Transporter Genes in Cucumber and Tomato [D]. Northwest A&F University, 2018. DOI:10.27409 / d.cnki.gxbnu.2018.000021. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0037] In the examples below, pGreenⅡ 0800-35S-5×GAL4-LUC, pGreenⅡ 62-SK-BD, and pGreenⅡ62-SK-BD-VP16 are products from the Plant Transcription Regulation Detection Kit, which was purchased from Coolaber, catalog number MH106.

[0038] The competent Escherichia coli DH5α, Agrobacterium tumefaciens strains GV3101 and GV3101 (pSoup-p19) in the following examples were preserved by the Henan Provincial Key Laboratory of Ion Beam Bioengineering and have been described in: Sun H, Duan Y, Mitani-UenoN, Che J, Jia J, Liu J, Guo J, Ma JF, Gong H. Tomato roots have a functional silicon influx transporter but not a functional silicon efflux transporter. Plant Cell Environ. 2020 Mar;43(3):732-744. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0039] The “Japanese Three Red” seeds used in the following examples were preserved for this experiment and have been documented in: Wu, J., Gao, F., Sun, H., Kong, X., Yan, X., Zhou, H., & Chen, L. (2025). Genome-wide identification and bioinformatics analysis of WD40 gene family in carrot (Daucus carota L.). Genetic Resources and Crop Evolution, 72 , 547 - 566. The biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of the present invention and may not be used for any other purpose.

[0040] Table 1. Experimental Reagents

[0041] The following examples used statistical software to process the data. Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used to analyze the expression level of the LOC108210899 gene in different growth and development stages and tissues of "Japanese Three Red" cultivar. Different letters indicate significant differences at the P < 0.05 level. Independent samples t-tests were used to analyze relative luciferase activity. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a highly significant difference, and P < 0.001 (***) indicated a highly significant difference.

[0042] Example 1 LOC108210899 Gene expression and construction of overexpression vectors 1. LOC108210899Acquisition of genes Carrot RNA was extracted using the Omega Plant RNA Extraction Kit (Omega Bio-Tek, R6827). First-strand cDNA was synthesized according to the instructions of the EX RT kit (gDNAremover) first-strand reverse transcription kit from Beijing Zhuangmeng International Biotechnology Co., Ltd.

[0043] Based on the results of carrot mGWAS analysis and the DH1 v3.0 genome database (RefSeq: GCF_001625215.2), the following data was retrieved: LOC108210899 The nucleotide sequence of the gene was determined, and cloning primers for the candidate gene were designed using the NCBI Primer-BLAST tool: LOC108210899-1F: 5'-GACGGGCAGCTATCTCTCAC-3'; LOC108210899-1R: 5'-AGTAGGCCTGCATCACTTGT-3'. The primers were then synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0044] Using cDNA from "Japanese Three Red" carrots as a template, the cells were cloned using the high-fidelity enzyme K5 HiFi 2×PCR Master Mix. LOC108210899 Gene. After amplification, 3 μL of the amplification product was taken and the band size was detected by 1.0% agarose gel electrophoresis. The correct amplification product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0045] LOC108210899 The coding sequence (CDS) of the gene in the carrot variety "Japanese Sanhong" is SEQ ID No:2, and the protein encoding the amino acid sequence of SEQ ID No:1 is LOC108210899. In the genomic DNA of "Japanese Sanhong", the genomic gene encoding the LOC108210899 protein is shown in SEQ ID No:3 of the sequence listing.

[0046] To verify the reliability of the changes in gene expression levels, qRT-PCR technology was used. LOC108210899 Gene expression pattern analysis. Specific primers were designed using the NCBI Primer-BLAST tool and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The extracted sample cDNA was diluted 7-fold and used as a template for... Actin and 5.8S rRNA As an internal reference gene, qRT-PCR was performed according to the instructions of the Zhuangmeng 2×HQ SYBR qPCR Mix (Wihthout ROX) kit.

[0047] Table 2. qRT-PCR primer sequences

[0048] right LOC108210899 The expression levels of the gene and its neighboring genes in materials with high cis-panthenol accumulation (B039, B049, B051) and low cis-panthenol accumulation (H209, H93, HT243) were analyzed, and the results are as follows: Figure 1 As shown: LOC108210899 The expression level in the low-accumulation material H209 was significantly higher than that in the other materials, and was 3.8 times higher than that in the high-accumulation material B039.

[0049] The qRT-PCR method was used to analyze the levels of certain substances in the roots and leaves of 'Japanese Three Red' variegated plants at 60, 80, 100, and 120 days after sowing. LOC108210899 The expression pattern, the result is as follows Figure 2 As shown, LOC108210899 Expression levels in roots were higher than in leaves, showing an overall upward trend, peaking at 100 days, approximately 2.04 times that at 60 days. Expression levels in leaves were lowest at 100 days, approximately 64% of those at 60 days, before returning to the same level at 120 days.

[0050] 2. Construction of overexpression vectors Based on the pCAMBIA1302 vector and target gene sequence information ( Figure 6 Specific primers were designed for 0899-GFP-F: 5'-CACGGGGGACGAGCTCGGTACCATGGAGGTGGCGAAAGTGG-3'; 0899-GFP-R: 5'-TGCTCACCATGTCGACTCTAGACTTCTTCCTCTTCTTTGGAG-3', and the target fragment was obtained by amplification using novizan high-fidelity enzyme P505.

[0051] The pCAMBIA1302-35S-eGFP vector was linearized using restriction endonucleases KpnI and XbaI and incubated in a 37°C metal bath for 3 h. The linearization results were detected by 1.0% agarose gel electrophoresis, and bands of the correct length were recovered from the gel.

[0052] The purified target fragment and the enzyme-digested linear plasmid were ligated using Biorun 2×EasyClone Mix (Wuhan Boyuan Biotechnology Co., Ltd., #RDA01L) according to the instructions.

[0053] The ligation product was transformed into *E. coli* DH5α competent cells. Transformation of *E. coli* was performed according to the Novizumab *E. coli* competent cell instructions. The correctly sequenced bacterial plasmid was extracted using the Novizumab Plasmid Extraction Kit (Nanjing Novizumab Biotechnology Co., Ltd., DC201-01), following the instructions, to obtain the recombinant plasmid pCAMBIA1302-0899-GFP.

[0054] The structure of the pCAMBIA1302-0899-GFP vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:2 between the KpnI and XbaI restriction sites of the starting vector pCAMBIA1302-35S-eGFP, while keeping other sequences of the vector pCAMBIA1302-35S-eGFP unchanged.

[0055] The expression vector pCAMBIA1302-0899-GFP was transformed into Agrobacterium tumefaciens GV3101. The transformation of Agrobacterium tumefaciens GV3101 was performed according to the instructions of Shanghai Weidi Biotechnology GV3101 Chemically Competent Cell, and positive Agrobacterium tumefaciens GV3101 / pCAMBIA1302-0899-GFP was obtained.

[0056] 3. Instantaneous transformation of tobacco Inoculate 10 mL of LB broth (containing 50 mg / L Kan + 25 mg / L Rif) with positive Agrobacterium GV3101 / pCAMBIA1302-0899-GFP and incubate overnight at 28°C and 180 rpm in a shaker. Transfer 1 mL of the overnight culture to 50 mL of LB broth (containing 50 mg / L Kan + 25 mg / L Rif) and incubate overnight at 28°C and 180 rpm in a shaker until the culture turns orange-yellow. Transfer the culture to a 50 mL centrifuge tube and centrifuge at 4°C and 4,000 rpm for 5 min, discarding the supernatant. Resuspend the cells in 25 mL of infection buffer (sterile water + 10 mM MES + 10 mM MgCl2 + 0.1 mM AS, pH 5.6), centrifuge at 4°C and 4,000 rpm for 5 min, discarding the supernatant. Resuspend the bacterial cells in 10 mL of infection solution, adjust the OD600 value to 0.6-0.8, and incubate at room temperature for 2-6 hours. Select healthy *Nicotiana benthamiana* plants with mCherry nuclear localization markers that are approximately 4 weeks old after transplanting. Inject the bacterial solution into the underside of the tobacco leaves using a 1 mL syringe (needle removed). Incubate the injected tobacco plants in a dark, artificial climate chamber for 2-3 days. Sampling and slide preparation are performed on the injected area, and the samples are observed and photographed using a laser confocal microscope.

[0057] The results are as follows Figure 3 As shown: the empty plasmid group consists of tobacco epidermal cells transformed with the empty plasmid pCAMBIA1302, and the LOC108210899 group consists of tobacco epidermal cells transformed with the transient expression vector LOC108210899. Compared with the empty plasmid group, the LOC108210899 group showed GFP green fluorescence signal only in the cell nucleus, which overlapped with the mCherry nuclear localization marker, proving that the LOC108210899 protein is localized in the cell nucleus.

[0058] 4. In situ hybridization Tissue fixation: 120 days after sowing 'Japanese Three Red', fleshy root tissues were collected, washed with PBS, and immediately placed in plant in situ hybridization fixative for at least 12 hours, followed by vacuum pump aeration. The tissues were then stored and transported at 4℃.

[0059] Dehydration and paraffin embedding: After tissue fixation, a tissue block of about 3 mm thickness was cut from the target area in a fume hood, dehydrated by gradient alcohol, cleared with xylene, and then embedded in paraffin.

[0060] Paraffin sections: 6 μm thick sections were cut from paraffin blocks of tissue using a microtome, picked up by a spreader, and baked in a 62℃ oven for 2 hours.

[0061] Dewaxing paraffin sections to water: The sections were placed in dewaxing and clearing solution I for 15 min, dewaxing and clearing solution II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and DEPC water for soaking.

[0062] Repair and digestion: Different repair solutions were selected based on tissue type, fixation time, and marker location. Repair was performed in a water bath at 80℃ for 30 min. After natural cooling, the target tissue area was circled with a gene pen, and proteinase K (20 μg / mL) was added according to tissue and marker characteristics for digestion at 40℃ for 5 min. After rinsing with pure water, the tissue was washed three times with PBS, 5 min each time.

[0063] Prehybridization: Add prehybridization solution and incubate at 65°C for 25 min.

[0064] Hybridization: Discard the pre-hybridization solution, add the hybridization solution containing the probe, and hybridize overnight in an incubator. The hybridization temperature is 40℃.

[0065] Washing after hybridization: Wash away the hybridization solution, wash with 2×SSC at 40℃ for 10 min, wash twice with 1×SSC at 40℃ for 5 min each time, and wash with 0.5×SSC at room temperature for 10 min. If there are many non-specific hybrids, formamide washing can be added.

[0066] Add the corresponding branch probe for hybridization: Gently shake the slide dry, add 60 μL of preheated hybridization solution of the corresponding branch probe (Table 3), and place horizontally in a humidified chamber for hybridization at 40°C for 45 min. During this process, add 50 mL of 2×SSC to the bottom of the humidified chamber to prevent the slide from drying out.

[0067] Washing after hybridization: Discard the hybridization solution and rinse the slides sequentially with 2×SSC, 1×SSC, 0.5×SSC, and 0.1×SSC preheated to 40℃ for 5 min at 40℃. If there are many nonspecific hybrids, the washing time and number of washes can be increased at this step, and the formamide concentration in the hybridization solution can be adjusted.

[0068] Add the corresponding signal probe: Add hybridization solution containing the signal probe (Table 3) at a dilution ratio of 1:400, incubate at 40℃ for 3 h, and then wash sequentially with the following SSCs: 2×SSC, wash at 40℃ for 10 min, 1×SSC, wash twice at 40℃ for 5 min each time, and 0.5×SSC, wash at 40℃ for 10 min.

[0069] Add blocking solution: Add normal rabbit serum blocking solution and block at room temperature for 30 min.

[0070] Add AP-labeled mouse anti-digoxin antibody (anti-DIG-AP): Discard the serum blocking solution and add anti-DIG-AP. Incubate at 40°C for 80 min (or overnight at 4°C). Then wash four times with TBST + 0.03% SDS at 40°C for 5 min each time.

[0071] BCIP / NBT chromogenic reagent development: Add BCIP / NBT chromogenic solution and observe for positive results under a microscope. Rinse with pure water afterwards.

[0072] Mounting: Glycerin gelatin mounting.

[0073] Microscopic examination, image acquisition and analysis.

[0074] Table 3. Information on in situ hybridization probes

[0075] The results are as follows Figure 4 As shown, the in situ hybridization signal of LOC108210899 RNA in the phloem is higher than that in the epidermis and xylem, and the expression level in the phloem adjacent to the epidermis is higher than that in the inner phloem, indicating that the expression of the LOC108210899 gene in the 120-day fleshy root of "Japanese Three Red" is mainly located in the phloem tissue.

[0076] Example 2: Assay of transcriptional activity of LOC108210899 1. Carrier Construction according to LOC108210899Recombinant primers with restriction enzyme sites were designed based on the gene, pGreenII 62-SK-BD, and pGreenII 62-SK-BD-VP16 vector information (Table 4). PCR amplification was performed using the subcellular localization vector as a template. pGreenII 62-SK-BD was linearized using restriction endonucleases EcoRI and BamHI; the pGreenII 62-SK-BD-VP16 vector was linearized using restriction endonucleases BamHI and PstI. Homologous recombination was used to ligate the amplified target fragment to the linearized vector.

[0077] Table 4. Primer sequences required for construction of dual-luciferase vectors

[0078] Recombinant vector pGreenII 62-SK-BD- 0899 The structure is described as follows: a recombinant vector is obtained by inserting a DNA fragment with sequence SEQ ID No:2 between the 5'-AAGCTGATCTCAGAGGAGGACCTG-3' and 5'-GCGGCCGCTGCAGGTCGACGGATC-3' fragments of the starting vector pGreenII 62-SK-BD, while keeping the other sequences of the starting vector pGreenII 62-SK-BD unchanged.

[0079] Recombinant vector pGreenII 62-SK-BD-VP16- 0899 The structure is described as follows: a recombinant vector is obtained by inserting a DNA fragment with sequence SEQ ID No:2 between the 5'-AGTACGGTGGGCCGGGGATC-3' and 5'-TACCTTATGCGGCCGCTGCA-3' fragments of the starting vector pGreenII 62-SK-BD-VP16, while keeping the other sequences of the starting vector pGreenII 62-SK-BD-VP16 unchanged.

[0080] 2. Tobacco Injection The detection of luciferin injected into tobacco was performed according to the instructions of the UElandy Dual Luminescent Enzyme Reporter Gene Detection Kit (Suzhou UYLandy Biotechnology Co., Ltd., F6075M).

[0081] The results are as follows Figure 5As shown: The BD group exhibited basal expression, showing a significant difference from the BD-VP16 group, indicating that the experimental system was stable and reliable. The luciferase signal in tobacco leaves from the BD-0899 group showed no significant difference from that of the BD group, indicating that the LOC108210899 protein itself does not possess transcriptional activation activity. Meanwhile, the relative luciferase activity of the VP16-0899 group was significantly lower than that of the BD-VP16 group, suggesting that although LOC108210899 lacks transcriptional activation function, it can significantly inhibit VP16-mediated transcriptional activation. The LOC108210899 protein may possess transcriptional repressive activity, or its sequence may contain a transcriptional repressive domain.

[0082] 3. Genetic transformation in Arabidopsis thaliana Using the subcellular localization vector pCAMBIA1302-0899-GFP as a template, the target gene was cloned using primers, digested with restriction endonucleases BamHI and SalI, and the target fragment was ligated into the pCAMBIA1302-35s vector and transformed into Escherichia coli DH5α. The correct recombinant plasmid was then extracted and transformed into Agrobacterium tumefaciens GV3101, following the same method as in Example 1.

[0083] The primers for identifying transgenic plants are At0899-F and At0899-R as follows: At0899-F: 5'-CCTTCGCAAGACCCTTCCTC-3'; At0899-R: 5'-TGCCGGAACCATTCTTTGAG-3'.

[0084] 1) Transformation of Colombian wild-type Arabidopsis thaliana plants using the flower-dipping method Positive colonies were picked and inoculated into 4 mL of LB liquid medium (containing 50 mg / L Kan + 25 mg / L Rif), and cultured at 28°C with shaking at 200 rpm for 2 days. 2 mL of the bacterial culture was then inoculated into 100 mL of LB liquid medium (containing 50 mg / L Kan + 25 mg / L Rif), and cultured at 28°C with shaking at 200 rpm for 16-24 h, until the OD600 value reached 1.5-2.0. The cells were collected by centrifugation at 4,000 rpm for 10 min at room temperature. The cells were resuspended in an equal volume of 1 / 2 MS liquid medium, centrifuged at 4,000 rpm for 10 min at room temperature, and collected. The cells were then resuspended in an equal volume of infection buffer (2.185 g / L 1 / 2 MS powder, 50 g / L sucrose, 60 μL / L Silwet L-77, and ddH2O to a final volume of 100 mL) and incubated at room temperature for 2-3 h. Remove the open flowers and pods of Arabidopsis thaliana, and place the remaining inflorescences in the infusion solution for 30 seconds. Repeat the infusion after 15 minutes. After infusion, water the inflorescences and cover them with plastic wrap. Remove the plastic wrap after 24 hours and place them in an artificial climate chamber for normal cultivation for one week. Perform a second infusion based on the growth status of Arabidopsis thaliana. Cultivate normally until the seeds mature, and the harvested seeds are the T0 generation.

[0085] 2) Preliminary screening of positive Arabidopsis seedlings using hygromycin B Arabidopsis seeds were sterilized in a clean bench. The seeds were placed in sterile 1.5 mL centrifuge tubes and washed with 75% anhydrous ethanol by inverting for 1 min, discarding the supernatant. 1 mL of 15% sodium hypochlorite (containing 0.1% Triton X-100) was added, vortexed for 5 min, centrifuged for 1 min, and the supernatant was discarded. 1 mL of sterile water was added, vortexed, centrifuged for 1 min, and the supernatant was discarded. This washing process was repeated 2-3 times. The sterilized seeds were then air-dried on sterile filter paper and evenly scattered onto 1 / 2 MS solid medium (containing 20 mg / L hygromycin B). After 2 days of dark culture at 4°C, the seeds were transferred to a climate chamber for 6 h of light culture, followed by 2 days of dark culture, and then 24 h of continuous light culture. Seedlings with slender hypocotyls and true leaves were preliminarily identified as positive offspring in the climate chamber (16 h light / 8 h dark). Arabidopsis thaliana with 4 true leaves were transplanted into sterilized culture soil (organic substrate: vermiculite = 2:1) for further cultivation.

[0086] After the Arabidopsis seedlings were transplanted and established, DNA was extracted from the tender green leaves using the CTAB method and then subjected to PCR detection. The primers were At0899-F: 5'-CCTTCGCAAGACCCTTCCTC-3' and At0899-R: 5'-TGCCGGAACCATTCTTTGAG-3'. The identified positive Arabidopsis plants were further cultured, and some plants were used for the detection of target metabolites. At least two transgenic lines for each target gene were retained and cultured to the T2 generation for subsequent experiments.

[0087] 3) Determination of target metabolite content in transgenic Arabidopsis thaliana Sample preparation: The sample was freeze-dried under vacuum for 63 h. It was then ground into powder using a grinder (30 Hz, 1.5 min). 30 mg of the sample powder was weighed and added to 1.5 mL of 70% methanol-water internal standard extraction buffer (pre-cooled at 20℃). The sample was vortexed every 30 min for 30 s each time, for a total of 6 vortexes. The sample was centrifuged at 12000 rpm for 3 min, and the supernatant was collected. The sample was filtered through a 0.22 μm microporous membrane and stored in a vial for UPLC-MS / MS analysis.

[0088] Liquid chromatography conditions: Agilent SB-C18 column, 1.8 µm, 2.1 mm × 100 mm; mobile phase: phase A was ultrapure water (with 0.1% formic acid added), phase B was acetonitrile (with 0.1% formic acid added); elution gradient: at 0.00 min, the proportion of phase B was 5%, and within 9.00 min, the proportion of phase B increased linearly to 95% and remained at 95% for 1 min. From 10.00 to 11.10 min, the proportion of phase B decreased to 5% and equilibrated at 5% for 14 min; flow rate: 0.35 mL / min; column temperature: 40℃; injection volume: 2 μL.

[0089] Mass spectrometry conditions: Electrospray ionization (ESI) temperature 500 °C; ion spray voltage (IS) 5500 V (+) / -4500 V (-); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and collision-induced ionization parameters were set to high. QQQ scans were performed using MRM mode with the collision gas (nitrogen) set to medium. DP and CE for each MRM ion pair were optimized through further declustering potential (DP) and collision energy (CE). A specific set of MRM ion pairs was monitored at each epoch based on the metabolites eluted within each epoch.

[0090] The results are as follows Figure 7As shown, the expression level of this gene in the overexpressing plants was significantly higher than that in the WT group, indicating that all overexpressing plants were positive.

[0091] The results are as follows Figure 8 As shown, compared with the WT group, the content of cis-panthenol in the LOC108210899 group was reduced by about 9%, indicating that overexpression of the LOC108210899 gene can reduce the content of cis-panthenol in Arabidopsis thaliana.

[0092] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a substance that regulates the content of protein LOC108210899 or regulates the expression of the gene encoding said protein in any of the following: 1) Application in regulating the synthesis of plant terpenoids; 2) Applications in the preparation of products that regulate the synthesis of plant terpenoids; 3) Applications in cultivating plants with altered terpene compound synthesis capabilities; 4) Applications in the preparation of products from plants whose ability to synthesize terpenoids has been altered; The protein is any of the following proteins: a1) A protein composed of the amino acid sequence shown in MEVAKVVTCAGETVVVMLLVALVNAHLSALLLRDFFQGFGEYPIHNEDDYISSATKVLDAEGVEDDDNDDDEDGDFDEGEEDPSSEDYGGKSNNKENSKNGSGKGDKQGNGDEEDEEEDDEDGDEDGDDQNNDDDEEEDDDDDEGDKTEEVEDKDEAEEEEEEEDEEIQPPKKRKK; a2) A protein that has the same function as the amino acid sequence defined in a1) by substitution and / or deletion and / or addition of one or more amino acid residues. a3) Proteins that share more than 80% identity with the amino acid sequence defined by a1) or a2) and have the same function; a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3).

2. The application according to claim 1, characterized in that: The substance is any one of the following: c1) A nucleic acid molecule encoding the protein of claim 1; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1); c5) Transgenic plant cell lines containing the nucleic acid molecules described in c1); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1); c7) Transgenic plant organs containing the nucleic acid molecules described in c1).

3. The application according to claim 2, characterized in that: c1) The nucleic acid molecule is any of the following DNA molecules. d1) The coding region sequence is the DNA molecule shown in SEQ ID No:2; d2) The nucleotide sequence is the DNA molecule shown in SEQ ID No:

3.

4. A method for breeding plants with reduced terpene compound synthesis capacity, characterized in that: This includes increasing the content of the protein described in claim 1 in the target plant, and / or increasing the expression level of the gene encoding the protein described in claim 1, to obtain a plant with reduced terpene compound synthesis capacity, wherein the terpene compound synthesis capacity of the plant with reduced terpene compound synthesis capacity is weaker than that of the recipient plant.

5. The method according to claim 4, characterized in that: Includes the following steps: (1) Construct a recombinant expression vector that expresses the gene encoding the protein described in claim 1; (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant whose ability to synthesize terpenoids is weaker than that of the recipient plant.

6. A method for reducing the ability of plants to synthesize terpenoids, characterized in that: The method is to increase the content of the protein described in claim 1 in the target plant, or / and increase the expression level of the gene encoding the protein described in claim 1, so as to reduce the plant's ability to synthesize terpenoids.

7. A method for improving the ability of plants to synthesize terpenoids, characterized in that: The method includes increasing the plant's ability to synthesize terpenoids by inhibiting the content of the protein described in claim 1 in the target plant, and / or by reducing the expression level of the gene encoding the protein described in claim 1.

8. The application according to any one of claims 1-3, and / or the method according to any one of claims 4-7, characterized in that: The plant in question is either carrot or Arabidopsis thaliana.

9. The application according to any one of claims 1-3, and / or the method according to any one of claims 4-7, characterized in that: The terpene compound is cis-panthenol.

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