Osmanthus fragrans transport protein OfABCG13 gene, transcription factor OfERF177 gene and application of Osmanthus fragrans transport protein OfABCG13 gene and transcription factor OfERF177 gene in increasing release amount of terpenoids of plant flowers

Abstract

The invention discloses an osmanthus fragrans transporter OfABCG13 gene, a transcription factor OfERF177 gene and application of the osmanthus fragrans transporter OfABCG13 gene and the transcription factor OfERF177 gene in increasing the release amount of terpenoids of plant flowers, and belongs to the field of plant molecular biology. The invention discloses a cassia formosana transport protein OfABCG13 gene. The nucleotide sequence of the cassia formosana transport protein OfABCG13 gene is shown as SEQ-ID-NO.1; the nucleotide sequence of the osmanthus fragrans transcription factor OfERF177 gene is shown as SEQ-ID-NO.3, and the osmanthus fragrans transcription factor OfERF177 gene is an OfABCG13 negative regulation factor. An OfABCG13 gene overexpression vector is constructed, and experiments on genetic transformation of tobacco grandiflora and instantaneous transformation of sweet-scented osmanthus show that the content of terpenoids in flower fragrance of a transgenic line is obviously increased compared with that of EV. A VIGS silencing vector of the OfERF177 gene is adopted to instantaneously convert osmanthus fragrans, so that the content of terpenoids in osmanthus fragrans petals is obviously increased compared with that of EV. The invention provides a high-quality candidate gene for cultivating new varieties of flowers with strong fragrance.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biology, and specifically relates to a cinnamomum camphora transporter OfABCG13 gene, a transcription factor OfERF177 gene and applications of the two in increasing the release of terpenoid compounds in plant flowers. Background Art

[0002] Floral aroma is a complex floral phenotype produced by different volatile organic compounds (VOCs) that serve as important ingredients in the food and aroma industries. The release of VOCs also plays an important role in the ornamental and flavor qualities of horticultural crops. Typically, these compounds are synthesized within cells and must cross the plasma membrane to be released into the environment. Previous studies have shown that transporters located on the cell membrane can play an important role in VOC release. However, the molecular mechanisms of VOC release in horticultural crops remain to be further revealed.

[0003] ABC transporters (ATP-binding-cassette-transporters) are important membrane proteins that rely on ATP hydrolysis to provide energy to promote the transmembrane transport of substrates in and out of the cell. They are not only involved in the accumulation and release of secondary metabolites in plants, but also protect plants from adverse external environmental stress. Flower aroma components are an important secondary metabolite of the interaction between plants and the external environment, and their transport is likely related to ABC transporters. ERF transcription factors are widely present in plants and are involved in various stress responses, plant floral organ growth and development, and the synthesis of secondary metabolites. However, the regulatory role of ERF-transcription factors in plant volatile organic compound emissions is still unclear.

[0004] Osmanthus fragrans is a popular aromatic tree species whose flowers are rich in terpenes and terpenoid derivatives. The fragrance of Osmanthus fragrans is of great economic importance due to its unique aroma and health benefits. However, the molecular mechanism and regulatory network of Osmanthus fragrans fragrance release remain unclear. Therefore, it is very important to identify key genes and reveal the regulatory mechanism of floral fragrance release, which will help improve the fragrance quality of Osmanthus fragrans. Summary of the invention

[0005] The present invention provides a Cinnamomum cassia transporter OfABCG13 gene. The present invention also provides a transcription factor OfERF177 gene. Finally, the present invention provides the use of the above two genes in increasing the release of terpenoid compounds in plant flowers.

[0006] In view of the above problems existing in the prior art, the technical solution adopted by the present invention is as follows:

[0007] A Osmanthus fragrans var. thunbergii transport protein OfABCG13 gene, the nucleotide sequence of which is shown in SEQ-ID-NO.1.

[0008] The expressed protein of the Osmanthus fragrans var. thunbergii transport protein OfABCG13 gene, the amino acid sequence of which is shown in SEQ-ID-NO.2.

[0009] The expression cassette, recombinant vector, recombinant bacterium or recombinant cell of the Osmanthus fragrans var. thunbergii transport protein OfABCG13 gene.

[0010] A Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene, the nucleotide sequence of which is shown in SEQ-ID-NO.3.

[0011] The expressed protein of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene, the amino acid sequence of which is shown in SEQ-ID-NO.4.

[0012] The expression cassette, recombinant vector, recombinant bacterium or recombinant cell containing the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene.

[0013] The application of the described Osmanthus fragrans var. thunbergii transport protein OfABCG13 gene and / or the described Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene in increasing the release amount of terpenoids in plant flowers, and the application includes: overexpressing the Osmanthus fragrans var. thunbergii transport protein OfABCG13 gene and / or reducing the expression of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene in plants, and the plants are Osmanthus fragrans or tobacco.

[0014] In some embodiments, the terpenoids are at least one of dihydro-β-ionone, linalool, trans-linalool oxide and cis-linalool oxide.

[0015] In some embodiments, the specific steps of the application include:

[0016] (1) Construct an overexpression vector of the Osmanthus fragrans var. thunbergii transport protein OfABCG13 gene and / or a silencing vector of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene;

[0017] (2) Transform the constructed overexpression vector of the Osmanthus fragrans var. thunbergii transport protein OfABCG13 gene and / or the silencing vector of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene into plants or plant tissues;

[0018] (3) Cultivate and screen to obtain transgenic plants or plant tissues with increased release amount of aroma substances, and the plants are Osmanthus fragrans or tobacco.

[0019] In some embodiments, the overexpression vector of the Osmanthus fragrans var. thunbergii OfABCG13 gene is the pSoup1300 vector, and the gene silencing vector of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene is the VIGS vector.

[0020] Compared with the prior art, the advantages of the present invention are as follows:

[0021] Based on previous research and bioinformatics analysis software, the present invention preliminarily predicted the function of the OfABCG13 gene, obtained the full length of the gene sequence by cloning, constructed an overexpression vector on this basis, and carried out genetic transformation of Nicotiana tabacum and transient transformation of Osmanthus fragrans by Agrobacterium-mediated method. The results showed that the content of terpenoids in the floral fragrance of the OfABCG13 transgenic lines was significantly increased compared with that of the EV (Empty-Vector). In addition, the transcription factor OfERF177 that negatively regulates the OfABCG13 gene was screened through yeast library screening. Transient transformation of the Osmanthus fragrans transcription factor OfERF177 using gene silencing technology found that silencing OfERF177 significantly increased the content of terpenoids in the transiently transformed petals of Osmanthus fragrans compared with that of the EV. This application provides high-quality candidate genes and specific application methods for cultivating new varieties with rich fragrance, and has good application prospects. Description of the Drawings

[0022] Figure 1 It is the PCR amplification electrophoresis diagram of the Osmanthus fragrans OfABCG13 gene;

[0023] Figure 2-1 It is the analysis diagram of the detection results of the expression level of OfABCG13 in different lines of the T1 generation of Nicotiana tabacum overexpressing Osmanthus fragrans OfABCG13. Among them, (a) is the electrophoresis detection diagram of the successful overexpression of OfABCG13 in transgenic tobacco; (b) is the PCA model diagram of the empty vector and the transgenic tobacco lines expressing OfABCG13;

[0024] Figure 2-2 It is the detection result diagram of the terpenoid volatiles of the flowers of the T1 generation of Nicotiana tabacum overexpressing Osmanthus fragrans OfABCG13 and the control flowers of the empty vector; among them, (c) is the comparison diagram of the content of terpenoid volatiles in the flowers of the empty vector and the transgenic tobacco lines overexpressing OfABCG13; (d) is the loading diagram of the OPLS-DA analysis of the terpenoid volatiles of the flowers of the empty vector and the transgenic tobacco lines overexpressing OfABCG13;

[0025] Figure 2-3 It is the analysis and comparison diagram of the content of terpenoid volatiles generated by the difference between transgenic tobacco and EV lines;

[0026] Figure 2-4 It is the analysis and comparison diagram of the GC-MS of the terpenoid volatiles generated by the difference between transgenic tobacco and EV lines;

[0027] Figure 3-1 Figure for measuring the expression level of OfABCG13 and the content of terpenoids in Osmanthus fragrans petals overexpressing OfABCG13 transiently; among them, (a) is a comparison figure of the qRT-PCR detection results of the expression level of the empty vector and OfABCG13 in transiently transformed Osmanthus fragrans petals; (b) is a PCA model figure showing that there are significant differences in the volatile organic compounds of OfABCG13 overexpressing flowers and EV flowers; (c) is a figure for measuring the content of terpenoids in Osmanthus fragrans petals overexpressing OfABCG13 transiently and the control; (d) is a loading figure of OPLS-DA analysis of terpenoid volatiles in Osmanthus fragrans transiently transformed with the empty vector and the OfABCG13 overexpression vector;

[0028] Figure 3-2 Figure for comparing the detection results of the content of terpenoids in Osmanthus fragrans petals overexpressing OfABCG13 transiently; (e) is a detection figure of trans-linalool oxide; (f) is a detection figure of dihydro-β-ionone; (g) is a detection figure of cis-linalool oxide;

[0029] Figure 4 Figure of PCR amplification electrophoresis of Osmanthus fragrans OfERF177 gene;

[0030] Figure 5 Figure of transcription factor OfERF177 binding to the OfABCG13 promoter and inhibiting its transcription; (a) is a figure for analyzing the cis-element structure of the promoter region of OfABCG13; (b) is a figure of the results of the yeast one-hybrid experiment of the interaction between OfERF177 and the OfABCG13 promoter in vitro; (c) is a figure of the detection results of the dual-luciferase reporter gene (DLR) of OfERF177 and the OfABCG13 promoter;

[0031] Figure 6-1 Figure for measuring the content of terpenoids in Osmanthus fragrans petals with transient silencing of transcription factor OfERF177; among them, (a) is the detection results of the expression levels of OfERF177 and OfABCG13 in Osmanthus fragrans petals transiently silenced by TRV2-OfERF177; (b) is a PCA model analysis figure of VOC samples in TRV2-empty and OfERF177-silenced flowers; (c) is a comparison figure of the detection of the release amount of terpenoid volatiles in OfERF177-silenced flowers and the control; (d) is a loading figure of OPLS-DA analysis of terpenoid volatiles in TRV2-empty and OfERF177-silenced flowers;

[0032] Figure 6-2Detection result graph of the contents of the terpenoids dihydro-β-ionone and linalool in the petals of Osmanthus fragrans with transient silencing of the transcription factor OfERF177; (e) Detection and comparison graph of the terpenoid volatile release amounts of the flowers with silenced OfERF177 and the empty vector control; (f) Detection and comparison graph of the GC-MS results of the flowers with silenced OfERF177 and the empty vector control. Detailed implementation manners

[0033] The present invention will be further described below in combination with specific embodiments. For the molecular biology experimental methods not specifically described in the following embodiments, they can be referred to the methods listed in the book "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook or the conventional methods in the art, or carried out according to the kits and product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0034] The materials used in this application are the flowers of Osmanthus fragrans cv. 'Rixianggui' grown in the National Germplasm Repository of Osmanthus fragrans. The flowers of Osmanthus fragrans cv. 'Rixianggui' are placed in a sterilized centrifuge tube, immediately frozen in liquid nitrogen, and then stored in a -80°C refrigerator. The Nicotiana benthamiana seedlings used are provided by the research group of Wang Lianggui from Nanjing Forestry University.

[0035] In this embodiment, the TIANGEN plant RNA extraction kit (DP432) is used to extract the total plant RNA. The TaKaRa-PrimeScript™-RT-Master-Mix (Perfect-Real-Time) reverse transcription kit is used to reverse transcribe the extracted RNA into cDNA, and the finally obtained cDNA is diluted 10 times with water and stored in a -20°C refrigerator.

[0036] Example 1: Acquisition and functional verification of the OfABCG13 gene

[0037] 1.1: Obtain the target gene and construct the overexpression vector of the Osmanthus fragrans OfABCG13 gene

[0038] (1) Obtain the target gene

[0039] According to the previously published Osmanthus fragrans whole-genome database of the research group, 1 gene sequence was screened and named OfABCG13.

[0040] Using the cDNA diluted 10 times as a template, PCR amplification was carried out. The BioXM software was used to analyze the restriction enzyme sites of the full-length nucleotide sequence of this gene, and Sma-I and Kpn-I enzymes were selected as two restriction enzymes to perform double digestion on the pSoup1300-vector. The CE-design software was used to design primers. The primers were forward primer F-aagcttctgcaggggcccgggATGGAAATAGAGGTGGCAAGTGG and reverse primer R-gcccttgctcaccatggtaccAGGAATCGGAGAGTTTAGACCTTCT. Gene amplification was carried out by PCR. The reaction conditions were: denaturation at 98°C for 10 s; annealing at 58°C for 15 s; extension at 72°C for 1 min, for 35 cycles; total extension at 72°C for 10 min; termination of the reaction at 16°C. The obtained amplification products were subjected to agarose gel electrophoresis, and the results were as Figure 1 shown, and then a gel extraction kit was used for gel extraction and recovery.

[0041] (2)Ligation and transformation

[0042] The recovered product of the target gene was ligated with the recovered product of the plasmid double digestion, and cultured in a water bath at 37°C for 30 min and on ice for 2 min. Transformation: In a laminar flow hood, 5 μL of the ligation product was taken with a pipette and added to 50 μL of Trelief-TM-5α competent cells, gently flicked to mix evenly, ice-bathed for 5 min, water-bathed at 42°C for 60 s, then ice-bathed for 2 min, and 250 μL of liquid LB (without Kana) was added. Incubate in a shaker at 37°C and 200 rppm for 30 min.

[0043] Plating: Take 200 μL of the incubated bacterial solution, evenly coat it on the LB solid medium (containing 50 mg / L of Kana) with a sterilized glass rod and let it dry. After covering with a sealing film, invert it and culture it in a constant temperature incubator at 37°C for 12 - 14 h.

[0044] (3)Positive single colony detection and sequencing

[0045] After the bacteria grew on the culture medium, single colony detection was carried out in a laminar flow hood. For each gene, 8 plump single colonies were picked, and backup was carried out in sequence on LB solid medium containing Kana resistance. The corresponding single colonies were picked and smeared into the following system for bacterial detection: The PCR reaction conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1 min, 35 cycles; total extension at 72°C for 10 min; termination reaction at 16°C. The obtained amplification products were subjected to agarose gel electrophoresis, and 3 correct positive colonies were picked for sequencing. The nucleotide sequence of the OfABCG13 gene was shown as SEQ-ID-NO.1, with a full length of 2094 bp, and its expressed protein amino acid sequence was shown as SEQ-ID-NO.2, consisting of 697 amino acids.

[0046] 1.2: Transient transformation of osmanthus petals with overexpression vector

[0047] (1) Transformation of recombinant plasmid into Agrobacterium

[0048] Take the GV3101 competent cells stored at -80°C to room temperature. When it was in an ice-water mixture state, 1 μL of pSoup1300-OfABCG13 plasmid was added, whipped and mixed evenly, and left standing on ice for 5 minutes. Then it was placed in liquid nitrogen for 5 minutes, water bath at 37°C for 5 minutes, ice bath for 5 minutes, 500 μL of LB medium was added, cultured on a shaker at 28°C for 1 hour, centrifuged at 5000 r / min for 5 minutes, and then spread on the corresponding antibiotic LB plate, and incubated in an inverted 28°C incubator for 2 - 3 days. Single colonies were picked and positive detection was carried out using the vector universal primer. After shaking the positive single colonies, the bacterial liquid and 50% glycerol were stored according to the volume ratio of 3:7, and stored in a -80°C refrigerator after quick freezing in liquid nitrogen.

[0049] (2) Preparation of infection solution

[0050] First, weigh 0.0196 g of acetosyringone powder (AS), dissolve it in dimethyl sulfoxide in a fume hood and make up the volume to 100 mL to prepare the stock solution. Take 15 mL of the stock solution and dilute it with 85 mL of sterile water to prepare a 150 μmol·L -1 AS solution. Subsequently, 0.2035 g of MgCl2 and 0.2135 g of MES were added to this solution. The frozen pSuper1300 empty vector, P19 auxiliary expression vector and the Agrobacterium bacterial liquid containing pSuper1300-OfABCG13 were taken out from -80°C, thawed in an ice bath, and inoculated in LB liquid medium containing Kana (10 μg·mL -1 ) at a ratio of 1:100, and cultured in the dark at 28°C and 200 rpm until OD 600It is 0.6 - 0.8. Centrifuge to collect the bacterial cells (at 4°C, 5000 rpm, for 10 min), resuspend them with freshly prepared buffer, mix them according to the ratio of P19 helper vector to target gene of 5:7 (V:V), and activate for 3 h after sufficient shaking.

[0051] (3)Infection

[0052] The selected plant material is fresh flower samples of Osmanthus fragrans in the full bloom period. First, use tweezers to remove the fresh Osmanthus fragrans flowers from the branches, remove the flower stalks, place them on a gauze, and place the gauze wrapped with petals in the infection solution. Use a vacuum pump to perform vacuum infiltration for 10 minutes under the condition of -0.8 KPa, and repeat the operation three times. At this time, most of the Osmanthus fragrans petals show a transparent state. Then, take out the vacuum-infiltrated Osmanthus fragrans petals with tweezers and place them on a 5% agar solid medium, and perform the determination of aroma components by GC-MS after 48 hours of dark culture.

[0053] 1.3: Infect Nicotiana tabacum with the overexpression vector and screen to obtain transgenic plants

[0054] (1)Explant disinfection: After picking the young leaves of Nicotiana tabacum, wash the ash layer on the leaf surface with dishwashing liquid, transfer it to the ultra-clean workbench for disinfection treatment after rinsing with running water for 30 min. First, pour 75% ethanol into a beaker, shake it to make the ethanol fully contact with the surface of the young leaves for 30 s, and wash it 3 times with sterile water. Then soak it in 5% NaClO for 10 min, rinse it 4 times with sterile water, and dry the water on the leaf surface with sterile filter paper. After disinfection treatment, use a sterile scalpel to cut off the leaf edges and veins, and then cut the remaining leaves into small pieces of 0.5×0.5 cm for infection.

[0055] (2)Bacterial shaking: Take out the bacterial liquid of the pSuper1300-OfABCG13 fusion expression vector that has been transferred into Agrobacterium tumefaciens GV3101 in advance from the -80°C refrigerator. After it melts on ice, perform streak plating on the LB solid medium with Kana resistance in the ultra-clean workbench. After culturing for 48 h, select the plump single colonies grown on the solid medium for bacterial inspection. Dip the corresponding positive single colonies from the backup plate into 20 ml of LB culture solution containing Kana resistance, and perform bacterial shaking on a shaker at 28°C - 200 rpm / min until the OD 600 value is between 0.4 - 0.6.

[0056] (3)Infection: Inoculate the cut tobacco leaves with sterile tweezers into the Agrobacterium suspension with an OD 600 between 0.4 - 0.6 for 10 - 15 min, and shake the conical flask every 2 min;

[0057] (4) Co-culture: Take out the infected leaves and lay them flat on sterile filter paper. After the bacterial solution dries slightly, lay the leaves flat in the symbiotic medium and culture them in the dark at 25°C for 3 days.

[0058] (5) Screening culture: After 3 days of co-culture, transfer the leaves to the screening medium for culture and change it every about 15 days until resistant callus and resistant buds grow out.

[0059] (6) Rooting culture: When the length of the germinated adventitious buds reaches more than 5 cm, cut them from the tissue and remove the callus connected to the stem tissue completely, and then transfer them to the rooting medium to induce root growth.

[0060] (7) Seedling hardening and transplantation: When the main root of the transgenic seedling elongates to about 5 cm and 7 - 8 leaves grow out, take out the tobacco seedlings from the medium. Under the condition of not damaging the roots, wash away the agar gel remaining between the root tissues and put them into a tissue culture bottle filled with deionized water for acclimation culture for 2 days. During the acclimation period, change the water regularly to prevent pollution from damaging the tobacco seedlings. After the acclimation is completed, mark the tobacco seedlings with numbers and transplant them into the sterilized substrate soil for continuous culture.

[0061] (8) Screening of transgenic plants: When the transgenic tobacco seedlings grow for 30 days, use a kit to extract RNA from the leaves and reverse transcribe it into cDNA. Then dilute the cDNA by 10 times and perform semi-quantitative PCR detection. After determining that the quality of the cDNA is qualified, perform qRT-PCR to detect the gene expression level, and select the transgenic seedlings with high expression level and consistent growth status for subsequent functional verification.

[0062] 1.4: Determination of terpenoids in Osmanthus fragrans and tobacco flowers

[0063] The content of terpenoids in tobacco and Osmanthus fragrans is determined by gas chromatography-mass spectrometry (GC-MS). Specifically:

[0064] (1) The sampling time of transgenic Nicotiana tabacum cv. 'K326' is fixed at 9:00 - 10:00 am on sunny days. When sampling, select tobacco flowers with fully opened petals and scattered pollen and good growth status. Use every two flowers as a repeated sample, weigh each repeated sample with an analytical balance and record it in time. Immediately put the weighed sample into a 30-milliliter extraction bottle. Before determination, first prepare the internal standard ethyl decanoate. Select methanol and ethyl decanoate. Add 3 microliters of ethyl decanoate to every 9 milliliters of methanol to obtain an internal standard diluted 3000 times. Before GC-MS determination, add the sample and 1 μL of diluted internal standard to the extraction bottle, seal it, insert a 65-μm-DB / 5MS extraction head, and place it in a water bath at 60°C for headspace extraction. After 30 minutes of extraction, insert the extraction head into the GC-MS instrument to determine the aroma content.

[0065] (2) Determination of Aroma Components in Instantaneously Transformed Osmanthus Flower Petals

[0066] When determining the aroma components of transgenic Osmanthus flower petals, four technical replicates were performed for each gene. For each replicate, 0.8 g of the Osmanthus flower petal samples after vacuum infiltration were weighed and placed into a 20 mL sampling bottle. The samples were hermetically sealed and placed on ice. Then, 1 μL of ethyl caprate diluted 3000 times was added. After sealing, a 65 μm-DB / 5MS extraction head was inserted, and headspace extraction was carried out in a 45 °C water bath for 30 min. After the extraction was completed, it was inserted into the GC-MS injection port, and after 3 min of desorption, the analysis was performed.

[0067] Chromatographic analysis conditions: The chromatographic column was a DB-5MS gas chromatography column; injection mode: injection volume 1.0 μL, injection port temperature 250 °C, carrier gas: helium (purity 99.999%); carrier gas flow rate 1.0 mL / min; temperature programming: initial temperature 40 °C for 2 min, rising to 60 °C at 2 °C / min and holding for 0 s, rising to 100 °C at 5 °C / min and holding for 0 s, then rising to 250 °C at 10 °C / min and holding for 5 min. Mass spectrometry conditions: Ionization mode: electron impact (EI), ionization energy 70 eV; solvent delay: 7 min; transfer line temperature: 280 °C; ion source temperature: 250 °C; scanning mode: selected reaction monitoring mode (SIM).

[0068] The chromatographic qualitative analysis of volatile organic compounds (VOCs) was retrieved and analyzed by the data system of the Thermo instrument. By analyzing the adjusted retention time of n-alkanes and combining with the Kovats retention index analysis method, the chromatographic qualitative analysis of VOCs was further confirmed. The content of each VOCs component was calculated according to the peak area normalization method of the total ion current chromatogram. The calculation formula:

[0069] The content of each VOCs component (μg / g) = [(peak area of each species × content of internal standard in the sample) / peak area of the internal standard] / sample mass (g).

[0070] The Excel software was used to summarize and analyze the qualitative and quantitative results of each VOCs component. The SPSS software (version 25.0) and Simca (version 14.1) were used to perform significant difference analysis and principal component analysis on the data respectively.

[0071] Figures 2-1 to 2-4 It is a graph showing the determination results of the expression level of OfABCG13 and the content of terpenoids in the T1 generation of Nicotiana tabacum overexpressing OfABCG13 in Osmanthus. As shown in the figure, OfABCG13 was successfully overexpressed in transgenic tobacco ( Figure 2-1 in (a)). According to the PCA model, the OfABCG13 transgenic lines were significantly separated from the EV ( Figure 2-1In (b)), transgenic lines of OfABCG13 (OE3, 9, 10) with high expression levels of the target gene were selected for subsequent analysis. Compared with the tobacco lines transformed with the empty vector (EV), the total terpene content in the flowers overexpressing OfABCG13 increased significantly ( Figure 2-2 In (c)). In addition, OPLS-DA analysis showed that the terpene volatiles (VIP > -1 and P < -0.05) responsible for the differences between the transgenic and EV lines were diterpene cembrene, sesquiterpene β-caryophyllene, and β-caryophyllene oxide ( Figure 2-2 In (d), Figure 2-3 , Figure 2-4 ).

[0072] Figure 3 shows the results of measuring the terpene compound content in the petals of Osmanthus fragrans with transient overexpression of OfABCG13. As shown, the relative expression level of OfABCG13 in the flowers of Osmanthus fragrans was significantly higher than that of EV ( Figure 3-1 In (a)). There were significant differences in the volatile organic compounds between the flowers overexpressing OfABCG13 and those of EV ( Figure 3-1 In (b)). Similar to the tobacco results, the relative content of total terpenes in the flowers overexpressing OfABCG13 also increased significantly ( Figure 3-1 In (c)), and terpene compounds such as dihydro-β-ionone, trans-linalool oxide, and cis-linalool oxide were identified as key floral scent substances ( Figure 3-1 In (d), Figure 3-2 In (e) to (g)).

[0073] Example 2: Obtaining and functional verification of the transcription factor OfERF177 gene

[0074] The transcription factor OfERF177 that negatively regulates the OfABCG13 gene was screened through a yeast library screening. Using gene silencing technology to transiently transform the Osmanthus fragrans transcription factor OfERF177, it was found that silencing OfERF177 significantly increased the terpene content in the transiently transformed petals of Osmanthus fragrans compared with EV. The specific content is as follows:

[0075] 2.1: Screening the transcription factor OfERF177 that negatively regulates the OfABCG13 gene through a yeast library screening-

[0076] (1) Construction of -pHIS2-OfABCG13 promoter vector and screening of 3-amino-1,2,4-triazole (3-AT) concentration. The 2000 bp coding region upstream of the OfABCG13 coding region was amplified and the PCR product was inserted into the pHIS2 plasmid. Subsequently, the recombinant plasmid was introduced into yeast Y187 by chemical transformation: 100 μL of competent cells were mixed with 1-2 μg of -pHIS2-OfABCG13 promoter plasmid, 10 μL of -Carrier-DNA (denatured at 95-100 °C), and 125 μL of -PEG / LiAc, incubated at 30 °C for 30 min, heat-shocked at 42 °C for 15 min, then centrifuged, washed, and resuspended, and spread on SD / -His plates for 3-5 days. In addition, yeast cells containing the pHIS2-OfABCG13 promoter vector could grow on SD-Trp / His, but could not grow on the medium of SD-Trp / His (100 mM - 3-AT), indicating that 100 mM - 3-AT is the suitable concentration for library screening.

[0077] (2) Screening of cDNA library

[0078] The yeast library transformation experiment was carried out according to the following steps: First, a single colony of Y187 yeast containing pHIS2-OfABCG13 was inoculated into 75 mL of SD / -Trp medium and cultured with shaking at 30 °C for 16 h (250 rpm); then 500 μL of the culture was transferred to 50 mL of YPDA medium and continued to be cultured until the OD600 reached 0.15 - 0.3 (about 10 h). After collecting the cells, they were resuspended in 200 mL of fresh YPDA and cultured until the OD600 reached 0.4 - 0.5 (4 - 6 h). The cells were washed twice with sterile water and then resuspended in 2 mL of 0.1 M - LiAc and kept on ice for use. At the same time, Carrier-DNA was treated by boiling at 95 °C and ice-bathing three times and reserved. 10 μg of library plasmid DNA, 100 μL of the treated Carrier-DNA were mixed with 5 mL of -LiAc & 50% - PEG3350, incubated at 30 °C for 45 min, then 600 μL of -DMSO was added, and heat-shocked at 42 °C for 20 min. The transformed cells were collected by centrifugation, resuspended in 10 mL of YPDAplus and recovered for 90 min, and finally resuspended in 0.9% - NaCl solution and spread on SD / -THL plates and cultured at 30 °C for 3 - 7 days. Positive clones were picked and transferred to SD / -THL screening medium for continued culture for 3 - 5 days.

[0079] (3) Identification and sequencing alignment of yeast positive clones -

[0080] After the transferred bacteria grew up, they were examined. The primer sequences are as follows: -

[0081] T7: 5'-TAATACGACTCACTATAGGGCGAGCGCCGCCATG-3',

[0082] ADR: 5'-GTGAACTTGCGGGGTTTTTCAGTATCTACGATT-3'.

[0083] To identify the corresponding genes of positive clones screened by the SD / THL plate, the following steps need to be carried out: First, amplify positive clones in yeast cells and perform DNA sequencing. Subsequently, screen out the gene with the highest homology through comparison with the osmanthus genome database, and conduct BLAST verification analysis in combination with the GenBank database, thereby initially obtaining protein information with the potential to interact with the OfABCG13 promoter. This includes a member of the AP2 / ERF family, OfERF177. The nucleotide sequence of the OfERF177 gene is shown as SEQ-ID-NO.3, with a full length of 1161bp, and the amino acid sequence of its expressed protein is shown as SEQ-ID-NO.4, consisting of 386 amino acids. Figure 4 - Electrophoresis map of PCR amplification of the full-length OfERF177 gene of osmanthus, with a length of 1161bp.

[0084] 2.2: Yeast one-hybrid experiment

[0085] (1) Clone the CDS of the OfERF117 gene into pGADT7, and insert the OfABCG13 promoter sequence into the pHIS2 vector;

[0086] (2) Use 3-AT to inhibit the self-activation of the pHIS2 plasmid;

[0087] (3) Culture the Y187 yeast cells containing the recombinant pGADT7 and pHIS2 plasmids on a solid medium lacking Trp, Leu, His and containing 3-AT to determine whether they can bind to each other.

[0088] 2.3: Dual-luciferase reporter gene assay

[0089] (1) Insert the CDS of the OfERF117 gene into the pGreen-SK plasmid, and insert the 2000bp sequence of the OfABCG13 promoter into pGreen0800-Luc;

[0090] (2) The recombinant plasmids are co-transformed into tobacco leaves by the Agrobacterium-mediated transient transformation method of GV3101 (pSoup). After 48h of infection, after pre-treating the samples with the Dual-Luciferase-Reporter-Assay-Kit, the fluorescence value is measured by an enzyme-labeling instrument. Using sea cucumber luciferase as an internal reference, the transient expression level of the target sequence activity is expressed as the LUC / REN ratio.

[0091] Figure 5 The transcription factor OfERF177 binds to the promoter of OfABCG13 and inhibits its transcription. Yeast one-hybrid assays showed that OfERF177 can interact with the promoter of OfABCG13 in vitro ( Figure 5 in (b) of Figure 5 ). Dual-luciferase reporter (DLR) assays showed that OfERF177 can inhibit the promoter activity of OfABCG13 ( Figure 5 in (c) of

[0092] ). Cis-element analysis found that the promoter region of OfABCG13 contains many ERF-binding motifs, such as ACCGAC, ATTCAAA, and CAACA (

[0093] in (a) of

[0094] ). These results support that OfERF177 inhibits the transcription of OfABCG13, probably by directly binding to the ERF-binding motifs in the promoter.

[0095]

[0096]

[0097]

[0098] Using Osmanthus fragrans cv. 'rixianggui' cDNA as a template, primers with homologous arms were designed, and the primer sequences are as follows:

[0098] TRV2-OfERF177-F 5'--3'-agaaggcctccatggggatccAAACCGCCAACAAATCAGTATAGC

[0095] TRV2-OfERF177-R 5'--3'-

[0096] tgtcttcgggacatgcccgggGGGCTTCATTTGGGACTCG

[0097] A specific fragment (330 bp) was amplified, and the sequence is shown as SEQ-ID-NO.5. The TRV2 vector was double-digested with BamHI and SmaI restriction endonucleases. After digestion at 37 °C for 1 h, the products were purified. The 330-bp specific fragment of OfERF177 was homologously recombined with the digested TRR2 vector fragment using homologous recombination. The recombinant products were transformed into Escherichia coli competent cells DH5α by heat shock. After growing on LB plates containing kanamycin resistance for 12 h, several monoclonal colonies were picked for bacterial inspection and sequencing. After the positive clones were sequenced correctly, the plasmids were extracted and further transformed into Agrobacterium tumefaciens competent cells GV3101. After growing on LB plates containing kanamycin and rifampicin resistance for 48 h, single colonies were selected for bacterial inspection. The target bands in the bacterial inspection were correct and had the same brightness. The bacterial solution was stored with 50% glycerol at a volume ratio of 3:7 and stored in a -80 °C ultra-low temperature freezer.

[0098] 2.5: Transient Transformation of Osmanthus fragrans with the VIGS Vector of OfERF177 Gene

[0099] (1)Transformation of Recombinant Plasmid into Agrobacterium

[0100] Take the GV3101 competent cells stored at -80 °C to room temperature. When it is in an ice-water mixture state, add 1 μL of -TRV2-OfERF177 plasmid, stir well, let it stand on ice for 5 minutes, then place it in liquid nitrogen for 5 minutes, in a 37 °C water bath for 5 minutes, and in an ice bath for 5 minutes. Add 500 μL of -LB medium, culture it on a shaker at 28 °C for 1 hour, centrifuge at 5000 r / min for 5 minutes, and then spread it on the corresponding antibiotic LB plate. Incubate it upside down in an incubator at 28 °C for 2 - 3 days. Pick monoclonal colonies and perform positive detection using the universal primers of the vector. After shaking the positive monoclonal colonies, store the bacterial liquid and 50% glycerol in a volume ratio of 3:7, quickly freeze it in liquid nitrogen, and store it in a -80 °C refrigerator.

[0101] (2)Preparation of Inoculum and Inoculation

[0102] The preparation method of the inoculum for transient transformation of the VIGS vector of the OfERF177 gene into Osmanthus fragrans and the inoculation method are the same as those in “(2) Preparation of Inoculum” and “(3) Inoculation” in “1.2: Transient Transformation of Osmanthus fragrans Petals with the Overexpression Vector”.

[0103] 2.6: Determination of the Content of Terpenoids in Osmanthus fragrans Petals with Transient Silencing of OfERF177

[0104] The method for determining the content of terpenoids in Osmanthus fragrans petals with transient silencing of OfERF177 is the same as that in “(2) Determination of the Aroma Components of Transiently Transformed Osmanthus fragrans Petals” in “1.4: Determination of Terpenoids in Osmanthus fragrans and Tobacco Flowers”.

[0105] Figure 6 is the determination chart of the content of terpenoids in Osmanthus fragrans petals with transient silencing of the transcription factor OfERF177. As shown in the figure, the expression of OfERF177 in Osmanthus fragrans was successfully down-regulated using the VIGS system ( Figure 6-1 in (a)). At the same time, in the flowers with silenced OfERF177, the expression of OfABCG13 was significantly up-regulated ( Figure 6-1 in (a)). According to the PCA results, the VOC samples in the TRV2-empty and OfERF177-silenced flowers could be easily separated ( Figure 6-1 in (b)). In addition, the terpenoid emission level in the OfERF177-silenced flowers was significantly increased ( Figure 6-1 in (c)), and the key differential terpenoid aroma substances identified were dihydro-β-ionone and linalool ( Figure 6-1 in (d), Figure 6-2 in (e) to (f)). -

[0106] The above embodiments of the present application show that: OfABCG13 is a transporter located on the cell membrane, and its expression level is highly positively correlated with the release level of terpene substances in osmanthus. Under the condition of overexpressing OfABCG13, the terpene release levels in osmanthus petals and tobacco flowers both increased significantly. In addition, through yeast one-hybrid screening and bimolecular luciferase experiments, it was identified that the osmanthus transcription factor OfERF177 can bind to the promoter of OfABCG13. Further gene silencing experiments clarified that OfERF177 can inhibit the activity of OfABCG13, thereby negatively regulating the release of floral fragrance substances in osmanthus. This application provides high-quality candidate genes for cultivating new varieties with rich fragrance and has good application prospects.

Claims

1. A Osmanthus fragrans var. thunbergii transporter OfABCG13 gene, whose nucleotide sequence is shown in SEQ-ID-NO.

1.

2. The expressed protein of the Osmanthus fragrans var. thunbergii transporter OfABCG13 gene as described in claim 1, whose amino acid sequence is shown in SEQ-ID-NO.

2.

3. An expression cassette, recombinant vector, recombinant bacterium or recombinant cell containing the Osmanthus fragrans var. thunbergii transporter OfABCG13 gene as described in claim 1.

4. A Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene, whose nucleotide sequence is shown in SEQ-ID-NO.

3.

5. The expressed protein of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene as described in claim 4, whose amino acid sequence is shown in SEQ-ID-NO.

4.

6. An expression cassette, recombinant vector, recombinant bacterium or recombinant cell containing the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene as described in claim 4.

7. Use of the Osmanthus fragrans Lour. transporter OfABCG13 gene according to claim 1 and / or the Osmanthus fragrans Lour. transcription factor OfERF177 gene according to claim 4 in increasing the release amount of terpenoids in plant flowers, characterized in that, The application includes: overexpressing the Osmanthus fragrans var. thunbergii transporter OfABCG13 gene and / or reducing the expression of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene in plants, and the plants are Osmanthus fragrans or tobacco.

8. The application according to claim 7, wherein The terpene compounds are at least one of dihydro-β-ionone, linalool, trans-linalool oxide and cis-linalool oxide.

9. The application according to claim 7, characterized in that The specific steps include: (1) Construct an overexpression vector of the Osmanthus fragrans var. thunbergii transporter OfABCG13 gene and / or a silencing vector of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene; (2) Transform the constructed overexpression vector of the Osmanthus fragrans var. thunbergii transporter OfABCG13 gene and / or the silencing vector of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene into plants or plant tissues; (3) Cultivate and screen to obtain transgenic plants or plant tissues with increased release amount of aroma substances, and the plants are Osmanthus fragrans or tobacco.

10. The application according to claim 9, characterized in that, The overexpression vector of the Osmanthus fragrans var. thunbergii transporter OfABCG13 gene is the pSoup1300 vector, and the silencing vector of the Osmanthus fragrans var. thunbergii transcription factor OfERF177 gene is the VIGS vector.

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