Method for synthesizing carnosic acid in vitro and application of carnosic acid in improvement of oxidation resistance

Abstract

The invention discloses a method for synthesizing carnosic acid in vitro and application of the carnosic acid in improvement of oxidation resistance, and belongs to the field of molecular biology. According to the application disclosed by the invention, SrCYP716A514 and SrCYP76AK7 genes positioned in endoplasmic reticulum are found, the SrCYP716A514 and SrCYP76AK7 genes are found to be capable of synthesizing the carnosic acid in vitro, and the content of the carnosic acid is obviously increased when the SrCYP716A514 and SrCYP76AK7 genes are overexpressed in rosemary, and the oxidation resistance of a rosemary extract can be enhanced. Through separation and functional identification analysis of the SrCYP716A514 gene and the SrCYP76AK7 gene, it is determined that the SrCYP716A514 gene and the SrCYP76AK7 gene can synthesize carnosic acid in vitro, the content of the carnosic acid is remarkably increased through overexpression in rosemary, and the oxidation resistance of a rosemary extract can be enhanced. According to the invention, synthesis of carnosic acid in plants can be accurately regulated and controlled, the carnosic acid content can be increased, the method can be used for cultivating transgenic plants, plants with high carnosic acid content can be obtained, and the method has a wide application prospect in the fields of food, medicines, cosmetics and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for synthesizing salvia acid in vitro and application thereof in improving antioxidant capacity. BACKGROUND

[0002] Rosemary, as a multifunctional plant, plays an important role in cooking, traditional medicine and modern industry. It is not only widely used in cooking to change and enhance the flavor of food, but also has a long history of application in traditional medicine (Achour et al., 2020) and is a highly praised medicinal plant (Nassazi et al., 2020). Studies have shown that rosemary has antioxidant (Rahmani et al., 2021), antibacterial (Saravani et al., 2021), anti-inflammatory (Wei et al., 2021), anti-tumor (El-Huneidi et al., 2021), anti-depression (Mirza et al., 2021), anti-choline (Fatemeh et al., 2021), weight loss (Sargin et al., 2021) and hypoglycemic (Mirza et al., 2021) functions, and is one of the most popular sources of natural bioactive compounds (R Af Ie, 2017).

[0003] Diterpenoid rosmaridic acid in rosemary leaves is the key component for its antioxidant effect. In addition to antioxidant capacity, the compound also has anti-cancer, neuroprotective, anti-inflammatory, metabolic regulation, and antibacterial physiological functions. At present, the mechanism of rosmaridic acid in anti-tumor and neuroprotection has been relatively systematic. Due to its natural source characteristics, rosmaridic acid is expected to be developed as an efficient and low-toxic anti-tumor and neuroprotective drug. At the same time, rosmaridic acid is widely used in medicine, health food, cosmetics, food additives, and edible oil industries. With its significant natural antioxidant activity, rosmaridic acid has become a key object in the current natural product research. Rosmaridic acid is a diterpenoid phenolic compound contained in rosemary. The ortho-diphenol hydroxyl and carboxyl groups in its molecular structure endow the component with various pharmacological activities. The synthesis of rosmaridic acid starts from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which are synthesized through the cytoplasmic mevalonate (MVA) pathway and the plastidic 2–C–methyl–D–erythritol–4–phosphate (MEP) pathway (Liao et al., 2016). IPP and DMAPP combine head-to-head or head-to-tail to form geranyl diphosphate (GPP), neryl pyrophosphate (NPP), or farnesyl diphosphate (FPP), which are then converted to monoterpenes and sesquiterpenes by terpene synthases (TPS) (Dong et al., 2022). In this process, cytochrome P450 oxygenase (CYP enzyme) is involved in the downstream oxidation reaction of abietene to synthesize rosmaridic acid.

[0004] At present, various CYP enzymes involved in the biosynthesis of Labiatae abietane diterpenes (AD) have been found, such as CYP76AH1 from Salvia miltiorrhiza and CYP76AH4, CYP76AH22, and CYP76AH23 from rosemary and CYP76AH1 from S. trichotoma. Salvia fruticosa SrCYP716A514CYP76AH24, etc., however, there are some problems in the catalytic process of these enzymes. Different enzymes have different catalytic functions, and some enzyme catalytic sites are not single, such as in the CYP76AH enzyme group, except for SmCYP76AH1, other enzymes will undergo a second hydroxylation to produce 11-hydroxy ferruginol; CYP76AH3 can oxidize ferruginol at C11 and C7 positions, which makes it difficult to accurately control the synthesis path of salvinolic acid, easily leading to the generation of non-target products, thereby affecting the synthesis efficiency and purity of salvinolic acid. In addition, although the enzymes catalyzing the oxidation of ADC20 position such as CYP76AK subfamily can accept a variety of substrates, the substrate specificity is not clear enough. This functional complexity makes it difficult to accurately predict and control the interaction between enzyme and substrate in practical application, which is not conducive to the efficient and targeted synthesis of salvinolic acid in vitro. Although some genes and enzymes are known to be involved in the synthesis of rosemary active ingredients, there is currently a lack of in-depth understanding of key genes and regulatory pathways for the specific mechanism of improving the antioxidant capacity of rosemary extract by regulating the expression of related genes, which greatly limits the development of methods for effectively improving the antioxidant capacity. SUMMARY

[0005] In view of the low efficiency of synthesizing salvinolic acid, the influence of by-products on purity, the poor substrate specificity of CYP enzymes, the uncontrollable catalysis, and the need to improve the antioxidant capacity of rosemary in the prior art, the present application aims to provide a rosemary SrCYP76AK7 and SrCYP716A514 The application of genes and their encoded proteins in the synthesis of salvinolic acid in vitro and the improvement of the antioxidant capacity of rosemary.

[0006] In order to achieve the above purpose, the technical scheme is adopted as follows: The application provides a gene combination for synthesizing salvinolic acid in vitro, which is composed of SrCYP76AK7 gene and SrCYP716A514 gene, the SrCYP76AK7 gene encodes SrCYP716A514 protein, the amino acid sequence of the SrCYP716A514 protein is shown as SEQ ID NO. 2, and the SrCYP716A514 gene encodes SrCYP76AK7 protein, and the amino acid sequence of the SrCYP76AK7 protein is shown as SEQ ID NO. 4.

[0007] The nucleotide sequence of the SrCYP76AK7 gene is shown as SEQ ID NO. 1; The nucleotide sequence of the SrCYP716A514 gene is shown as SEQ ID NO. 3.

[0008] The nucleotide sequence of the SrCYP76AK7The gene also includes a nucleotide sequence having more than 90% homology with the sequence shown in SEQ ID No. 1 and encoding a protein having the function of the elicitor protein. The gene also includes a nucleotide sequence having more than 90% homology with the sequence shown in SEQ ID No. 3 and encoding a protein having the function of the elicitor protein. SrCYP716A514 The gene also includes a nucleotide sequence having more than 90% homology with the sequence shown in SEQ ID No. 3 and encoding a protein having the function of the elicitor protein.

[0009] The SrCYP76AK7 protein also includes a protein derived from the sequence shown in SEQ ID No. 4 by substitution and / or deletion and / or addition of one or more amino acid residues and having equivalent activity. The SrCYP76AK7 protein also includes a protein derived from the sequence shown in SEQ ID No. 4 by substitution and / or deletion and / or addition of one or more amino acid residues and having equivalent activity.

[0010] The present application provides a recombinant expression vector containing the gene combination.

[0011] The present application provides a host cell containing the recombinant expression vector.

[0012] The present application provides a recombinant bacterium containing the gene combination.

[0013] The present application provides the use of the gene combination, or the expression vector, or the host cell, or the recombinant bacterium in the in vitro synthesis of salvinic acid.

[0014] The present application provides a method for in vitro synthesis of salvinic acid, comprising: transforming a recombinant expression vector containing the gene combination into a host cell, adding the transformed host cell to ferruginol for induction and incubation, extraction, and obtaining salvinic acid.

[0015] Preferably, the host cell is any one of yeast, Escherichia coli, HEK293 cells, and Halobacterium.

[0016] Further preferably, the host cell is yeast.

[0017] Preferably, the recombinant expression vector containing the gene combination is transformed into the host cell by freeze-thaw method.

[0018] Preferably, the ferruginol induction and incubation conditions are: ferruginol final concentration of 80-120 μM, 27-30°C, 150-250 rpm shaking culture for 36-48 hours.

[0019] Preferably, the solvent used in the extraction is ethyl acetate.

[0020] The gene combination, the expression vector, the host cell, or the recombinant bacteria are used for improving the oxidation resistance of plants.

[0021] Preferably, the gene combination, the expression vector, the host cell, or the recombinant bacteria are used for plant breeding.

[0022] Compared with the prior art, the present application has the following technical effects: The gene combination for synthesizing salvinic acid in vitro provided by the present application comprises SrCYP76AK7 a gene and SrCYP716A514 a gene, and the specific amino acid sequences of the proteins encoded by the two genes are determined, which provides the key genes and protein basis for synthesizing salvinic acid in vitro, and by limiting the precise amino acid sequences of the two proteins, the spatial conformation of the catalytic site and the substrate specificity binding ability are ensured, so that the directional catalysis of ferruginol to salvinic acid is realized; the construction of such a double-enzyme system avoids the accumulation of intermediate products produced by traditional single-enzyme catalysis, and cuts off the generation path of non-target products from the reaction path.

[0023] Further, the SrCYP716A514 protein and the SrCYP76AK7 protein also include derivative proteins that still have equivalent activity after substitution, deletion or addition of one or more amino acid residues. This increases the diversity of the proteins, expands the selection range of proteins that can be used for synthesizing salvinic acid in vitro, improves the flexibility and practicability of the gene combination, and helps to meet the needs of different experimental conditions and application scenarios. Further, SrCYP76AK7 a gene and SrCYP716A514 a gene include nucleotide sequences that can hybridize with specific sequences and nucleotide sequences that have more than 90% homology and encode proteins with the function of elicitor proteins, which widens the gene resources that can be used for synthesizing salvinic acid, allows variation and selection of genes within a certain range while ensuring the function of the encoded proteins, helps to screen out more efficient genes that are more suitable for specific experimental conditions, and improves the efficiency and success rate of salvinic acid synthesis.

[0024] The expression vector provided by the present application can realize the stable existence and efficient expression of the gene in the host cell by integrating the gene combination into the vector, which provides a necessary vector platform for subsequent synthesis of salvinic acid using host cells, and improves the convenience and repeatability of gene operation.

[0025] The host cell provided by the present application contains a recombinant expression vector, and the expression system of the host cell is used to realize SrCYP76AK7 and SrCYP716A514The co-expression of the two key enzymes enables the genes to be transcribed and translated in the cell to produce the proteins that regulate the production of salvinic acid. The host cell acts as a biological reactor, and by maintaining the optimal expression ratio and active conformation of the two enzymes, the efficiency and specificity of the catalytic reaction are ensured. Based on the synergistic expression system of the host cell, the technical bottlenecks of enzyme activity maintenance and multi-enzyme system coordination in traditional in vitro enzyme catalytic system are broken through, and a stable biosynthesis platform is provided for the directed synthesis of high-purity salvinic acid.

[0026] The present application provides a recombinant bacteria, which has the advantages of rapid growth, easy cultivation and operation, etc. By precisely controlling the synergistic expression of the two enzymes, the accumulation of intermediate products or non-target oxidation reactions during single enzyme catalysis is avoided, thereby improving the specificity and efficiency of salvinic acid synthesis. The present application can produce salvinic acid on a large scale, reduce production costs, improve production efficiency, and provide the possibility for the industrial production of salvinic acid.

[0027] The application provided by the present application realizes the in vitro efficient synthesis of salvinic acid and improves the antioxidant capacity through the synergistic effect of the combination of the genes and the related biological carriers. SrCYP76AK7 The combination of the genes and the related biological carriers. SrCYP716A514 The present application provides a method for in vitro synthesis of salvinic acid, which comprises the following steps: transforming a recombinant expression vector containing the combination of the genes into a host cell, and inducing incubation with ferruginol, and finally extracting salvinic acid. The combination of the genes and the related biological carriers.

[0028] The present application provides a method for in vitro synthesis of salvinic acid, which comprises the following steps: transforming a recombinant expression vector containing the combination of the genes into a host cell, and inducing incubation with ferruginol, and finally extracting salvinic acid. SrCYP76AK7 The combination of the genes and the related biological carriers. SrCYP716A514 The present application provides a method for in vitro synthesis of salvinic acid, which comprises the following steps: transforming a recombinant expression vector containing the combination of the genes into a host cell, and inducing incubation with ferruginol, and finally extracting salvinic acid. SrCYP76AK7 The combination of the genes and the related biological carriers. Figure 1 The present application provides a method for in vitro synthesis of salvinic acid, which comprises the following steps: transforming a recombinant expression vector containing the combination of the genes into a host cell, and inducing incubation with ferruginol, and finally extracting salvinic acid. The combination of the genes and the related biological carriers.

[0029] The application provided by the present application is not only limited to the synthesis of salicin acid, but also combined with improving the antioxidant capacity of plants, so that the synthesis efficiency of the key antioxidant component salicin acid in the plant body is significantly improved, and the interference of non-target products is avoided, thereby improving the overall antioxidant capacity of the plant, providing a new way and method for improving the stress resistance and quality of the plant, and helping to solve the problem that the antioxidant capacity of rosemary needs to be improved in the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0030] SrCYP716A514 Figure 1 shows the expression levels of SrCYP76AK7 and SrCYP716A514 Figure 2 shows the basic characterization, wherein (a) the effect of UV-B treatment on the transcription level of SrCYP76AK7 and SrCYP716A514 Figure 3 shows the effect of PEG6000 treatment on the transcription level of SrCYP76AK7 and SrCYP716A514 Figure 4 shows the comparison of the expression levels of SrCYP76AK7 and SrCYP716A514 Figure 5 shows the sequence alignment of (c) SrCYP76AK7 and SrCYP716A514 Figure 6 shows the sequence alignment of (d) SrCYP76AK7 and SrCYP716A514 Figure 7 shows the subcellular localization of SrCYP76AK7 and Figure 2 Figure 8 shows the subcellular localization of SrCYP716A514 Figure 9 shows the comparison of the expression levels of SrCYP76AK7 and Figure 3 Figure 10 shows the subcellular localization of

[0031] SrCYP716A514 Figure 11 shows the comparison of the expression levels of SrCYP716A514 Figure 12 shows the comparison of the expression levels of SrCYP716A514 Figure 13 shows the comparison of the expression levels of SrCYP716A514 Figure 14 shows the comparison of the expression levels of SrCYP716A514 Figure 15 shows the comparison of the expression levels of SrCYP716A514 Figure 16 shows the comparison of the expression levels of SrCYP716A514 Figure 17 shows the comparison of the expression levels of Figure 4 Figure 18 shows the comparison of the expression levels of SrCYP716A514 Figure 19 shows the comparison of the expression levels of Figure 5Figure 8 shows the gene and the trans-empty vector PBI121 rosemary plant multi-index comparative analysis diagram, wherein A is the trans

[0032] SrCYP716A514 Figure 9 shows the overexpression Figure 6 Figure 10 shows the rosemary antioxidant capacity and related index influence comparison diagram, wherein (a) is the DPPH free radical scavenging capacity comparison column diagram of different treatment groups of rosemary; (b) is the ABTS free radical scavenging capacity comparison column diagram of different treatment groups of rosemary; (c) is the FeSO4 equivalent content comparison column diagram of different treatment groups of rosemary; (d) is the APC index average comparison column diagram of different treatment groups of rosemary.

[0033] SrCYP76AK7 Figure 11 shows the trans SrCYP76AK7 Figure 12 shows the gene and the trans-empty vector PBI121 rosemary plant multi-index comparative analysis diagram, wherein A is the trans SrCYP76AK7 Figure 13 shows the gene and the trans-empty vector PBI121 rosemary plant appearance form comparison; B is the trans SrCYP76AK7 Figure 14 shows the gene and the trans-empty vector PBI121 rosemary plant fluorescence detection diagram; C is the trans SrCYP76AK7 Figure 15 shows the gene and the trans-empty vector PBI121 rosemary plant PCR detection electrophoretogram; D is the trans SrCYP76AK7 Figure 16 shows the gene and the trans-empty vector PBI121 rosemary plant local morphology display diagram; E is the trans SrCYP76AK7 Figure 17 shows the gene and the trans-empty vector PBI121 rosemary plant multi-index comparative analysis diagram; F is the trans Figure 7 Figure 18 shows the gene and the trans-empty vector PBI121 rosemary related index content comparison diagram; SrCYP76AK7 Figure 19 shows the trans Figure 8Gene and empty vector transfection rosemary multiple metabolic indicators (ACCT-892, mvk-3213, ACCT-3036, HMGS-455, PMK-1518, PMK-1680, MVD-4980, MVD-3280, DXR-3254, DXR-4527, DXS-776, MCT-4, CMK-2825, CMK-1569, MDS-1646, MCT-2716, IDI-20, KSL-5669 and SrCYP716A514) content of the bar chart.

[0034] SrCYP76AK7 The overexpression of the gene is shown Figure 9 The impact of rosemary antioxidant capacity and related indicators are shown in the comparison chart, wherein (a) is the ABTS free radical scavenging capacity of rosemary in different treatment groups; (b) is the FeSO4 equivalent content of rosemary in different treatment groups; (c) is the DPPH free radical scavenging capacity of rosemary in different treatment groups; (d) is the average of the APC index of rosemary in different treatment groups.

[0035] SrCYP716A514 The overexpression of the gene is shown SrCYP76AK7 and Figure 10 The reaction of ferruginol in vitro enzyme activity generates salvinic acid.

[0036] SrCYP716A514 The overexpression of the gene is shown Srcyp716A514 The gene after mutation of one base SrCYP716A514 , SrCYP76AK7 respectively and SrCYP716A514 The comparison of salvinic acid generated by the reaction of ferruginol in vitro enzyme activity is shown, wherein A is the chromatogram of salvinic acid standard, B is Srcyp716A514 The chromatographic details of the reaction catalyzed by the mutant gene SrCYP716A514 respectively are compared, C is Srcyp716A514 The mutant gene SrCYP716A514 catalyzed reaction to generate salvinic acid content bar chart. DETAILED DESCRIPTION

[0037] To illustrate the technical content of the present application in detail, the following specific examples are provided. It should be noted that the following specific embodiments are intended to further illustrate the present application, but the scope of the methods and protection of the present application is not limited thereto.

[0038] The reagents and methods used in the following examples are prepared and operated according to conventional methods unless otherwise specified; the reagents used can be obtained from commercial channels.

[0039] DPPH method: it is a commonly used method for evaluating the antioxidant capacity of substances, the principle is that DPPH free radical is purple in organic solvent, has an absorption peak, when antioxidant exists, DPPH free radical is reduced, the absorption peak disappears or weakens, the antioxidant capacity of DPPH free radical can be calculated by measuring the change of absorbance.

[0040] ABTS method: ABTS [2,2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt] is oxidized to generate stable blue-green cationic free radical ABTS + , antioxidant can make the color of ABTS + lighter, and the antioxidant capacity can be measured by measuring the change of absorbance.

[0041] FRAP method: it is based on the reduction of Fe 3+ -TPTZ (tripyridyltriazine) complex to blue Fe 2+ -TPTZ by antioxidant, and the total antioxidant capacity of the sample is evaluated by measuring the absorbance change of the blue complex.

[0042] The carrier pBM16K (Perryman, P-PR1196), PBI121 (Fenghui Biology, ZT588), and PHSE-HIS (Shanghai Zeye Biology Technology Co., Ltd., ZY1770) are used in the application.

[0043] Example 1 SrCYP76AK7 and Figure 1 Gene acquisition I. Transcriptome data acquisition Select several rosemary potted seedlings with good growth conditions and uniform size, ensure that the plants are healthy and free of pests and diseases, and divide them into three groups for different treatments.

[0044] (1) UV-B irradiation treatment group and control group One group of rosemary potted seedlings is treated with UV-B irradiation, the light intensity is 1 W / m 2 , and the irradiation lasts for 12 h; another group of rosemary potted seedlings is placed in a light incubator and given normal light irradiation for 12 h; after irradiation, similar leaves in size and position are selected from the rosemary plants in the UV-B treatment group and the control group, frozen in liquid nitrogen, quickly ground into powder, and the ground powder is quickly transferred to RNase-free centrifuge tubes and stored at -80℃ for standby.

[0045] (2) PEG6000 treatment group and control group A group of rosemary potted seedlings were soaked in 10% PEG6000 solution, ensuring that the roots were in contact with the solution, for 7 days; the control group was watered normally, and 7 days after treatment; after the treatment, the leaves were taken, frozen in liquid nitrogen, ground into powder and stored at -80℃ for standby.

[0046] (3) Collection of young glandular hairs and mature glandular hairs of rosemary leaves Young glandular hairs and mature glandular hairs on rosemary leaves were selected, and the collected young glandular hairs and mature glandular hairs were placed in different RNase-free centrifuge tubes, frozen in liquid nitrogen, and stored at -80℃ for standby.

[0047] According to the Magen total RNA extraction kit (ZP437, Zhuangmeng Biological), the total RNA of the above samples was extracted, and after detection, it was sent to Nuohuo Biological for transcriptome sequencing, and the differential genes of the treatment group and the control group were analyzed. The results are shown in the accompanying Figure 1 .

[0048] According to the accompanying SrCYP716A514 data, the differential genes SrCYP76AK7 and SrCYP716A514 increased in expression after UV-B irradiation, which was consistent with the trend of cannabichromene; after PEG6000 treatment, the content of cannabichromene decreased, SrCYP76AK7 and SrCYP716A514 , and the expression of SrCYP76AK7 and SrCYP716A514 was higher in young glandular hairs, suggesting that the gene may be involved in the synthesis of cannabichromene.

[0049] CYPAH1 The similarity of the identified cannabichromene pathway Salvia pomifera was as high as 81.3%, and the similarity of the gene to the CYP716A96 gene in SrCYP716A514 was as high as 91.85%. SrCYP716A514 belongs to the CYP716A family. Based on the above similarity, it is identified that the gene SrCYP76AK7 may have the function of CYPAH family. Through the screening of transcriptome differential genes and the construction of CYP450 phylogenetic tree, the gene CYP76AK6-8 was screened, which has high similarity with the identified SrCYP716A514 , and is predicted to be able to catalyze the conversion of 11-hydroxyironol to cannabichromene.

[0050] Two, SrCYP76AK7 and SrCYP716A514 genes are obtained Total RNA was extracted from rosemary (from the National Key Laboratory of Plant Diversity and Specialty Economic Crops), and cDNA was obtained by reverse transcription. Using cDNA as a template, PCR amplification was performed using primers SrCYP716A514F / SrCYP76AK7F and SrCYP716A514R / SrCYP76AK7 (as shown in SEQ ID NO.5~SEQ ID NO.8). The PCR amplification products were detected by agarose gel electrophoresis, and the cDNA was successfully amplified. SrCYP76AK7 and ​ Gene fragments were extracted and recovered via gel extraction, ligated into the cloning vector pBM16K, transformed into *E. coli* DH5α, and positive single colonies were detected by PCR and sequencing. The resulting colonies were then stored in 50% glycerol at -80°C for later use. Primer sequences are as follows:

[0051] PCR reaction system

[0052] PCR reaction procedure

[0053] Sequencing results showed that the nucleotide sequences of the PCR products were as shown in SEQ ID No. 1 and SEQ ID No. 3. SrCYP716A514 and SrCYP76AK7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2 and SEQ ID No. 4.

[0054]

[0055] three, SrCYP716A514 and SrCYP76AK7 Protein localization 1. Recombinant vector PBI121- SrCYP716A514 / SrCYP76AK7 Build The sequences are as shown in SEQ ID No. 1 and SEQ ID No. 3. SrCYP716A514 and SrCYP76AK7 Using genes as templates, primer P was designed. SrCYP716A514 -F / P SrCYP76AK7 -F and P SrCYP716A514 -R / P SrCYP76AK7 -R was used for PCR amplification to obtain PCR products, and homologous recombination was used to... SrCYP716A514 and SrCYP76AK7 The full-length cDNA was ligated into the expression vector PBI121 to generate the recombinant vector (35S::SrCYP716A514 / SrCYP76AK7-GFP); the primer sequences are as follows:

[0056] The empty vector (PBI121) and recombinant vector (35S::SrCYP716A514 / SrCYP76AK7-GFP) were transformed into Agrobacterium rhizogenes GV3101 using the freeze-thaw method. The specific steps are as follows: (1) Agrobacterium GV3101 competent cells stored at -80°C were streaked on LB solid plates containing 25 μg / mL rifampicin and cultured upside down at 28°C for 2 days; (2) Single colonies of Agrobacterium GV3101 were picked from the cultured LB plates and inoculated into 5 mL of LB liquid medium containing 25 μg / mL rifampicin; (3) 50 μL of the overnight culture was transferred to 50 mL of LB liquid medium containing 25 μg / mL rifampicin and cultured until OD. 600 The value is approximately 0.5 (approximately 8 h); (4) Place the cultured bacterial solution on ice for 30 min, then centrifuge at 4°C and 4000 rpm for 10 min to collect the bacterial cells and discard the supernatant; (5) Add 5 mL of pre-cooled 20 mM CaCl2 solution to resuspend the bacterial cells; (6) Centrifuge at 4°C and 4000 rpm for 5 min to collect the bacterial cells and discard the supernatant; (7) Add 1 mL of pre-cooled 20 mM CaCl2 solution, mix thoroughly, and then dispense; (8) During transformation, place the Agrobacterium competent cells on ice to thaw, take 30 µL of competent cells and 2 µL of plasmid to be transformed, gently mix, and incubate on ice for 30 min; after quick freezing in liquid nitrogen for 1 min, immediately transfer to a 37°C water bath for heat shock for 1 min, and then quickly place on ice to cool for 2 min; add 800 μL of antibiotic-free LB liquid medium and culture at 28°C and 200 rpm for 3 h with shaking; finally, take the bacterial solution and spread it on a medium containing 25 μg / mL rifampin and 50 μg / mL kanamycin on LB solid plates.

[0057] 2. Subcellular localization Single colonies of Agrobacterium rhizogenes GV3101 containing recombinant vector (35S::SrCYP716A514 / SrCYP76AK7-GFP) and empty vector (PBI121) were picked and inoculated into 5 mL of YEB liquid medium containing the corresponding antibiotics. The cultures were incubated overnight at 28°C with shaking. The bacterial cells were collected, and the inoculation solution was added. The bacterial culture was mixed by pipetting, and the OD value was adjusted to 0.6. The cultures were then incubated at 28°C for 2 h.

[0058] Composition of infection buffer

[0059] Select healthy, 3-4 week old tobacco plants (Tobacco Benedictine var. benzo[i]) Nicotiana benthamiana Plant, OD drawn from sterile syringe 600= 0.6~0.8 of Agrobacterium liquid, Agrobacterium liquid containing 35S:GFP, 35S:P890 (SrCYP716A514 fusion GFP) and 35S:P2734 (SrCYP76AK7 fusion GFP) respectively, and Agrobacterium liquid containing endoplasmic reticulum marker mcherry was prepared, and each Agrobacterium liquid was cultured to OD 600 = 0.6-0.8, P890 and P2734 encode SrCYP716A514 and SrCYP76AK7 proteins respectively), Agrobacterium liquid containing different fusion protein genes was mixed with Agrobacterium liquid containing endoplasmic reticulum marker mcherry in a certain proportion (1:1); the mixed liquid was gently injected from the back of the tobacco leaf using a needle-free syringe, and the tobacco plants after injection were cultured for 3 days, then the tobacco leaves were taken out and analyzed on a confocal laser scanning microscope (Leica TCS SP5), and the tobacco leaves injected with empty vector (PBI121) were used as controls to observe the cell background fluorescence, and the results are shown in the following Figure 2 .

[0060] As shown in the following Figure 2 , for the 35S:GFP sample, the GFP fluorescence showed a relatively uniform distribution in the whole cell, and as a control, GFP itself had no specific subcellular localization signal and would diffuse freely in the cell; for the 35S:P890 and 35S:P2734 groups, the GFP fluorescence showed a specific network-like or membrane-like structure distribution, which was consistent with the morphological characteristics of the endoplasmic reticulum. The fluorescence signal of mcherry as an endoplasmic reticulum marker protein clearly outlined the structure of the endoplasmic reticulum, showing a typical endoplasmic reticulum network morphology. After merging the fluorescence images of GFP and mcherry, it can be seen that the GFP fluorescence in the 35S:P890 and 35S:P2734 groups has a good overlap with the mcherry fluorescence. This indicates that the localization of SrCYP716A514-GFP and SrCYP76AK7-GFP fusion proteins is consistent with that of the endoplasmic reticulum marker protein mcherry, so it can be concluded that SrCYP716A514 and SrCYP76AK7 the proteins are localized in the endoplasmic reticulum.

[0061] The above experiment clearly SrCYP716A514 and SrCYP76AK7 the subcellular localization of the proteins is endoplasmic reticulum, which helps to further study the functions of the two proteins in the cell and provides important cell localization information for in-depth exploration of their roles in plant physiological processes.

[0062] Example 2: SrCYP716A514 Analysis of transgenic plants for obtaining and regulating the function of salvinic acid I. Transgenic plant acquisition 1. Transformation The recombinant bacteria GV3101 obtained in step 1 in example 1 was used to transform the empty vector PBI121 recombinant bacteria GV3101 as a control, and Agrobacterium was used to infect tobacco (Nicotiana tabacum) N. tabacum ), and the specific steps are as follows: (1) The GV3101 Agrobacterium containing the recombinant vector was shaken to OD 600 =1, and the bacterial liquid was enriched, and the supernatant was discarded.

[0063] (2) The enriched bacterial liquid was suspended in 1 / 2 MS containing 200 mM acetosyringone (AS), and cultured at 28°C in the dark for 2-3 h to obtain the infection liquid.

[0064] (3) The prepared whole rosemary plant was placed in the infection liquid for 15-20 min, and shaken every 5 min to ensure that each part of the plant was in contact with the infection liquid.

[0065] (4) After infection, the infected rosemary plant was placed on a clean bench, the excess bacterial liquid was absorbed with sterile filter paper, and was placed in MS medium in the dark for 3 d; wherein, the composition of the co-culture medium is: MS 4.43 g / L, glucose 30 g / L, agar 7 g / L, 6-benzyladenine (6-BA) 1 mg / L, ddH2O supplemented to 1000 mL.

[0066] (5) After co-culture, the rosemary plant was washed with sterile water twice, the excess water was absorbed with sterile filter paper, and the rosemary plant was placed in MS (Timentin, 100 mg / L; K + , 100 mg / L) medium containing antibiotics, and cultured under light at 25+1°C, and the medium was replaced every 2 weeks.

[0067] 2. Identification The plants that sprouted new shoots were observed under a fluorescence microscope to detect positive seedlings, and the leaf DNA was extracted for PCR to identify positive seedlings again, and 5 positive seedlings were selected for qPCR verification.

[0068] II. Overexpression SrCYP716A514 Increase the content of salvinic acid in rosemary 1. Salvinic acid content detection The transgenic rosemary leaves were ground in liquid nitrogen, 50 mg of ground transgenic rosemary leaf sample was taken into a centrifuge tube, 500 μL of 80% methanol was added, and after ultrasonic treatment for 60 min, the supernatant was used for determination of salvinic acid content.

[0069] 2. Product identification According to the peak time comparison of the salvinic acid standard, it is determined whether the sample contains salvinic acid.

[0070] 3. Calculation of carnosic acid content The peak area of the identified carnosic acid was calculated by the standard curve of carnosic acid content and peak area. The results are shown in the following table. Figures 3-4

[0071] The results are shown in the following table. Figure 3 Figure 4 From the data, it can be concluded that SrCYP716A514 Overexpression of the gene in rosemary not only changed the appearance and local morphology of the plants, but also significantly promoted the synthesis and accumulation of carnosic acid, and had a wide impact on the plant metabolome. These results provide a favorable experimental basis for the application of the gene in improving the production of carnosic acid in rosemary. SrCYP716A514 Overexpression of the gene in rosemary not only changed the appearance and local morphology of the plants, but also significantly promoted the synthesis and accumulation of carnosic acid, and had a wide impact on the plant metabolome. These results provide a favorable experimental basis for the application of the gene in improving the production of carnosic acid in rosemary. SrCYP716A514 Overexpression of the gene in rosemary not only changed the appearance and local morphology of the plants, but also significantly promoted the synthesis and accumulation of carnosic acid, and had a wide impact on the plant metabolome. These results provide a favorable experimental basis for the application of the gene in improving the production of carnosic acid in rosemary.

[0072] Example 3: SrCYP716A514 Increasing the antioxidant capacity of rosemary extract This example aims to explore the differences in antioxidant capacity between the overexpression SrCYP716A514 rosemary extract and the control group (CK and PBI121). Three different antioxidant capacity determination methods were used, namely DPPH free radical scavenging capacity determination, ABTS free radical scavenging capacity determination, and FRAP determination. Finally, the overall evaluation was made by comprehensive antioxidant index APC. The specific experimental process is as follows: 1. Determination of DPPH free radical scavenging capacity According to the total antioxidant capacity determination kit, the rapid DPPH method (BAIGEN, Hubei, China) was used to react the overexpression SrCYP716A514 rosemary extract, control group (CK and PBI121) samples with DPPH solution under specific conditions, and then measure the absorbance and other parameters of the reaction system. According to the standard curve, the DPPH free radical scavenging capacity of each sample was calculated.

[0073] 2. Determination of ABTS free radical scavenging capacity Using the total antioxidant capacity determination kit, the rapid ABTS method (Beyotime, Shanghai, China) was used to measure the ABTS free radical scavenging capacity.

[0074] 3. Determination of FRAP Using the total antioxidant capacity determination kit of FRAP method (Beyotime, Shanghai, China) to measure FRAP.

[0075] 4. Calculation of comprehensive antioxidant index APC Based on the results of the previous three determination methods, the comprehensive antioxidant index APC was calculated to evaluate the overall antioxidant capacity of the overexpression SrCYP716A514 ​​The difference in antioxidant capacity between the rosemary extract and the control group.

[0076] See the attached document for test results. Figure 5 As shown.

[0077] DPPH radical scavenging ability: overexpression SrCYP716A514 Rosemary extract ( SrCYP716A514 The DPPH radical scavenging capacity of the group was significantly higher than that of the control group (CK and PBI121), indicating that overexpression of PBI121... SrCYP716A514 It can significantly enhance the DPPH free radical scavenging ability of rosemary extract.

[0078] ABTS free radical scavenging ability: SrCYP716A514 The ABTS radical scavenging ability of the overexpression group was also significantly higher than that of the CK group and the PBI121 group; this further supports the overexpression group's ability to scavenge free radicals. SrCYP716A514 Enhancement of the antioxidant capacity of rosemary extract.

[0079] FRAP determination: SrCYP716A514 The FeSO4 equivalent concentration (i.e., antioxidant capacity) in the overexpression group was significantly higher than that in the CK group and the PBI121 group. This further demonstrates the effectiveness of overexpression. SrCYP716A514 It can enhance the antioxidant capacity of rosemary extract.

[0080] The overall antioxidant index (APC) was significantly higher in the SrCYP716A514 group than in the control group. The results of the combined measurements by DPPH, ABTS, and FRAP methods fully demonstrate that the overall antioxidant capacity of the rosemary extract overexpressing SrCYP716A514 is higher than that of the control rosemary extract.

[0081] In summary, overexpression SrCYP716A514 This finding significantly enhances the antioxidant capacity of rosemary extract. It is significant for developing rosemary extracts with stronger antioxidant properties, potentially applicable in food, health supplements, or cosmetics to provide better antioxidant protection.

[0082] Example 4: SrCYP76AK7 Analysis of Acquisition and Regulation of Sage Acid Function in Transgenic Plants I. Acquisition of genetically modified plants 1. Transformation Using the recombinant bacteria GV3101 obtained in step 1 of Example 1, and with the empty vector PBI121 recombinant bacteria GV3101 as a control, Agrobacterium was used to infect tobacco ( N. tabacum The specific steps are as follows: (1) Inoculate GV3101 Agrobacterium containing the recombinant vector into OD. 600 =1, enrich the bacterial solution, and discard the supernatant.

[0083] (2) The enriched bacterial solution was suspended in 1 / 2 MS containing 200 mM AS and kept at 28°C in the dark for 2-3 hours.

[0084] (3) Place the prepared rosemary plants in the inoculum for 15-20 minutes, shaking once every 5 minutes.

[0085] (4) Co-culture: In a clean bench, use sterile filter paper to absorb excess bacterial solution, place it in MS medium, and incubate in the dark for 3 days.

[0086] (5) Screening culture: After 3 days of dark incubation, rinse twice with sterile water, blot dry with sterile filter paper, and place on MS (Timentin, 100 mg / L; K) + (100 mg / L), light, 25±1℃, change the culture medium every 2 weeks.

[0087] 2. Identification DNA was extracted from the newly grown shoots to identify positive seedlings. The extracted DNA was then used for PCR and fluorescence microscopy to detect positive seedlings. Five positive seedlings were selected for qPCR verification.

[0088] II. Overexpression SrCYP76AK7 Increase the content of sarsaparilla acid in rosemary 1. Detection of sarsaparilla acid content Transgenic rosemary leaves were ground in liquid nitrogen, and 50 mg was placed in a centrifuge tube. Then 500 μL of 80% methanol was added, and the mixture was sonicated for 60 min. The supernatant was then used to determine the content of oxalic acid.

[0089] 2. Product Identification Substances are identified by comparing the elution times of oxalic acid standards.

[0090] 3. Calculation of oxalic acid content The peak area of ​​the identified oxalic acid was used to calculate the content of oxalic acid by using a standard curve of oxalic acid content and peak area.

[0091] The results are as follows Figures 6-7 As shown, overexpression SrCYP76AK7 The plants that showed fluorescence signals exhibited fluorescence signals, while the plants transfected with the empty vector and the control group (CK) showed weak or no fluorescence signals, indicating that... SrCYP76AK7 The gene has been successfully transferred into rosemary plants and expressed, as fluorescent signals are typically associated with gene expression; overexpression SrCYP76AK7 The selected plant (P2734) exhibited a specific band, while the control group (CK) and the empty vector-transformed plant (PBI121) did not, further confirming this at the molecular level. SrCYP76AK7 Successful introduction and presence of the gene in rosemary plants. Overexpression.SrCYP76AK7 The content of cannabichromene in the rosemary plants (P2734) was significantly higher than that in the plants transformed with the empty vector (PBI121), indicating that the overexpression of the gene promoted the synthesis or accumulation of cannabichromene in rosemary. SrCYP76AK7 The content of FeSO4 equivalent in the P2734 group was significantly higher than that in the PBI121 group, indicating that the overexpression of the gene enhanced the antioxidant-related properties of rosemary. The content of P2734 group was generally higher than that of PBI121 group under different indicators (MVA, MBP, etc.), indicating that the overexpression of the gene had an impact on the various metabolic or physiological processes of rosemary, involving the regulation of multiple metabolic pathways. SrCYP76AK7 The content of FeSO4 equivalent in the P2734 group was significantly higher than that in the PBI121 group, indicating that the overexpression of the gene enhanced the antioxidant-related properties of rosemary. The content of P2734 group was generally higher than that of PBI121 group under different indicators (MVA, MBP, etc.), indicating that the overexpression of the gene had an impact on the various metabolic or physiological processes of rosemary, involving the regulation of multiple metabolic pathways. SrCYP76AK7 The content of FeSO4 equivalent in the P2734 group was significantly higher than that in the PBI121 group, indicating that the overexpression of the gene enhanced the antioxidant-related properties of rosemary. The content of P2734 group was generally higher than that of PBI121 group under different indicators (MVA, MBP, etc.), indicating that the overexpression of the gene had an impact on the various metabolic or physiological processes of rosemary, involving the regulation of multiple metabolic pathways.

[0092] Overall, these experimental results show that the overexpression of the gene has a significant impact on the gene expression, metabolic product synthesis, and physiological functions of rosemary, providing an important basis for further studying the function and application of the gene. SrCYP76AK7 Overall, these experimental results show that the overexpression of the gene has a significant impact on the gene expression, metabolic product synthesis, and physiological functions of rosemary, providing an important basis for further studying the function and application of the gene.

[0093] Example 5: SrCYP76AK7 Increasing the antioxidant capacity of rosemary extract 1. Measurement of DPPH free radical scavenging capacity According to the total antioxidant capacity assay kit, the DPPH free radical scavenging capacity was measured by the rapid DPPH method (BAIGEN, Hubei, China).

[0094] As shown in the results, the antioxidant capacity of rosemary extract overexpressing the gene was higher than that of the control. Figure 8 SrCYP76AK7 2. Measurement of ABTS free radical scavenging capacity Using the total antioxidant capacity assay kit, the ABTS free radical scavenging capacity was measured by the rapid ABTS method (Beyotime, Shanghai, China).

[0095] As shown in the results, the antioxidant capacity of rosemary extract overexpressing the gene was higher than that of the control.

[0096] 3. Measurement of FRAP Figure 8 Using the total antioxidant capacity assay kit of FRAP method (Beyotime, Shanghai, China), FRAP was measured. SrCYP76AK7 As shown in the results, the antioxidant capacity of rosemary extract overexpressing the gene was higher than that of the control.

[0097] In summary, the comprehensive antioxidant index APC indicates that the overexpression of the gene has a significant impact on the antioxidant capacity of rosemary extract.

[0098] In summary, the comprehensive antioxidant index APC indicates that the overexpression of the gene has a significant impact on the antioxidant capacity of rosemary extract. Figure 8 SrCYP76AK7 In summary, the comprehensive antioxidant index APC indicates that the overexpression of the gene has a significant impact on the antioxidant capacity of rosemary extract.

[0099] In summary, the comprehensive antioxidant index APC indicates that the overexpression of the gene has a significant impact on the antioxidant capacity of rosemary extract. SrCYP76AK7 ​​​The antioxidant capacity of the rosemary extract was higher than the control.

[0100] Example 6: SrCYP716A514 and SrCYP76AK7 In vitro enzyme activity assay 1. Construction of recombinant vector PHSE-HIS-SrCYP716A514 / SrCYP76AK7 PCR amplification was performed using primers pSrCYP716A514-F / pSrCYP76AK7-F and pSrCYP716A514-R / pSrCYP76AK7-R (as shown in SEQ ID NO. 13~SEQ ID NO. 16) with the genes shown in SEQ ID No. 1 and SEQ ID No. 3 as templates to obtain PCR products. The full-length cDNA was ligated to the expression vector PHSE-HIS by homologous recombination to generate a recombinant vector. The empty vector (PHSE-HIS) and the recombinant vector were transformed into yeast WATII by freeze-thaw method. The primer sequences are as follows: SrCYP716A514 SrCYP76AK7 SrCYP716A514 SrCYP76AK7 The full-length cDNA was ligated to the expression vector PHSE-HIS by homologous recombination to generate a recombinant vector. The empty vector (PHSE-HIS) and the recombinant vector were transformed into yeast WATII by freeze-thaw method. The primer sequences are as follows:

[0101] 2. CYP substrate feeding The above bacterial liquid was streaked on SD-His solid plates for activation, and the plates were incubated in a 30°C incubator for 3-5 days. Medium-sized single colonies were picked from the activated plates and inoculated into 10 mL of SD-His+glucose culture solution. The inoculated culture solution was incubated in a shaker at 30°C and 200 rpm for 24 hours. After 24 hours of incubation, the bacterial liquid was centrifuged at room temperature at 1500 g for 5 minutes, and the supernatant was discarded. The bacterial cells were collected and washed with 5 mL of sterilized deionized water, and then centrifuged again. The bacterial cells were resuspended in SD-His+galactose culture solution to a bacterial liquid concentration of OD 600 =1.2. 1 mL of the adjusted bacterial liquid was taken in a glass tube, and the substrate ferruginol was added to a final concentration of 100 μM. The mixture was incubated at 30°C with shaking at 200 rpm for 24 hours. After the reaction was completed, 500 μL of the bacterial liquid was taken in a 2 mL centrifuge tube, and an equal volume of ethyl acetate was added. The mixture was vortexed thoroughly, ultrasonicated for 20 minutes, and the reaction mixture was extracted. The two phases were separated by centrifugation at 12,000 rpm for 2 minutes. The upper organic phase was concentrated using a vacuum rotary concentrator, and the concentrated precipitate was redissolved in 200 μL of chromatographic methanol for HPLC analysis. The results are shown in the accompanying Figure 9

[0102] The accompanying​​​​Figure 9 Data shows that, under the catalysis of the combination of SrCYP716A514 and SrCYP76AK7 , Ferruginol can be converted into Carnosic acid. This indicates that the enzyme system composed of SrCYP716A514 and SrCYP76AK7 has specific catalytic activity on Ferruginol and can convert it into Carnosic acid. SrCYP716A514 or SrCYP76AK7 alone cannot effectively catalyze Ferruginol to Carnosic acid, and only when both of them exist together can the conversion from Ferruginol to Carnosic acid be realized. This indicates that SrCYP716A514 and SrCYP76AK7 have a synergistic effect in the catalytic reaction and may each play a key function in different steps of the reaction to complete the catalytic conversion of Ferruginol together. In the chromatogram, the peak area of Carnosic acid in the sample of SrCYP716A514 + SrCYP76AK7 is relatively large, indicating that under the catalysis of the enzyme combination, more Ferruginol is converted into Carnosic acid. Compared with other samples in which only a single enzyme exists with the substrate, the peak area of Carnosic acid is smaller or even nonexistent, further proving the necessity of the synergistic effect of the two enzymes.

[0103] In addition, it is verified whether the alleles of SrCYP716A514 Srcyp716A514 can also catalyze Ferruginol to Carnosic acid together with SrCYP76AK7 , SrCYP716A514 and Srcyp716A514 have one amino acid difference, and the results are shown in the accompanying Figure 10 . The experimental results show that the difference of one amino acid does not affect the action of SrCYP716A514 , Srcyp716A514 and SrCYP76AK7 can also catalyze Ferruginol to Carnosic acid, and the catalytic effect is similar to that of the catalytic system of SrCYP716A514 and SrCYP76AK7 , that is, the difference of one amino acid does not affect the original catalytic effect of SrCYP716A514 .

[0104] In summary, SrCYP716A514 and SrCYP76AK7 can catalyze the substrate Ferruginol to Carnosic acid.

[0105] The above content only illustrates the technical idea of the present application and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.​

Claims

1. A combination of genes for in vitro synthesis of j eronicanth acid, characterized in that, The gene combination is composed of SrCYP716A514 a gene and SrCYP76AK7 a gene, wherein SrCYP716A514 the gene encodes a SrCYP716A514 protein, the amino acid sequence of the SrCYP716A514 protein is shown as SEQ ID NO. 2, and SrCYP76AK7 the gene encodes a SrCYP76AK7 protein, the amino acid sequence of the SrCYP76AK7 protein is shown as SEQ ID NO.

4.

2. The gene combination for in vitro synthesis of oxalic acid according to claim 1, characterized in that, The SrCYP716A514 The nucleotide sequence of the gene is shown as SEQ ID NO. 1; The SrCYP76AK7 The nucleotide sequence of the gene is shown as SEQ ID NO.

3.

3. The combination of genes according to claim 2, wherein, The SrCYP716A514 The gene also includes a nucleotide sequence having more than 90% homology with the sequence as shown in SEQ ID NO. 1 and encoding a protein having the function of the elicitor protein. The SrCYP76AK7 The gene also includes nucleotide sequences having more than 90% homology with the sequence as shown in SEQ ID NO. 3 and encoding a protein having the function of an elicitor protein.

4. The gene combination for in vitro synthesis of oxalic acid according to claim 1, characterized in that, The SrCYP716A514 protein also includes a protein derived from the sequence as shown in SEQ ID No. 2 by substitution and / or deletion and / or addition of one or several amino acid residues and having equivalent activity to the amino acid sequence as shown in SEQ ID No. 2; The SrCYP76AK7 protein also includes a protein derived from the sequence as shown in SEQ ID No. 4 by substitution and / or deletion and / or addition of one or several amino acid residues and having equivalent activity to the amino acid sequence as shown in SEQ ID No.

4.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the gene combination of any one of claims 1-4.

6. A host cell, characterized in that, The host cell contains the recombinant expression vector of claim 5.

7. A recombinant bacterium, characterized in that, The recombinant bacteria contain the gene combination of any one of claims 1-4.

8. Use of the gene combination of claims 1-4, or the expression vector of claim 5, or the host cell of claim 6, or the recombinant bacteria of claim 7 in the synthesis of salvinic acid in vitro.

9. A method of synthesizing in vitro chrysanthemic acid, characterized by, Comprising: The recombinant expression vector containing the gene combination of any one of claims 1-4 is transformed into a host cell, the transformed host cell is added to ferruginol induction incubation, extraction, and salvinic acid is obtained.

10. Use of the gene combination of claims 1-4, or the expression vector of claim 5, or the host cell of claim 6, or the recombinant bacteria of claim 7, or the method of claim 9 in enhancing the antioxidant capacity of plants.

Images