Key enzyme genes EjCYP716A1 / 2 and EjCYP716C1 / 2 for loquat triterpenic acid synthesis and their application

Abstract

The present invention discloses loquat triterpenic acid synthesis key enzyme genes EjCYP716A1 / 2 and EjCYP716C1 / 2 and applications. The present invention first reveals the function of a new oxidized squalene cyclase gene EjOSC2 in synthesizing α / β-amyrin in the process of triterpenic acid synthesis; clarifies the function of gene EjCYP716A1 / 2 in catalyzing oxidation reaction at C-28 of α / β-amyrin to convert it into ursolic acid and oleanolic acid; and clarifies the function of EjCYP716C1 / 2 in catalyzing hydroxylation reaction at C-2 of ursolic acid / oleanolic acid to convert it into corosolic acid / maslinic acid. These five genes can provide gene resources for regulating the accumulation level of ursane-type and oleanolic acid-type triterpenic acids in tissues such as leaves of medicinal plants loquat, and also lay the foundation for batch production of natural active triterpenic acid components such as loquat ursolic acid and corosolic acid through environmentally friendly production processes such as gene editing or fermentation engineering.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more specifically to loquat triterpenic acid synthesis key enzyme genes EjCYP716A1 / 2 and EjCYP716C1 / 2 and applications thereof. Background Art

[0002] Loquat is an important evergreen fruit tree that can be used for both medicine and food. In addition to its delicious fruit, which is loved by consumers, its dry leaves are one of the main donors of some important natural products. They are not only widely used in traditional Chinese medicine, but also attract much attention in the development of modern biomedicine. Triterpenic acid is the most abundant natural active compound in loquat leaves. With the continuous development of detection technology, researchers have identified dozens of triterpenic acid components from loquat leaves in the past two decades. Bioactivity evaluation and pharmacological studies have shown that major triterpenic acids such as ursolic acid and corosolic acid play an important role in the treatment of diseases such as cancer cell inhibition, bactericidal and anti-inflammatory, and hypoglycemic, and have been widely valued and used in the fields of medicine and food. For loquat, its commonly used medicinal leaves are collected from fruit production trees. The production of various components requires the separation of individual triterpenic acid components from a large number of leaves through cumbersome process flows. The use of traditional preparation processes affects the production of fresh loquat fruit. At the same time, the extraction process consumes a lot of manpower and material resources, and is prone to industrial pollution. Using engineered bacteria or plants as biological chassis to express genes related to metabolite synthesis can not only reduce the impact of leaf picking on fruit production, but also produce target components in batches and in a targeted manner. It is an environmentally friendly way to synthesize compounds.

[0003] Identifying the structural genes for triterpenic acid biosynthesis and establishing an expression system is the key to ensuring biosynthesis and directional production of target components. Although researchers have reported several fragments of genes related to triterpenic acid synthesis in loquat, these studies have not identified the functions of related synthetic genes under blank controls. Patent CN110938640A discloses an oxidosqualene cyclase gene EjAAS1 for synthesizing α-amyrin, but other homologous genes with similar functions have not been identified; the synthase genes for triterpenic acids such as ursolic acid and corosolic acid, as well as oleanolic acid and hawthorn acid, which are high in leaves, have not yet been identified and mined. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide loquat triterpenic acid synthesis key enzyme genes EjCYP716A1 / 2 and EjCYP716C1 / 2 and applications.

[0005] The first object of the present invention is to provide a gene with a nucleotide sequence as shown in SEQ ID NO.1-2, and / or a protein with an amino acid sequence as shown in SEQ ID NO.3-4, for use in promoting the production of triterpene acids in loquat.

[0006] The second object of the present invention is to provide a gene with a nucleotide sequence as shown in SEQ ID NO.1-2, and / or a protein with an amino acid sequence as shown in SEQ ID NO.3-4, for use in promoting the production of ursolic acid from α-amyrin, the production of corosolic acid from α-amyrin, the production of oleanolic acid from β-amyrin, and / or the production of hawthorn acid from β-amyrin.

[0007] The third object of the present invention is to provide a method for increasing the yield of ursane-type and / or oleanane-type triterpene acids.

[0008] The fourth object of the present invention is to provide a gene having a nucleotide sequence as shown in SEQ ID NO.5 to 6, and / or a protein having an amino acid sequence as shown in SEQ ID NO.7 to 8, for use in promoting loquat to produce ursane-type and / or oleanane-type triterpene acids.

[0009] The fifth object of the present invention is to provide the use of any gene with nucleotide sequences as shown in SEQ ID NO.5-6 or any protein with amino acid sequences as shown in SEQ ID NO.7-8 in promoting the production of corosolic acid from ursolic acid and / or promoting the production of maslinic acid from oleanolic acid.

[0010] The sixth object of the present invention is to provide a method for increasing the yield of corosolic acid and / or maslinic acid.

[0011] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0012] The present invention cloned a loquat oxidosqualene cyclase gene EjOSC2 from the 'Jiefangzhong' loquat, whose nucleotide sequence is shown in SEQ ID NO.9, comprising an open reading frame of 2283 bp, encoding 760 amino acids, and its encoded amino acid sequence is shown in SEQ ID NO.10. The gene can catalyze the synthesis of α-amyrin and β-amyrin when transiently expressed in tobacco leaves.

[0013] At the same time, two loquat α / β-amyrin C-28 oxidase genes EjCYP716A1 and EjCYP716A2 were cloned, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Both contain an open reading frame of 1455 bp and encode 484 amino acids. The amino acid sequences encoded by them are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. These two genes are transiently co-expressed with the gene EjOSC2 in tobacco leaves, and both can catalyze the formation of ursolic acid and oleanolic acid.

[0014] The present invention also cloned two loquat ursolic acid / oleanolic acid C-2 hydroxylase genes EjCYP716C1 and EjCYP716C2, whose nucleotide sequences are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively, both of which contain an open reading frame of 1443 bp and encode 480 amino acids, and the amino acid sequences encoded by them are shown as SEQ ID NO.7 and SEQ ID NO.8 respectively, and the two genes are transiently co-expressed with genes EjOSC2-EjCYP716A1 and / or EjOSC2-EjCYP716A2 in tobacco leaves, and both can catalyze the formation of corosolic acid and hawthorn acid.

[0015] Therefore, the present invention claims the following:

[0016] Use of any gene with nucleotide sequences as shown in SEQ ID NO.1-2, and / or any protein with amino acid sequences as shown in SEQ ID NO.3-4 in promoting the production of triterpene acids in loquat.

[0017] The triterpene acids are ursane type and / or oleanane type triterpene acids.

[0018] The ursane type triterpene acid is ursolic acid and / or corosolic acid, and the oleanane type triterpene acid is oleanolic acid and / or maslinic acid.

[0019] The use of any gene with nucleotide sequences as shown in SEQ ID NO.1-2, and / or any protein with amino acid sequences as shown in SEQ ID NO.3-4, in promoting α-amyrin to produce ursolic acid, α-amyrin to produce corosolic acid, β-amyrin to produce oleanolic acid, and / or β-amyrin to produce hawthorn acid.

[0020] A method for increasing the yield of ursane-type and / or oleanane-type triterpene acids, overexpressing one or two genes with nucleotide sequences as shown in SEQ ID NOs. 1-2, and / or increasing the expression of one or two proteins with amino acid sequences as shown in SEQ ID NOs. 3-4.

[0021] The ursane type triterpene acid is ursolic acid and / or corosolic acid, and the oleanane type triterpene acid is oleanolic acid and / or maslinic acid.

[0022] Preferably, the method comprises: preparing a recombinant engineered bacterium containing a gene with a nucleotide sequence such as SEQ ID NO.1 and / or SEQ ID NO.2, and introducing the gene into an organism capable of producing α-amyrin and / or β-amyrin to obtain a recombinant organism; culturing the recombinant organism to overexpress a protein with an amino acid sequence such as SEQ ID NO.3 and / or SEQ ID NO.4.

[0023] The use of any gene with nucleotide sequences as shown in SEQ ID NOs.5 to 6, and / or any protein with amino acid sequences as shown in SEQ ID NOs.7 to 8 in promoting loquat to produce ursane-type and / or oleanane-type triterpene acids.

[0024] Preferably, the ursane-type triterpene acid is corosolic acid, and the oleanane-type triterpene acid is maslinic acid.

[0025] The use of any gene with nucleotide sequence as shown in SEQ ID NO.5-6 or any protein with amino acid sequence as shown in SEQ ID NO.7-8 in promoting the production of corosolic acid from ursolic acid and / or promoting the production of maslinic acid from oleanolic acid.

[0026] A method for increasing the yield of corosolic acid and / or maslinic acid, overexpressing one or two genes with nucleotide sequences as shown in SEQ ID NOs. 5-6, and / or increasing the expression of one or two proteins with amino acid sequences as shown in SEQ ID NOs. 7-8.

[0027] Preferably, the method comprises: preparing a recombinant engineered bacterium containing a gene having a nucleotide sequence such as SEQ ID NO.5 and / or SEQ ID NO.6, and introducing the gene into an organism capable of producing ursolic acid and / or oleanolic acid to obtain a recombinant organism; culturing the recombinant organism to overexpress a protein having an amino acid sequence such as SEQ ID NO.7 and / or SEQ ID NO.8.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The invention establishes an accumulation model of main triterpenic acids in loquat, clones a new α-amyrin synthase gene EjOSC2 for the first time, and can synthesize α-amyrin and a small amount of β-amyrin when transiently expressed in tobacco leaves; simultaneously, two loquat α / β-amyrin C-28 oxidase genes EjCYP716A1 and EjCYP716A2 are cloned, and both can catalyze the formation of ursolic acid and oleanolic acid when transiently expressed in tobacco; and two loquat ursolic acid / oleanolic acid C-2 hydroxylase genes EjCYP716C1 and EjCYP716C2 are cloned, and both can catalyze the formation of corosolic acid and hawthorn acid when transiently expressed in tobacco. The expression of these genes can promote the synthesis of loquat α-amyrin, β-amyrin, ursolic acid, oleanolic acid, corosolic acid and maslinic acid and the in vitro biosynthesis of α-amyrin, β-amyrin, ursolic acid, oleanolic acid, corosolic acid and maslinic acid and other ursane-type and oleanane-type triterpenoid acids derived from them. The present invention lays a foundation for obtaining important natural active compounds of loquat in batches through environmentally friendly technologies such as structural gene regulation and co-expression of key genes in metabolic synthesis pathways, and provides theoretical support for the modification of triterpenoid acid-specific functional components and product development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The GC-MS chromatographic detection results of triterpenic acid components in loquat leaves at five different developmental stages; A is the GC-MS chromatogram, and B is the proportion of triterpenic acid components in mature loquat leaves.

[0031] Figure 2 This is the phylogenetic tree of EjOSC2 and OSC proteins from other plants.

[0032] Figure 3 This is the clustering evolutionary tree of EjCYP716A1 / 2, EjCYP716C1 / 2 and CYP716 proteins of other plants.

[0033] Figure 4 These are the GC-MS detection chromatograms and mass spectra of the transient expression product of EjOSC2 in tobacco; in Figure A, the pSAK277 empty vector was used as a blank control, EjAAS1 was used as a positive control, triterpene acid standards were used as external standards, and coprostanol was used as an internal standard; Figure B is the mass spectrometry ion fragment comparison of α-amyrin and β-amyrin standards and the EjOSC2 expression product.

[0034] Figure 5These are the GC-MS detection chromatograms and mass spectra of the tobacco transient expression products of EjCYP716A1 and EjCYP716A2; in Figure A, the pSAK277 empty vector was used as a blank control, EjOSC2 was expressed to provide α-amyrin and β-amyrin substrates for EjCYP716A1 / 2, triterpene acid standards were used as external standards, and coprostanol was used as an internal standard; Figure B is the mass spectrometry ion fragment comparison of ursolic acid / oleanolic acid standards and EjCYP716A1 or EjCYP716A2 expression products.

[0035] Figure 6 The GC-MS chromatograms and mass spectra of the transient expression products of EjCYP716C1 and EjCYP716C2 in tobacco; In Figure A, the pSAK277 empty vector was used as a blank control. The expression of EjOSC2 can provide α-amyrin and β-amyrin substrates for EjCYP716A, and the expression of EjOSC2-EjCYP716A1 or EjOSC2-EjCYP716A2 can provide ursolic acid and oleanolic acid substrates for EjCYP716C. Triterpenic acid standards were used as external standards, and coprostanol was used as internal standards; Figure B shows the mass spectrometry ion fragment comparison of corosolic acid / maslinic acid standards and the expression products of EjCYP716C1 or EjCYP716C2.

[0036] Figure 7 This is a metabolic pathway for synthesizing the main triterpene acids in loquat with EjOSC2 as the fulcrum. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0038] Example 1 Detection of triterpenic acid components in loquat leaves

[0039] 1. Experimental Methods

[0040] 1. Sample collection

[0041] Leaf samples of 'Jiefangzhong' loquat were collected from the germplasm resource nursery of loquat plants at South China Agricultural University at five developmental stages, from the newly sprouted young leaves (stage 1) to the fully mature leaves (stage 5) (three biological replicates for each sample). The fresh samples were quickly frozen with liquid nitrogen and stored in a -80℃ refrigerator for later use.

[0042] 2. Extraction and detection of triterpene acid components

[0043] The loquat leaf samples were freeze-dried overnight in a Frerzone 12L freeze dryer (Labconco, USA) at -80°C and 0.1 MPa. The freeze-dried material was chopped and ground into fine powder using a plant grinder. Weigh 5.0 mg of sample dry powder into a 1.5 mL centrifuge tube and add 500 μL of ethanol mixture (volume ratio of ethanol: water: KOH = 9:1:1); shake vigorously and place in a water bath at 70°C for 1 hour; evaporate the ethanol in a microconcentrator (Eppendorf, Germany) at 40°C for 2 hours; add 500 μL of ethyl acetate and shake vigorously for 1 min; add 500 μL of water and continue to shake vigorously for 1 min; centrifuge at 16000g for 5 min; take the upper ethyl acetate layer; take 50 μL of ethyl acetate extraction layer and dry it with nitrogen in a nitrogen blower; add 30 μL of methoxyamine hydrochloride pyridine solution to dissolve the dried material and place in a water bath at 37°C for 2 hours; add 20 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide and incubate at 37°C for 0.5 hour to carry out silanization derivatization of triterpene acid extract.

[0044] The derivatives were transferred into injection bottles, and the triterpenic acid components in the extract were detected using an Agilent 7890A / 5975C GC-MS (CA, USA). The chromatographic conditions for the determination were as follows: HP-5M (30m×0.25mm×0.25μm) chromatographic column, programmed temperature rise to 40°C for 1 min, then temperature rise to 320°C at 20°C / min, and maintained for 15 min; splitless injection, injection volume of 1μL, helium flow rate of 1mL / min; mass spectrometry conditions were as follows: interface temperature of 250°C, ion source temperature of 200°C, quadrupole temperature of 150°C, solvent delay of 15 min, voltage of 1500V, and mass scan range of 60-800m / s. β-amyrin (CAS No.: 559-70-6), α-amyrin (CAS No.: 508-04-3), oleanolic acid (CAS No.: 508-02-1), ursolic acid (CAS No.: 77-52-1), corosolic acid (4547-24-4), and maslinic acid (CAS No.: 4373-41-5) were used as standards (Sigma-Aldrich), and coprostanol (CAS No.: 360-68-9) was used as an internal standard, and all were detected under the same conditions.

[0045] 2. Experimental Results

[0046] The results are as follows Figure 1 As shown in the figure, GC-MS analysis found that triterpenic acids could be detected in leaves of all five stages; only a small amount of α-amyrin and β-amyrin could be detected in samples of the first stage (newly sprouted young leaves), followed by a gradual increase in the content of ursolic acid and oleanolic acid, and the beginning of the appearance of corosolic acid and hawthorn acid; the content of major triterpenic acids such as corosolic acid and ursolic acid in leaves approaching maturity reached the highest peak ( Figure 1A). Further comparison of the proportion of triterpenic acids in mature leaves revealed that loquat leaves accumulated two types of triterpenic acids: ursane-type and oleanane-type; ursane-type accounted for about 76% of the total triterpenic acids, of which 55% were corosolic acid and 20% were ursolic acid; of the 24% oleanane-type triterpenic acids, nearly 18% were maslinic acid and about 3.4% were oleanolic acid ( Figure 1 B). Component ratio analysis indicates that corosolic acid, ursolic acid, and maslinic acid, which account for the majority of the components, may be the core components that play a key role in the use of loquat as a traditional Chinese medicine. The discovery of the biosynthetic genes of these components is conducive to the development and utilization of related compounds.

[0047] Example 2 Loquat EjOSC2, EjCYP716A1 / 2, EjCYP716C1 / 2 gene cloning and vector construction

[0048] 1. Experimental Methods

[0049] 1. Sample collection

[0050] The leaf samples were collected and preserved according to the method of Example 1.

[0051] 2. RNA Extraction

[0052] Mature leaves stored at -80°C were ground into powder under liquid nitrogen. 100 mg of the powder was transferred to a 1.5 mL centrifuge tube, 1.0 mL of RLT lysis buffer was added, and RNA was extracted according to the instructions of the EASYspin Plus Plant RNA Rapid Extraction Kit (Adlai, China), as follows: the lysis buffer in each tube with sample was vigorously shaken on an oscillator for 20 seconds, and then lysed in a 55°C water bath for 20 minutes; the lysate was centrifuged at 13000 rpm for 10 minutes; the supernatant was taken and transferred to a new centrifuge tube, and half the volume of anhydrous ethanol was added, and the mixture was gently pipetted and mixed; the mixture was added to the RA adsorption column, and after standing for 30 seconds, it was centrifuged at 13000 rpm for 2 minutes; the waste liquid was discarded , repeat the previous step and filter all the mixtures through the RA adsorption column; discard the waste liquid, add 700μl of deproteinization solution RW1 to the adsorption column, let it stand for 1min, and centrifuge at 13000rpm for 30s; discard the waste liquid, add 500μl of rinsing solution RW to the adsorption column, let it stand for 1min, and centrifuge at 13000rpm for 30s; discard the waste liquid, add rinsing solution RW and repeat; discard the waste liquid, put the adsorption column back into the empty tube and centrifuge at 13000rpm for 2min; take out the adsorption column, put it into a new RNase free centrifuge tube, add 50μl RNase free water in the middle of the adsorption membrane, let it stand at room temperature for 2min, and centrifuge at 12000rpm for 1min; aspirate the eluted RNA back to the middle of the original adsorption membrane, let it stand at room temperature for another 2min, and centrifuge at 12000rpm for 1min to obtain total RNA.

[0053] 3. Reverse transcription to synthesize cDNA

[0054] The RNA concentration was detected using NanoDrop2000 ultra-micro-volume spectrophotometer (Thermo Fisher Scientific, USA). RNA was diluted to 100 ng·μL with RNase-free water. -1 , by PrimeScript TM The first chain of cDNA was synthesized according to the instruction manual of RT reagent Kit with gDNA Eraser reverse transcription system (TaKaRa, Japan). The reaction system for removing DNA included 2μL gDNA Eraser buffer, 1μL gDNA Eraser and 7μL RNA. After mixing, gDNA was removed at 42°C. After the reaction, the following components were added to each system: 1μL PrimeScript RT Enzyme Mix I + 1μL RT Primer Mix + 4μL 5× PrimeScript Buffer 2 + 4μL RNase Free dH2O, mixed, and incubated on a PCR instrument at the following temperature: 37°C, 15min→85°C, 10s→4°C to complete the reverse transcription reaction.

[0055] 4. Amplification of genes related to triterpenic acid synthesis

[0056] The full-length coding region sequences of OSC2, CYP716A1 / 2 and CYP716C1 / 2 genes annotated in the loquat genome were retrieved, and primers were designed using Primer Premier 5.0. Homologous sequence linkers on both sides of the restriction sites (EcoRI and XhoI) on the expression vector pSAK277 were added to the upstream and downstream primers, respectively. Finally, the primers were commissioned to Shanghai Bioengineering Co., Ltd. to synthesize the full-length amplification primer pairs of the sequences. The amplification primer pairs are shown in Table 1.

[0057] Table 1. Amplification primer sequences

[0058]

[0059]

[0060] use HS DNA Polymerase (TaKaRa, Japan) was used to amplify the target gene. The amplification system was 20 μl, including: 10 μl high-fidelity DNA Polymerase Mix, 1 μl forward primer, 1 μl reverse primer, 1 μl leaf cDNA, 7 μl ddH2O; the amplification program was 95°C, 5 min; 95°C, 30 s; 56°C, 30 s; 72°C, 1 min 30 s to 2 min 30 s; run 34 cycles; 72°C post-extension, 10 min; 12°C, storage.

[0061] Linearization of pSAK277 vector: 5μl CutSmart Buffer (NEB, USA), 1μl EcoRI, 1μl XhoI, 1μg plasmid, add water to 10μl. Use a pipette to carefully aspirate and mix, digest at 37℃ for 3h, and inactivate the endonuclease in a 65℃ water bath for 20min. Agarose gel electrophoresis to recover the target band.

[0062] Hieff The recovered PCR products were connected to the linearized pSAK277 expression vector using the Plus One Step Cloning Kit (Shanghai Yisheng Biotechnology, China), specifically: 150 ng of purified and recovered PCR amplification products, 200 ng of linearized pSAK277 vector, 5 μl Hieff Enzyme Premix, add ddH2O to 10μl. Gently pipette and mix, react at 50℃ for half an hour, incubate on ice for 5min, then take 5μl of ligation product and transfer into 50μl E. coli DH5α competent cells; kanamycin screens positive E. coli clones, selects positive clones detected by PCR and submits them to Shanghai Bioengineering Co., Ltd. for Sanger sequencing. The plasmid returned after correct sequencing is the corresponding expression vector.

[0063] 2. Experimental Results

[0064] 1. Using the designed specific primer pairs, a single EjOSC2 sequence can be isolated from leaves, and its coding region length is 2283bp (the nucleotide sequence is shown in SEQ ID NO.9), and the sequence encodes 760 amino acid residues (the amino acid sequence is shown in SEQ ID NO.10); specific EjCYP716A1 and EjCYP716A2 sequences were isolated, and their coding regions were both 1455bp in length (the nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively), and both encode 484 amino acid residues (the amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively); specific EjCYP716C1 and EjCYP716C2 sequences were isolated, and their coding regions were both 1443bp in length (the nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively), and both encode 480 amino acid residues (the amino acid sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively).

[0065] 2. Use MEGA6 software to construct the evolutionary trees of OSC and CYP716 homologous genes respectively. The results are as follows Figure 2 and Figure 3 As shown in the figure, loquat EjOSC2 was found to be clustered with apple MdOSC1-MdOSC3 and Arabidopsis AtBAS. EjOSC2 and the previously identified EjAAS1 were clustered with apple MdOSC1 / 3 in a small branch. It was most closely related to apple MdOSC3, with a 98.95% identity of the encoded amino acids; the identities of the amino acids encoded by EjAAS1 and apple MdOSC1 were 96.32% and 96.84%, respectively, and the sequence identity with Arabidopsis AtBAS was 63% ( Figure 2 ). Both EjCYP716A1 / 2 and EjCYP716C1 / 2 are in the large branch of the plant CYP716 subfamily, but they are distributed in different branches. EjCYP716A1 / 2 is closest to MdCYP716A175, among which the amino acid identity between EjCYP716A1 and MdCYP716A175 reaches 98.97%, and the amino acid identity between EjCYP716A2 and MdCYP716A175 is 97.73%. EjCYP716C1 / 2 and two known functional CYP716Cs are clustered together to form another small branch, but they are most similar to each other, with an amino acid sequence identity of 93.33%; their sequences with the amino acid encoded by Lagerstroemia indica LsCYP716C55 are 75.67% and 76.08% respectively ( Figure 3 ).

[0066] Example 3 Transient expression of EjOSC2 gene in tobacco and detection of triterpene acid products

[0067] 1. Experimental Methods

[0068] The construction of the vector refers to Example 2, and the amplified full-length EjOSC2 is inserted into the pSAK277 expression vector to construct the pSAK277-EjOSC2 recombinant plasmid. The constructed expression vector was transferred into GV3101 (pSoup) Agrobacterium, and the pSAK277 vector without any gene inserted was transformed as a blank control. The positive clone bacterial solution was added with glycerol and stored in a -80°C refrigerator for standby use. Cultivate Nicotiana benthamiana in a greenhouse. After 5 to 6 true leaves of the tobacco are fully unfolded, take the glycerol bacteria and activate them by streaking on a plate containing kanamycin and rifampicin antibiotics. Pick the colony into 600 μL liquid culture medium, shake at 200 rpm at 30°C for 6 hours, and perform bacterial solution PCR detection. Take 400 μL of the newly activated bacterial solution of the positive clone and inoculate it into 10 mL of YEP liquid culture medium containing 50 μL Kan (50 μM) and 100 mL Rif (50 μM), shake overnight, until the bacterial solution A 600 The value reaches 0.8~1.0. Use 30mL MES (100mM), 30mL MgCl2 (100mM), 150μL acetosyringone (Acetosyringone concentration is 100mM), add sterile water to 150mL, mix well to make MMA working solution. Take 5mL bacterial solution, centrifuge at 5000rpm for 10min; discard the supernatant, add 2mL MMA working solution, suspend the bacteria; centrifuge at 5000rpm for 10min; discard the supernatant, repeat suspension-centrifugation once; add 2mL MMA working solution, suspend the bacteria and transfer to a 15mL test tube; add MMA working solution to dilute the bacterial solution to A 600 = about 0.2; incubate the diluted bacterial solution in dark and room temperature for about 3 hours. Use a 1mL syringe without a needle to inject the diluted bacterial solution into the back of the leaf. The leaf injected with the pSAK277 empty vector bacterial solution is used as a control. After injection, move the tobacco to low light conditions for 1 day, and then transfer to normal light conditions for culturing.

[0069] After 3 days, a small amount of leaf samples were collected, and RNA was extracted and reverse transcribed into cDNA using the method of Example 2 to detect gene expression. After 1 week, tobacco leaves were collected, quickly frozen with liquid nitrogen, transferred to a freeze dryer, and dried at -80°C and 0.1 MPa until the leaves were balanced. The triterpenic acid components in the tobacco leaves were extracted using the method of Reference Example 1, and the triterpenic acid products transiently expressed by EjOSC2 were detected by GC-MS under the same chromatographic conditions.

[0070] 2. Experimental Results

[0071] The results are as follows Figure 5As shown in Figure 2, after transient expression of EjOSC2, a large amount of α-amyrin and a small amount of β-amyrin were detected in the leaf extracts of Nicotiana benthamiana, while no corresponding products were detected in the control leaves treated with the empty vector ( Figure 4 A). After homogenization with coprostanol, the chromatographic peak areas of α-amyrin and β-amyrin were compared and it was found that the ratio of α-amyrin to β-amyrin in the EjOSC2 expression product was 89:11. Afterwards, the total ion fragments (TIC) of the EjOSC2 expression product and the standard (α-amyrin / β-amyrin) were compared and it was found that the ion fragment structures of the two products detected after EjOSC2 expression were the same as those of the standard β-amyrin and α-amyrin, respectively ( Figure 4 B). The results of GC-MS double detection of triterpenic acid product retention time and product total ion fragment comparison showed that EjOSC2 is a synthetic gene of triterpenic acid precursors, and the protein it encodes can catalyze the production of α-amyrin and β-amyrin.

[0072] Example 4 Transient expression of EjCYP716A1 and EjCYP716A2 genes in tobacco and detection of triterpene acid products

[0073] 1. Experimental Methods

[0074] The construction of the vector was referred to Example 2. The amplified full-length EjCYP716A1 and EjCYP716A2 were inserted into the pSAK277 expression vector to construct the pSAK277-EjCYP716A1 and pSAK277-EjCYP716A2 recombinant plasmids. The constructed expression vector was transferred into GV3101 (pSoup) Agrobacterium, and the pSAK277-EjOSC2 glycerol bacteria of Example 3 were activated to provide α / β-amyrin catalytic substrate for the target gene encoding oxidase. The target gene was transiently expressed in tobacco according to Example 3. After 1 week, tobacco leaves were collected and triterpene acid products were detected according to Example 3.

[0075] 2. Experimental Results

[0076] Transient expression of EjOSC2 can continuously provide α-amyrin and β-amyrin substrates. When EjCYP716A1 or EjCYP716A2 was injected simultaneously with EjOSC2, new products, oleanolic acid and ursolic acid, were detected at around 19.6 and 20.0 min, respectively. Figure 5 As shown, the ratios of ursolic acid to oleanolic acid in the co-expression products of EjOSC2-EjCYP716A1 and EjOSC2-EjCYP716A2 were 81:19 and 88:12, respectively. Figure 5A). Comparison of the total ion fragments (TIC) of the expression products of EjOSC2-EjCYP716A1 and EjOSC2-EjCYP716A2 and the standards (ursolic acid and oleanolic acid) revealed that the ion fragment structures of the two products detected after the expression of EjOSC2-EjCYP716A1 and EjOSC2-EjCYP716A2 were the same as those of the standards ursolic acid / oleanolic acid ( Figure 5 B). The results of GC-MS double detection of triterpenic acid product retention time and total ion fragment comparison showed that EjCYP716A1 and EjCYP716A 2 are genes related to triterpenic acid synthesis, and the proteins encoded by them can catalyze the production of ursolic acid and oleanolic acid.

[0077] Example 5 Transient expression of EjCYP716C1 and EjCYP716C2 genes in tobacco and detection of triterpene acid products

[0078] 1. Experimental Methods

[0079] The vector was constructed with reference to Example 2, and the amplified full-length sequences of the EjCYP716C1 and EjCYP716C2 coding regions were connected to the pSAK277 expression vector, respectively, to construct the pSAK277-EjCYP716C1 and pSAK277-EjCYP716C2 recombinant plasmids. The constructed expression vector was transferred into GV3101 (pSoup) Agrobacterium, and the pSAK277-EjOSC2 of Example 3 and the pSAK277-EjCYP716A1 / 2 glycerol bacteria of Example 4 were activated at the same time, so as to provide α / β amaranth and ursolic acid / oleanolic acid substrates for the target gene encoding catalytic enzyme. The target gene was transiently expressed with reference to Example 3. After 1 week, tobacco leaves were collected with reference to Example 3 and triterpenic acid products were detected.

[0080] 2. Experimental Results

[0081] Transient expression of EjOSC2 can continuously provide α-amyrin and β-amyrin substrates, and expression of EjOSC2-EjCYP716A1 or EjOSC2-EjCYP716A2 can continuously provide ursolic acid and oleanolic acid substrates. When EjCYP716C1 or EjCYP716C2 is injected on the basis of EjOSC2-EjCYP716A1 or EjOSC2-EjCYP716A2, maslinic acid and corosolic acid are detected at 21.2 and 21.5 minutes. The results are as follows Figure 6As shown, the ratio of corosolic acid to maslinic acid in the expression products of the four combinations of EjOSC2-EjCYP716A1-EjCYP716C1, EjOSC2-EjCYP716A1-EjCYP716C2, EjOSC2-EjCYP716A2-EjCYP716C1, and EjOSC2-EjCYP716A2-EjCYP716C2 was between 85:15 and 86:14 ( Figure 6 A). Comparison of the total ion fragments (TIC) of the expression products of EjOSC2-EjCYP716A1-EjCYP716C1 / 2 and EjOSC2-EjCYP716A2-EjCYP716C1 / 2 and the standards (corosolic acid and maslinic acid) revealed that the ion fragment structures of the two products detected after the expression of EjOSC2-EjCYP716A1-EjCYP716C1 / 2 and EjOSC2-EjCYP716A2-EjCYP716C1 / 2 were the same as those of the standards ( Figure 6 B). The results of GC-MS double detection of triterpenic acid product retention time and product total ion fragment comparison showed that EjCYP716C1 / 2 is a gene related to triterpenic acid synthesis, and the protein encoded by it can catalyze the production of corosolic acid and hawthorn acid.

[0082] Through the research of Examples 3, 4, and 5, the present invention systematically reports the synthesis pathways of the main triterpenic acids of loquat: EjOSC2 is an oxidosqualene cyclase gene, which can catalyze the synthesis of α-amyrin and β-amyrin; EjCYP716A1 and EjCYP716A2 are α / β-amyrin C-28 oxidase genes, which can catalyze α-amyrin to form ursolic acid and β-amyrin to form oleanolic acid; EjCYP716C1 and EjCYP716C2 are ursolic acid / oleanolic acid C-2 hydroxylase genes, which can catalyze ursolic acid to form corosolic acid and catalyze oleanolic acid to form hawthorn acid, as shown in the following example. Figure 7 shown.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention. Sequence Listing <120> Key enzyme genes EjCYP716A1 / 2 and EjCYP716C1 / 2 for loquat triterpenic acid synthesis and their application <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1455 <212> DNA <213> Eriobotrya japonica (Thunb.) Lindl <400> 1 atggagcact tctatctgac cctcctcttg gggtttgtct ccttcatcac tctttctctc 60 tccgtgctct tttaccggca cagatcgcag ttcgtcggca ccaacctgcc gcctggcaaa 120 gttggctacc cagtgatcgg cgagacctac cagttcctag ccacaggatg gaaaggtcac 180 ccggagaagt tcatcttcga ccgcatgacc aagtactcct ccgaagtgtt caagacctca 240 ctcatgggcg agaaggccgc catcttctgc ggtgcagcct gcaacaagtt cttgttctcc 300 aacgagaaca agctcgtcac tgcatggtgg cccagctccg tcaacaaggt cttcccttct 360 tctatggaga cttccgccaa ggaggaggcc aagaagatga gaaagatgct tcccaacttc 420 atgaagcccg aagctctcca gcgatacatc ggcatcatgg acaccgtcgc ccgacgccac 480 ttcgctgagg gctgggaaaa caagaaggaa gtggaagtct ttcccctcgc caagaactac 540 accttttggc ttgctgcacg gttgtttgtg agcctggagg actcggttga gatcgccaag 600 ctaggcgatc cgttcgcagt ttggcctcg ggaatcatat cgatgcctct ggatttcccg 660 gggactccgt tttacaaagc gatcaggcg tccaacttca tcagggagga gctgacgaag 720 atcatcaagc agaggaagat agacttggcg gagggcaagg cgtcgccgac gcaagacata 780 ttgtcacaca tgttgttgct gtgcgacgag cacggaagtc acatgaagga acacgatatc 840 gcggataaga ttttggggct gctgatcggt ggccatgaca ccgctagcgc tacctgcact 900 tttattgtca agtatcttgc tgagcttcct cacatttg acgaagtcta CAggagcaa 960 atggaggtcc taagcgcaa agcaccaggg gagttgttga actgggatga cctacagaag 1020 atgaaatact catggaacgt agctcaggaa gttctcagat tggctcacc tctcagga 1080 gcattcaggg aagccttgtc cgacttgtc ttcaatggtt tcaccattcc aaaggctgg 1140 aagttgtatt ggagtgcaaa ttcacat aagaacgcag cttactccc ggaaccattc 1200 aaattcgacc ctacaagatt cgaggaat ggtccagcac catacacgtt tgtgcccttt 1260 ggaggaggtc ctaggatgtg ccccggaaaa gagtacgccc gattggaat cctagtgttc 1320 atgcacaact tggtgaagag gttcaaatgg gaaaaagttc ttcccgacga gcagatcgta 1380 gttgacccac tgcccatgcc cgccaaggga ctccccgtcc gccttttctc tcaccctaag 1440 acagccacag cttaa 1455 <210> 2 <211> 1455 <212> DNA <213> Eriobotrya japonica (Thunb.) Lindl <400> 2 atggagcact tctatctgac cctcctctta gggtttgtct cttcatcac tctttctctc 60 tccgtgctct tttaccggca cagatcgcag ttcgtcggca ccaacctgcc gcctggcaaa 120 gttggctacc cggtgatcgg tgagacctac cagttcctgg ccacaggatg gaaaggtcac 180 ccagagaagt tcatcttcga ccgcatgagc aagtactctt ccgaagtgtt caagacctcc 240 cttatgggcg agaaggccgc catcttctgc ggcgcagcct gcaacaagtt cttgttctcc 300 aacgagaaca agctcgtcac tgcatggtgg cccagctccg tcaacaaggt cttcccttct 360 tctatggaga cttccgccaa ggaggaggcc aagaagatga gaaagatgct tcccaacttc 420 atgaagcccg aagctctcca gcgatacatc ggcatcatgg acaccgtcgc ccgacgccac 480 540 accttttggc ttgctgcacg gttgtttgtg agcctggacg actcggttga aatcgccaag 600 ctaggcgacc cgttcgcagt tttggcctct ggaatcatat cgatgcctct ggatttcccg 660 ggaactccgt tttacaaagc gatcaaggca tctaacttca tcagggagga gctgacgaag 720 atcatcaagc agagaagat agacttggcg gagggcaagg cgtcgccgac gcaagacata 780 ttgtcacaca tgttgttgct gtgcgacgag cacggaagtc acatgaagga acacgatatc 840 gccgataaga ttttggggct gctgattggt ggccatgaca ccgccagcgc tacctgcact 900 tttattgtca agtatcttgc cgaacttcct catatttatg acgaagtcta caaggagcaa 960 atggaggtcc taagcgcaaa agccccaggg gagttgttga attgggatga cctgcagaag 1020 atgaaatact catggaacgt agctcaggaa gttctcagat tggctccacc tcttcaagga 1080 gctttcaggg aagccttgtc tgactttgtc ttcaatggtt tcaccattcc aaagggctgg 1140 aagttatatt ggagcgcaaa ctcaacgcac aagaacgcag cttacttccc ggaaccattc 1200 aaattcgacc cttcagatt cgaggaaaa ggcccaccac catacacgtt tgtgccctc 1260 ggaggaggtc ctaggatgtg ccccggcaaa gagtacgccc gattggagat cctagtgttc 1320 atgcacaact tggtgagag gttcaaatgg gagaaagtcc ttcccaacga gcagatcgta 1380 gttgacccac tgcccatgcc cgccaaggga ctccctgtcc gccttttcc tcacctaag 1440 accgccgcag cttaa 1455 <210> 3 <211> 484 <212> PRT <213> Eryobotrya japonica (Left.) Lindl <400> 3 Met Glu His Phe Tyr Leu Thr Leu Leu Leu Gly Phe Val Ser Phe Ile 1 5 10 15 Thr Leu Ser Leu Ser Val Leu Phe Tyr Arg His Arg Ser Gln Phe Val 20 25 30 Gly Thr Asn Leu Pro Gly Lys Val Gly Tyr Pro Val Ile Gly Glu 35 40 45 Thr Tyr Gln Phe Leu Ala Thr Gly Trp Lys Gly His Pro Glu Lys Phe 50 55 60 Ile Phe Asp Arg Met Thr Lys Tyr Ser Ser Glu Val Phe Lys Thr Ser 65 70 75 80 Leu Met Gly Glu Lys Ala Ala Ile Phe Cys Gly Ala Ala Cys Asn Lys 85 90 95 Phe Leu Phe Ser Asn Glu Asn Lys Leu Val Thr Ala Trp Trp Pro Ser 100 105 110 Ser Val Asn Lys Val Phe Pro Ser Ser Met Glu Thr Ser Ala Lys Glu 115 120 125 Glu Ala Lys Lys Met Arg Lys Met Leu Pro Asn Phe Met Lys Pro Glu 130 135 140 Ala Leu Gln Arg Tyr Ile Gly Ile Met Asp Thr Val Ala Arg Arg His 145 150 155 160 Phe Ala Glu Gly Trp Glu Asn Lys Lys Glu Val Glu Val Phe Pro Leu 165 170 175 Ala Lys Asn Tyr Thr Phe Trp Leu Ala Ala Arg Leu Phe Val Ser Leu 180 185 190 Glu Asp Ser Val Glu Ile Ala Lys Leu Gly Asp Pro Phe Ala Val Leu 195 200 205 Ala Ser Gly Ile Ile Ser Met Pro Leu Asp Phe Pro Gly Thr Pro Phe 210 215 220 Tyr Lys Ala Ile Lys Ala Ser Asn Phe Ile Arg Glu Glu Leu Thr Lys 225 230 235 240 Ile Ile Lys Gln Arg Lys Ile Asp Leu Ala Glu Gly Lys Ala Ser Pro 245 250 255 Thr Gln Asp Ile Leu Ser His Met Leu Leu Leu Cys Asp Glu His Gly 260 265 270 Ser His Met Lys Glu His Asp Ile Ala Asp Lys Ile Leu Gly Leu Leu 275 280 285 Ile Gly Gly His Asp Thr Ala Ser Ala Thr Cys Thr Phe Ile Val Lys 290 295 300 Tyr Leu Ala Glu Leu Pro His Ile Tyr Asp Glu Val Tyr Lys Glu Gln 305 310 315 320 Met Glu Val Leu Ser Ala Lys Ala Pro Gly Glu Leu Leu Asn Trp Asp 325 330 335 Asp Leu Gln Lys Met Lys Tyr Ser Trp Asn Val Ala Gln Glu Val Leu 340 345 350 Arg Leu Ala Pro Pro Leu Gln Gly Ala Phe Arg Glu Ala Leu Ser Asp 355 360 365 Phe Val Phe Asn Gly Phe Thr Ile Pro Lys Gly Trp Lys Leu Tyr Trp 370 375 380 Ser Ala Asn Ser Thr His Lys Asn Ala Ala Tyr Phe Pro Glu Pro Phe 385 390 395 400 Lys Phe Asp Pro Thr Arg Phe Glu Gly Asn Gly Pro Ala Pro Tyr Thr 405 410 415 Phe Val Pro Phe Gly Gly Gly Pro Arg Met Cys Pro Gly Lys Glu Tyr 420 425 430 Ala Arg Leu Glu Ile Leu Val Phe Met His Asn Leu Val Lys Arg Phe 435 440 445 Lys Trp Glu Lys Val Leu Pro Asp Glu Gln Ile Val Val Asp Pro Leu 450 455 460 Pro Met Pro Ala Lys Gly Leu Pro Val Arg Leu Phe Ser His Pro Lys 465 470 475 480 Thr Ala Thr Ala <210> 4 <211> 484 <212> PRT <213> Eriobotrya japonica (Thunb.) Lindl <400> 4 Met Glu His Phe Tyr Leu Thr Leu Leu Leu Gly Phe Val Ser Phe Ile 1 5 10 15 Thr Leu Ser Leu Ser Val Leu Phe Tyr Arg His Arg Ser Gln Phe Val 20 25 30 Gly Thr Asn Leu Pro Pro Gly Lys Val Gly Tyr Pro Val Ile Gly Glu 35 40 45 Thr Tyr Gln Phe Leu Ala Thr Gly Trp Lys Gly His Pro Glu Lys Phe 50 55 60 Ile Phe Asp Arg Met Ser Lys Tyr Ser Ser Glu Val Phe Lys Thr Ser 65 70 75 80 Leu Met Gly Glu Lys Ala Ala Ile Phe Cys Gly Ala Ala Cys Asn Lys 85 90 95 Phe Leu Phe Ser Asn Glu Asn Lys Leu Val Thr Ala Trp Trp Pro Ser 100 105 110 Ser Val Asn Lys Val Phe Pro Ser Ser Met Glu Thr Ser Ala Lys Glu 115 120 125 Glu Ala Lys Lys Met Arg Lys Met Leu Pro Asn Phe Met Lys Pro Glu 130 135 140 Ala Leu Gln Arg Tyr Ile Gly Ile Met Asp Thr Val Ala Arg Arg His 145 150 155 160 Phe Ala Asp Gly Trp Glu Asn Lys Lys Glu Val Glu Val Phe Pro Leu 165 170 175 Ala Lys Asn Tyr Thr Phe Trp Leu Ala Ala Arg Leu Phe Val Ser Leu 180 185 190 Asp Asp Ser Val Glu Ile Ala Lys Leu Gly Asp Pro Phe Ala Val Leu 195 200 205 Ala Ser Gly Ile Ile Ser Met Pro Leu Asp Phe Pro Gly Thr Pro Phe 210 215 220 Tyr Lys Ala Ile Lys Ala Ser Asn Phe Ile Arg Glu Glu Leu Thr Lys 225 230 235 240 Ile Ile Lys Gln Arg Lys Ile Asp Leu Ala Glu Gly Lys Ala Ser Pro 245 250 255 Thr Gln Asp Ile Leu Ser His Met Leu Leu Leu Cys Asp Glu His Gly 260 265 270 Ser His Met Lys Glu His Asp Ile Ala Asp Lys Ile Leu Gly Leu Leu 275 280 285 Ile Gly Gly His Asp Thr Ala Ser Ala Thr Cys Thr Phe Ile Val Lys 290 295 300 Tyr Leu Ala Glu Leu Pro His Ile Tyr Asp Glu Val Tyr Lys Glu Gln 305 310 315 320 Met Glu Val Leu Ser Ala Lys Ala Pro Gly Glu Leu Leu Asn Trp Asp 325 330 335 Asp Leu Gln Lys Met Lys Tyr Ser Trp Asn Val Ala Gln Glu Val Leu 340 345 350 Arg Leu Ala Pro Pro Leu Gln Gly Ala Phe Arg Glu Ala Leu Ser Asp 355 360 365 Phe Val Phe Asn Gly Phe Thr Ile Pro Lys Gly Trp Lys Leu Tyr Trp 370 375 380 Ser Ala Asn Ser Thr His Lys Asn Ala Ala Tyr Phe Pro Glu Pro Phe 385 390 395 400 Lys Phe Asp Pro Ser Arg Phe Glu Gly Lys Gly Pro Ala Pro Tyr Thr 405 410 415 Phe Val Pro Phe Gly Gly Gly Pro Arg Met Cys Pro Gly Lys Glu Tyr 420 425 430 Ala Arg Leu Glu Ile Leu Val Phe Met His Asn Leu Val Lys Arg Phe 435 440 445 Lys Trp Glu Lys Val Leu Pro Asn Glu Gln Ile Val Val Asp Pro Leu 450 455 460 Pro Met Pro Ala Lys Gly Leu Pro Val Arg Leu Phe Pro His Pro Lys 465 470 475 480 Thr Ala Ala Ala <210> 5 <211> 1443 <212> DNA <213> Eriobotrya japonica (Thunb.) Lindl <400> 5 atggagaccc tttaccttat attttctgtg ggtgctgctt tcttagccct taccatcttt 60 gcactcaaag gcaaatcaga tgatggcaaa aaccttccac cagggagcat ggggtggcct 120 attgtgggtg aatcaatcga gttcttgttc gggaagccgg aaaattttgt gttcaatagg 180 atgaagaagt actctcctga catcttcaag acctatattc ttggagagaa aactgctgtg 240 atctgtggtc ctagtggaca taaatttctg ttctccaatg aaaaagt cttcacagct 300 tttcgaccac attcgatgca aaagatgttt cgatcataca aggctgccgc ccccactgct 360 tctgctgctc ccgctgtggc acaaccatct cgcgaatgaag aatcgaaagt tataagatcg 420 ccggggtttt tgaagccgga agcattggtg aggtacttgg ggaaatgga ctctatcact 480 caggaaaaa tgaaggcgta ttgggaaggc aaagatgagg tcaaggtcta ccctttggcc 540 aagacactca ctctaagtct tgcctgcaga ttcttcttgg ggatcgatga ttccgagcga 600 attgcaaggc tggtgagcaa ttttgatgat gttaccgttg ggatgcattc acttattatc 660 aacttcccag gaacaacatt ctataaggca accaaagcag ccgatgcgct gcgaaaggag 720 ttgaggattg tgattcagga gaagaggct gcaatggccg caggaggtcc catgcatgat 780 atattgtcac atatgatcgt ggctagtgat ccatccggca aacacatgcc cgaggctgag 840 gttgctgaca agatcatggg tttattgact gcaggatata gcaccgtggc cactgctatg 900 actttcttca tgaaatatgt cggagaaagg ccagacattt atgccaaggt cctagctgag 960 cacaaggaga ttgcagactc gaagaagcca ggagactttt tggagtggga agacatcaac 1020 aaaatgaagt actcgtggaa tgtgttgtat gaagtgatga gattcacgcc gccacttcaa 1080 gggacattca gagaggcctt gacagatttt acctacgccg gttacaccat cccaaagggc 1140 tggaaggtat attggactgt gagtacaaca aacatgaacc cacagtactt ccccaacccc 1200 gaaaagtttg atccatcaag atatgaggac ctaaatgcat tcccggcttt caacacttgtt 1260 ccatttggag gagggccaag aatgtgcccc gggaaagagt acgctcgcct agcgatactc 1320 actttcgttc acaatgtggt gaaggtc gaatgggaag ttctgtttcc caaggaag 1380 atcacaggtg atatgatgcc aacaccggag aaagggcttc cagttcgcct tcgatgtcac 1440 go to 1443 <210> 6 <211> 1443 <212> DNA <213> Eriobotrya japonica (Thunb.) Lindl <400> 6 atggaaaccc tttaccttgt tttatctttg ggagctgctt tcttagcttt tatcatcttt 60 gcattcaaag gcaaatcaga cgatggcaaa aaccttccac cagggagtat ggggtggcct 120 attgtgggtg aatcaatcga gttcctgttc gggaagccgg aaaatttgtgt attcaagagg 180 atgagaaggt actctcctga aatcttcaag acctatattc ttggagagaa aactgcagtt 240 atttgtggtc ctagtggaca caaattcctg ttctccaatg aacaaaagta cttcacagct 300 ttcgaccac attcgatgca aaagatgttt cgatcataca aggctgccgc ccccactgct 360 tctgctgttc ctgctgtggc acaaccttct cgtgatgaag aagcgaaagt aataagatct 420 ccagggtttc tgaagccgga agcattggtg aggtacttgg ggaaaatgga ctctatcact 480 caggaacaga tgaaggccta ttgggaagga aaagatgagg tccaggtcta ccccttggcc 540 aagaccctca ctctaggtct tgcttgcaga ttcttcatgg gtgtcgacga acccgggcga 600 attgcaaggc ttgtgagcaa ttttgacgat gttaccgttg ggatgcattc gcttattatc 660 gacttcccag gaacaacatt ctacaaggca accaaagcgg ccgatgcgct gcgaaaggag 720 ttgaggactg tgattcagga gaagaggct gcaatggccg caggaggtcc cttgcatgat 780 atattgtcgc acatgattgt ggctagtgac ccatctggca aacacatgcc tgaggctgag 840 gttgcagata agatcatggg tttgctcaca gcaggatata gcactgtggc tactgccatg 900 actttcttca tgaaatatgt tggagagagg ccagacatct atgccaaggt tctagctgag 960 caaatggaga ttgcaagcgc caagaagcca ggagactttt tggaatggga agacatcaac 1020 aaaatgaagt actcatggaa tgtgttgtat gaagtgatga gattcactcc accacttcag 1080 gggacattca gagaggcttt gactgatttc acctatgctg gttacaccat cccaaagggc 1140 tggaaggtat attggactgt tagcacaaca aacatgaacc cagagtactt tcccaaccca 1200 gaaaagtttg acccatcgag atatgatgac ctaaatgcat tcccagcttt cacattcgtt 1260 ccatttggag gagggccaag aatgtgccca ggcaaagagt acgctcgcct agcaatcctc 1320 actttcgttc acaacgtggt caagaggtc aaatgggaag tgttatttcc tagggaagaag 1380 atcaccggtg acatgatgcc aacaccggag aaggactc cagttcgcct taccgtcac 1440 tag 1443 <210> 7 <211> 480 <212> PRT <213> Eryobotrya japonica (Left.) Lindl <400> 7 Met Glu Thr Leu Tyr Leu Ile Phe Ser Val Gly Ala Ala Phe Leu Ala 1 5 10 15 Leu Thr Ile Phe Ala Leu Lys Gly Lys Ser Asp Asp Gly Lys Asn Leu 20 25 30 Pro Pro Gly Ser Met Gly Trp Pro Ile Val Gly Glu Ser Ile Glu Phe 35 40 45 Leu Phe Gly Lys Pro Glu Asn Phe Val Phe Asn Arg Met Lys Lys Tyr 50 55 60 Pro Asp Ile Phe Lys Thr Tyr Ile Leu Gly Glu Lys Thr Ala Val 65 70 75 80 Ile Cys Gly Pro Ser Gly His Lys Phe Leu Phe Ser Asn Gln Lys 85 90 95 Tyr Phe Thr Ala Phe Arg Pro His Ser Met Gln Lys Met Phe Arg Ser 100 105 110 Tyr Lys Wing Wing Pro Thr Wing Ser Wing Pro Wing Val Wing Gln 115 120 125 Pro Ser Arg Asp Glu Glu Ser Lys Val Ile Arg Ser Pro Gly Phe Leu 130 135 140 Lys Pro Glu Ala Leu Val Arg Tyr Leu Gly Lys Met Asp Ser Ile Thr 145 150 155 160 Gln Glu Gln Met Lys Ala Tyr Trp Glu Gly Lys Asp Glu Val Lys Val 165 170 175 Tyr Pro Leu Ala Lys Thr Leu Thr Leu Ser Leu Ala Cys Arg Phe Phe 180 185 190 Leu Gly Ile Asp Asp Ser Glu Arg Ile Ala Arg Leu Val Ser Asn Phe 195 200 205 Asp Asp Val Thr Val Gly Met His Ser Leu Ile Ile Asn Phe Pro Gly 210 215 220 Thr Thr Phe Tyr Lys Ala Thr Lys Ala Ala Asp Ala Leu Arg Lys Glu 225 230 235 240 Leu Arg Ile Val Ile Gln Glu Lys Lys Ala Ala Met Ala Ala Gly Gly 245 250 255 Pro Met His Asp Ile Leu Ser His Met Ile Val Ala Ser Asp Pro Ser 260 265 270 Gly Lys His Met Pro Glu Ala Glu Val Ala Asp Lys Ile Met Gly Leu 275 280 285 Leu Thr Ala Gly Tyr Ser Thr Val Ala Thr Ala Met Thr Phe Phe Met 290 295 300 Lys Tyr Val Gly Glu Arg Pro Asp Ile Tyr Ala Lys Val Leu Ala Glu 305 310 315 320 His Lys Glu Ile Ala Asp Ser Lys Lys Pro Gly Asp Phe Leu Glu Trp 325 330 335 Glu Asp Ile Asn Lys Met Lys Tyr Ser Trp Asn Val Leu Tyr Glu Val 340 345 350 Met Arg Phe Thr Pro Pro Leu Gln Gly Thr Phe Arg Glu Ala Leu Thr 355 360 365 Asp Phe Thr Tyr Ala Gly Tyr Thr Ile Pro Lys Gly Trp Lys Val Tyr 370 375 380 Trp Thr Val Ser Thr Thr Asn Met Asn Pro Gln Tyr Phe Pro Asn Pro 385 390 395 400 Glu Lys Phe Asp Pro Ser Arg Tyr Glu Asp Leu Asn Ala Phe Pro Ala 405 410 415 Phe Thr Leu Val Pro Phe Gly Gly Gly Pro Arg Met Cys Pro Gly Lys 420 425 430 Glu Tyr Ala Arg Leu Ala Ile Leu Thr Phe Val His Asn Val Val Lys 435 440 445 Arg Phe Glu Trp Glu Val Leu Phe Pro Lys Glu Lys Ile Thr Gly Asp 450 455 460 Met Met Pro Thr Pro Glu Lys Gly Leu Pro Val Arg Leu Arg Cys His 465 470 475 480 <210> 8 <211> 480 <212> PRT <213> Eriobotrya japonica (Thunb.) Lindl <400> 8 Met Glu Thr Leu Tyr Leu Val Leu Ser Leu Gly Ala Ala Phe Leu Ala 1 5 10 15 Phe Ile Ile Phe Ala Phe Lys Gly Lys Ser Asp Asp Gly Lys Asn Leu 20 25 30 Pro Pro Gly Ser Met Gly Trp Pro Ile Val Gly Glu Ser Ile Glu Phe 35 40 45 Leu Phe Gly Lys Pro Glu Asn Phe Val Phe Lys Arg Met Arg Arg Tyr 50 55 60 Ser Pro Glu Ile Phe Lys Thr Tyr Ile Leu Gly Glu Lys Thr Ala Val 65 70 75 80 Ile Cys Gly Pro Ser Gly His Lys Phe Leu Phe Ser Asn Glu Gln Lys 85 90 95 Tyr Phe Thr Ala Phe Arg Pro His Ser Met Gln Lys Met Phe Arg Ser 100 105 110 Tyr Lys Ala Ala Ala Pro Thr Ala Ser Ala Val Pro Ala Val Ala Gln 115 120 125 Pro Ser Arg Asp Glu Glu Ala Lys Val Ile Arg Ser Pro Gly Phe Leu 130 135 140 Lys Pro Glu Ala Leu Val Arg Tyr Leu Gly Lys Met Asp Ser Ile Thr 145 150 155 160 Gln Glu Gln Met Lys Ala Tyr Trp Glu Gly Lys Asp Glu Val Gln Val 165 170 175 Tyr Pro Leu Ala Lys Thr Leu Thr Leu Gly Leu Ala Cys Arg Phe Phe 180 185 190 Met Gly Val Asp Glu Pro Gly Arg Ile Ala Arg Leu Val Ser Asn Phe 195 200 205 Asp Asp Val Thr Val Gly Met His Ser Leu Ile Ile Asp Phe Pro Gly 210 215 220 Thr Thr Phe Tyr Lys Ala Thr Lys Ala Ala Asp Ala Leu Arg Lys Glu 225 230 235 240 Leu Arg Thr Val Ile Gln Glu Lys Lys Ala Ala Met Ala Ala Gly Gly 245 250 255 Pro Leu His Asp Ile Leu Ser His Met Ile Val Ala Ser Asp Pro Ser 260 265 270 Gly Lys His Met Pro Glu Ala Glu Val Ala Asp Lys Ile Met Gly Leu 275 280 285 Leu Thr Ala Gly Tyr Ser Thr Val Ala Thr Ala Met Thr Phe Phe Met 290 295 300 Lys Tyr Val Gly Glu Arg Pro Asp Ile Tyr Ala Lys Val Leu Ala Glu 305 310 315 320 Gln Met Glu Ile Ala Ser Ala Lys Lys Pro Gly Asp Phe Leu Glu Trp 325 330 335 Glu Asp Ile Asn Lys Met Lys Tyr Ser Trp Asn Val Leu Tyr Glu Val 340 345 350 Met Arg Phe Thr Pro Pro Leu Gln Gly Thr Phe Arg Glu Ala Leu Thr 355 360 365 Asp Phe Thr Tyr Ala Gly Tyr Thr Ile Pro Lys Gly Trp Lys Val Tyr 370 375 380 Trp Thr Val Ser Thr Thr Asn Met Asn Pro Glu Tyr Phe Pro Asn Pro 385 390 395 400 Glu Lys Phe Asp Pro Ser Arg Tyr Asp Asp Leu Asn Ala Phe Pro Ala 405 410 415 Phe Thr Phe Val Pro Phe Gly Gly Gly Pro Arg Met Cys Pro Gly Lys 420 425 430 Glu Tyr Ala Arg Leu Ala Ile Leu Thr Phe Val His Asn Val Val Lys 435 440 445 Arg Phe Lys Trp Glu Val Leu Phe Pro Lys Glu Lys Ile Thr Gly Asp 450 455 460 Met Met Pro Thr Pro Glu Lys Gly Leu Pro Val Arg Leu Thr Arg His 465 470 475 480 <210> 9 <211> 2283 <212> DNA <213> Eryobotrya japonica (Left.) Lindl <400> 9 atgtggagga ttaagtttgg agaaggtgca aacgacccct tgttgttcag cacaacaac 60 ttccacggaa ggcagacatg ggagttcgac ccagatgcag gtaccgagga ggaacgagca 120 gaagttgaag ctgctcgtga gcattctac caaatcgct tcaggtcac gcctagcagt 180 gacctcctat ggcgttttca gattttaaga gaaaaaact tcaacaaga aattcctcca 240 gtaagaattg gcgagggtga ggagattaca tatgatcaag ccacagctgc attcaggagg 300 gctgctacct tctggaatgc cttgcaatca ccccatggac attggcctgc tgaaaatgct 360 ggcccaaact tttacttccc tcccttggtc atggctgcat acattccagg atatctcaat 420 gttattttct cagctgagca caagaaggaa atcttgcggt atacatacaa ccatcagaat 480 gaagatggtg ggtggggact gcacatatca ggccctagta tgatgtttac tacatgcctt 540 aactattgta tgatgcgtat tctcggagag ggccctgatg gtgggcgtga caatgcatgc 600 gcaagggcac gaaagtggat tcttgatcgt ggtggtgctt actattctgc atcatgggga 660 aaaacttgga tggcaattct tggtgtgtat gactgggaag gcagcaaccc aatgcccccg 720 gaattctgga ctgggtctac actgcttcct tttcatccat caaaaatgtt ctgctactgt 780 aggttgactt acctacctat gtcttacttt tacgccacaa gatttgttgg cccgatcact 840 cctctagttg aggaattgag acaggaaatt tactgtgaac cttacaacga aattaactgg 900 cctaaagtgc gccattggtg tgcaccggag gataactact atccccatgg tcgtgtacaa 960 cgttttatgt gggatggttt ttacaatatc gctgagcctc tcctaaaacg ctggccctta 1020 aagagatca gagacaatgc cattcaattc acaattgacc aaattcatta cgaagatgag aacagtcgct acattacaat cggatgtgtg gaaaagccat taatgatgct tgcttgctgg gccgaggatc ctagtggaga agctttcaag aagcatattc ctagtagttac tgattatatc tggctcggag aggatggat caagatgcag agttttgga gccagtcatg ggattgtgct cttgtgattc aagctttgct tgctggcaat ctaaatactg aaatggcacc tacccttaag caagcacacg aatttctcaa gatatctcag gtgagggtga atacttctgg tgactaccta gctcatttcc gtcacgtttc taagggcgca tggacattct ctgaccgcga ccatggatgg 1440 caagtgtcgg attgtactgc agaagcactg aggtgttgct gcattttcgc aaatatgtcc ccagaacttg ttggtgagcc aatggatgct gagtgtatgt atgatgccgt caatgtcatc ttgacccttc aggtccaaa tggtggtgta tcagcctggg agccaacagg agcaccaaaa 1620 tggttggagt ggctcaaccc tgtggagttt cttgaggacc tagtcattga gtacgagtac 1680 atcgaatgca cttcatcttc gatccaggcc ttaactttgt ttaggaagtt gtaccctggc catagaagga aggagatcaa caatttcatc acgagagcag cagactacat tgaagacata cagtaccctg atggctcatg gtatggaaac tggggaatct gctttgtgta tggtacatgg ttcgcaatca aagggctgga ggccgctggt aggacgtaca acaactgcga ggcagtgcgc aaaggtgttg actttttgct caaaacacaa agggcagatg gtggctgggg agagcactac acctcatgca caaacaaga atatacagct caagatagca caaatttggt ccaaactgca ctcggattaa tgggtctgat tcatggtcga caggctgaga gagatccaac tcctattcac cgtgctgctg cggtgttaat gaaggtcag ttggatgatg gcgatttccc acaacaggaa ctgatgggag tttttatgag gaatgctatg ttgcactatg cagcatacag aaacatcttc ccattgtggg ctcttggaga ataccgcact ctggtttcgt tgcctataaa aaggattgct 2280 from <210> 10 <211> 760 <212> PRT <213> Eriobotrya japonica (Thunb.) Lindl <400> 10 Met Trp Arg Ile Lys Phe Gly Glu Gly Ala Asn Asp Pro Leu Leu Phe 1 5 10 15 Ser Thr Asn Asn Phe His Gly Arg Gln Thr Trp Glu Phe Asp Pro Asp 20 25 30 Ala Gly Thr Glu Glu Glu Arg Ala Glu Val Glu Ala Ala Arg Glu His 35 40 45 Phe Tyr Gln Asn Arg Phe Lys Val Thr Pro Ser Ser Asp Leu Leu Trp 50 55 60 Arg Phe Gln Ile Leu Arg Glu Lys Asn Phe Lys Gln Glu Ile Pro Pro 65 70 75 80 Val Arg Ile Gly Glu Gly Glu Glu Ile Thr Tyr Asp Gln Ala Thr Ala 85 90 95 Ala Phe Arg Arg Ala Ala Thr Phe Trp Asn Ala Leu Gln Ser Pro His 100 105 110 Gly His Trp Pro Ala Glu Asn Ala Gly Pro Asn Phe Tyr Phe Pro Pro 115 120 125 Leu Val Met Ala Ala Tyr Ile Pro Gly Tyr Leu Asn Val Ile Phe Ser 130 135 140 Ala Glu His Lys Lys Glu Ile Leu Arg Tyr Thr Tyr Asn His Gln Asn 145 150 155 160 Glu Asp Gly Gly Trp Gly Leu His Ile Ser Gly Pro Ser Met Met Phe 165 170 175 Thr Thr Cys Leu Asn Tyr Cys Met Met Arg Ile Leu Gly Glu Gly Pro 180 185 190 Asp Gly Gly Arg Asp Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile Leu 195 200 205 Asp Arg Gly Gly Ala Tyr Tyr Ser Ala Ser Trp Gly Lys Thr Trp Met 210 215 220 Ala Ile Leu Gly Val Tyr Asp Trp Glu Gly Ser Asn Pro Met Pro Pro 225 230 235 240 Glu Phe Trp Thr Gly Ser Thr Leu Leu Pro Phe His Pro Ser Lys Met 245 250 255 Phe Cys Tyr Cys Arg Leu Thr Tyr Leu Pro Met Ser Tyr Phe Tyr Ala 260 265 270 Thr Arg Phe Val Gly Pro Ile Thr Pro Leu Val Glu Glu Leu Arg Gln 275 280 285 Glu Ile Tyr Cys Glu Pro Tyr Asn Glu Ile Asn Trp Pro Lys Val Arg 290 295 300 His Trp Cys Ala Pro Glu Asp Asn Tyr Tyr Pro His Gly Arg Val Gln 305 310 315 320 Arg Phe Met Trp Asp Gly Phe Tyr Asn Ile Ala Glu Pro Leu Leu Lys 325 330 335 Arg Trp Pro Leu Lys Lys Ile Arg Asp Asn Ala Ile Gln Phe Thr Ile 340 345 350 Asp Gln Ile His Tyr Glu Asp Glu Asn Ser Arg Tyr Ile Thr Ile Gly 355 360 365 Cys Val Glu Lys Pro Leu Met Met Leu Ala Cys Trp Ala Glu Asp Pro 370 375 380 Ser Gly Glu Ala Phe Lys Lys His Ile Pro Arg Val Thr Asp Tyr Ile 385 390 395 400 Trp Leu Gly Glu Asp Gly Ile Lys Met Gln Ser Phe Gly Ser Gln Ser 405 410 415 Trp Asp Cys Ala Leu Val Ile Gln Ala Leu Leu Ala Gly Asn Leu Asn 420 425 430 Thr Glu Met Ala Pro Thr Leu Lys Gln Ala His Glu Phe Leu Lys Ile 435 440 445 Ser Gln Val Arg Val Asn Thr Ser Gly Asp Tyr Leu Ala His Phe Arg 450 455 460 His Val Ser Lys Gly Ala Trp Thr Phe Ser Asp Arg Asp His Gly Trp 465 470 475 480 Gln Val Ser Asp Cys Thr Ala Glu Ala Leu Arg Cys Cys Cys Ile Phe 485 490 495 Ala Asn Met Ser Pro Glu Leu Val Gly Glu Pro Met Asp Ala Glu Cys 500 505 510 Met Tyr Asp Ala Val Asn Val Ile Leu Thr Leu Gln Ser Pro Asn Gly 515 520 525 Gly Val Ser Ala Trp Glu Pro Thr Gly Ala Pro Lys Trp Leu Glu Trp 530 535 540 Leu Asn Pro Val Glu Phe Leu Glu Asp Leu Val Ile Glu Tyr Glu Tyr 545 550 555 560 Ile Glu Cys Thr Ser Ser Ser Ile Gln Ala Leu Thr Leu Phe Arg Lys 565 570 575 Leu Tyr Pro Gly His Arg Arg Lys Glu Ile Asn Asn Phe Ile Thr Arg 580 585 590 Ala Ala Asp Tyr Ile Glu Asp Ile Gln Tyr Pro Asp Gly Ser Trp Tyr 595 600 605 Gly Asn Trp Gly Ile Cys Phe Val Tyr Gly Thr Trp Phe Ala Ile Lys 610 615 620 Gly Leu Glu Ala Ala Gly Arg Thr Tyr Asn Asn Cys Glu Ala Val Arg 625 630 635 640 Lys Gly Val Asp Phe Leu Leu Lys Thr Gln Arg Ala Asp Gly Gly Trp 645 650 655 Gly Glu His Tyr Thr Ser Cys Thr Asn Lys Lys Tyr Thr Ala Gln Asp 660 665 670 Ser Thr Asn Leu Val Gln Thr Ala Leu Gly Leu Met Gly Leu Ile His 675 680 685 Gly Arg Gln Ala Glu Arg Asp Pro Thr Pro Ile His Arg Ala Ala Ala 690 695 700 Val Leu Met Lys Gly Gln Leu Asp Asp Gly Asp Phe Pro Gln Gln Glu 705 710 715 720 Leu Met Gly Val Phe Met Arg Asn Ala Met Leu His Tyr Ala Ala Tyr 725 730 735 Arg Asn Ile Phe Pro Leu Trp Ala Leu Gly Glu Tyr Arg Thr Leu Val 740 745 750 Ser Leu Pro Ile Lys Arg Ile Ala 755 760

Claims

1. Use of any gene with a nucleotide sequence as shown in SEQ ID NO.1-2, and / or any protein with an amino acid sequence as shown in SEQ ID NO.3-4, characterized in that: The application is to promote loquat to produce triterpene acid.

2. The use according to claim 1, characterized in that: The triterpene acids are ursane type and / or oleanane type triterpene acids.

3. The use according to claim 2, characterized in that: The ursane type triterpene acid is ursolic acid and / or corosolic acid, and the oleanane type triterpene acid is oleanolic acid and / or maslinic acid.

4. Use of any gene with a nucleotide sequence as shown in SEQ ID NO.1-2, and / or any protein with an amino acid sequence as shown in SEQ ID NO.3-4, characterized in that: The application is to promote α-amyrin to generate ursolic acid, α-amyrin to generate corosolic acid, β-amyrin to generate oleanolic acid and / or β-amyrin to generate hawthorn acid.

5. A method for increasing the yield of loquat ursane-type and / or oleanane-type triterpene acids, characterized in that: Overexpress one or two genes with nucleotide sequences as shown in SEQ ID NO.1-2, and / or increase the expression level of one or two proteins with amino acid sequences as shown in SEQ ID NO.3-4.

6. The method according to claim 4, characterized in that The ursane type triterpene acid is ursolic acid and / or corosolic acid, and the oleanane type triterpene acid is oleanolic acid and / or maslinic acid.

7. Use of any gene with a nucleotide sequence as shown in SEQ ID NO.5-6, and / or any protein with an amino acid sequence as shown in SEQ ID NO.7-8, characterized in that: The application is to promote loquat to produce ursane type and / or oleanane type triterpene acids.

8. The use according to claim 6, characterized in that: The ursane type triterpene acid is corosolic acid, and the oleanane type triterpene acid is maslinic acid.

9. Use of any gene having a nucleotide sequence as shown in SEQ ID NO.5-6, and / or any protein having an amino acid sequence as shown in SEQ ID NO.7-8, characterized in that: The application is to promote ursolic acid to generate corosolic acid and / or oleanolic acid to generate maslinic acid.

10. A method for increasing the yield of corosolic acid and / or maslinic acid in loquat, characterized in that: Overexpress one or two genes with nucleotide sequences as shown in SEQ ID NOs. 5 to 6, and / or increase the expression level of one or two proteins with amino acid sequences as shown in SEQ ID NOs. 7 to 8.

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