CN115074374B - Apple triterpenic acid synthesis key enzyme genes MdTYP 716A2 and MdTYP 716C1 and application thereof - Google Patents

Abstract

The invention discloses apple triterpenic acid synthesis key enzyme genes MdTYP 716A2 and MdTYP 716C1 and application thereof. The invention clones apple triterpenic acid synthesis key enzyme genes MdTYP 716A2 and MdTYP 716C1 from the leaves for the first time, and the nucleotide sequences of the apple triterpenic acid synthesis key enzyme genes are respectively shown as SEQ ID NO.1 and SEQ ID NO. 3; the transient expression related genes of the tobacco leaves show that: mdTYP 716A2 expression can catalyze alpha/beta-amyrin oxidation in tobacco to form ursolic acid/oleanolic acid; mdTYP 716C1 expression catalyzes the hydroxylation of ursolic acid/oleanolic acid to form corosolic acid/crataegolic acid in tobacco. The two genes can promote the synthesis of important triterpenic acid such as apple ursolic acid, corosolic acid and the like, and promote the biosynthesis of ursolic acid, corosolic acid and other ursane-type and oleanane-type triterpenic acid derived from the ursolic acid and the corosolic acid in vitro. The invention lays a foundation for the application of the synthetic biological methods such as gene tandem expression, a biological generator and the like in apple metabolism research, and provides application support for fruit functional variety improvement and triterpenic acid component modification.

Description

Apple triterpenic acid synthesis key enzyme genes MdTYP 716A2 and MdTYP 716C1 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to apple triterpenic acid synthesis key enzyme genes MdTYP 716A2 and MdTYP 716C1 and application thereof.

Background

Apples, one of the biggest fresh food fruits, play an important role in improving dietary structure and providing rich nutritional components. The apple fruit is reported to contain more than 40 kinds of triterpenic acid such as ursane type, oleanane type, lupin alkane type and the like, wherein the ursolic acid is an important natural active product for resisting cancer, inhibiting bacteria and reducing blood sugar. In addition to the effects of diminishing inflammation, resisting cancer and the like to a certain extent, researches on related species such as loquat and the like also find that corosolic acid is a natural hypoglycemic drug, and the utilization of the corosolic acid on fresh food products is favorable for developing more fresh food fruits which are convenient for hyperglycemia and diabetics to directly eat. Although the apple peel contains ursolic acid, it lacks corosolic acid accumulated in the leaves; at the same time we have found that the majority of the triterpenic acid in the fruit accumulates on the pericarp rather than the pulp, which is easily ignored by the consumer and causes a significant waste of the natural active ingredient. In the reported research, scientific researchers have focused on the identification of biosynthesis genes of ursolic acid and precursor alpha-amyrin, but the main work is focused on peel comparison among varieties, and the biosynthesis of corosolic acid is not focused on lack of excavation of important genes in leaves.

Identification of triterpenic acid biosynthesis structural genes and establishment of an expression system are key to guaranteeing biosynthesis and targeted production of target components. Patent CN110938640 a discloses an oxidation squalene cyclase gene ejias 1 for synthesizing alpha-amyrin, but does not identify other homologous genes with similar functions; the synthase genes of triterpenic acids such as ursolic acid, corosolic acid, oleanolic acid, crataegolic acid and the like with high content in apple leaves have not been identified and mined.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide apple triterpenic acid synthesis key enzyme genes MdTYP 716A2 and MdTYP 716C1 and application thereof.

The first object of the invention is to provide the application of the gene with the nucleotide sequence shown as SEQ ID NO.1 or the protein with the amino acid sequence shown as SEQ ID NO.2 in promoting the generation of triterpenic acid by apples.

The second purpose of the invention is to provide the application of the gene with the nucleotide sequence shown as SEQ ID NO.1 or the protein with the amino acid sequence shown as SEQ ID NO.2 in promoting alpha-amyrin to generate ursolic acid, alpha-amyrin to generate corosolic acid, beta-amyrin to generate oleanolic acid and/or beta-amyrin to generate crataegolic acid.

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

The fourth object of the invention is to provide the application of the gene with the nucleotide sequence shown as SEQ ID NO.3 and/or the protein with the amino acid sequence shown as SEQ ID NO.4 in promoting the generation of the ursane type and/or oleanane type triterpenic acid by apples.

The fifth purpose of the invention is to provide the application of the gene with the nucleotide sequence shown as SEQ ID NO.3 and/or the protein with the amino acid sequence shown as SEQ ID NO.4 in promoting the production of the corosolic acid from the ursolic acid and/or promoting the production of the crataegolic acid from the oleanolic acid.

It is a sixth object of the present invention to provide a method for increasing the yield of corosolic acid and/or crataegolic acid.

In order to achieve the above object, the present invention is realized by the following technical scheme:

the invention clones an apple alpha/beta-amyrin C-28 bit oxidase gene MdTYP 716A2 from the apple leaf of 'golden crown', the nucleotide sequence of which is shown as SEQ ID NO.1 and comprises an open reading frame of 1455 bp; encode 484 amino acids, whose encoded amino acid sequence is shown in SEQ ID NO. 2. Meanwhile, an apple ursolic acid/oleanolic acid C-2 hydroxylase gene MdTYP 716C1 is obtained by cloning, the nucleotide sequence of which is shown as SEQ ID NO.3 and comprises an open reading frame of 1443 bp; 480 amino acids are encoded, and the encoded amino acid sequence is shown as SEQ ID NO. 4; through transient expression of tobacco leaves and GC-MS analysis, the MdTYP 716A2 expression can catalyze the oxidation of the C-28 position of the alpha/beta-amygdalus arborescens to form ursolic acid/oleanolic acid, and the MdTYP 716C1 expression can catalyze the hydroxylation of the ursolic acid/oleanolic acid to form corosolic acid/crataegolic acid. The two genes can promote the synthesis of important triterpenic acid such as apple ursolic acid, corosolic acid and the like, and promote the biosynthesis of ursolic acid, corosolic acid and other ursane-type and oleanane-type triterpenic acid derived from the ursolic acid and the corosolic acid in vitro.

Accordingly, the present invention claims the following:

the application of the gene with the nucleotide sequence shown as SEQ ID NO.1 and/or the protein with the amino acid sequence shown as SEQ ID NO.2 in promoting the generation of triterpenic acid by apples.

The triterpene acid is ursane type and/or oleanane type triterpene acid.

The ursolic acid and/or corosolic acid are/is ursolic acid and/or corosolic acid, and the oleanane-type triterpene acid is oleanolic acid and/or crataegolic acid.

The nucleotide sequence of the protein is shown as SEQ ID NO.1, or the amino acid sequence of the protein is shown as SEQ ID NO.2, and the protein is applied to the aspects of promoting alpha-amyrin to generate ursolic acid, alpha-amyrin to generate corosolic acid, beta-amyrin to generate oleanolic acid and/or beta-amyrin to generate crataegolic acid.

A method for improving the yield of ursane type and/or oleanane type triterpene acid comprises over-expressing the gene with nucleotide sequence shown as SEQ ID NO.1, and/or improving the expression level of protein with amino acid sequence shown as SEQ ID NO. 2.

The ursolic acid and/or corosolic acid are/is ursolic acid and/or corosolic acid, and the oleanane-type triterpene acid is oleanolic acid and/or crataegolic acid.

Preferably, the method comprises: preparing recombinant engineering bacteria containing genes with nucleotide sequences shown as SEQ ID NO.1, and introducing the recombinant engineering bacteria into organisms capable of producing alpha-amyrin and/or beta-amyrin to obtain recombinant organisms; culturing the recombinant organism, and over-expressing the protein with the amino acid sequence shown as SEQ ID NO. 2.

The application of the gene with the nucleotide sequence shown as SEQ ID NO.3 and/or the protein with the amino acid sequence shown as SEQ ID NO.4 in promoting the generation of ursane type and/or oleanane type triterpene acid by apples.

The application of the gene with the nucleotide sequence shown as SEQ ID NO.3 and/or the protein with the amino acid sequence shown as SEQ ID NO.4 in promoting the production of the corosolic acid from the ursolic acid and/or promoting the production of the crataegolic acid from the oleanolic acid.

A method for improving the yield of corosolic acid and/or crataegolic acid by over-expressing the gene with nucleotide sequence shown as SEQ ID NO.3 and/or improving the expression level of protein with amino acid sequence shown as SEQ ID NO. 4.

The ursane-type triterpene acid is corosolic acid, and the oleanane-type triterpene acid is crataegolic acid.

Preferably, the method comprises: preparing recombinant engineering bacteria containing a gene with a nucleotide sequence shown as SEQ ID NO.3, and introducing the recombinant engineering bacteria into organisms capable of producing ursolic acid and/or oleanolic acid to obtain recombinant organisms; culturing the recombinant organism, and over-expressing the protein with the amino acid sequence shown as SEQ ID NO. 4.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the component difference of the triterpenic acid in the apple leaves and the fruit is compared and analyzed, and the triterpenic acid in the leaves is found to have stronger synthesis capability, so that the unknown genes specifically expressed in the leaves can be excavated to provide new application for regulating the synthesis and distribution of natural active compounds in the fruit. The invention clones the key enzyme genes MdTYP 716A2 and MdTYP 716C1 for synthesizing triterpenic acid from apple leaves for the first time, and the transient expression related genes of tobacco leaves show that: mdTYP 716A2 expression can catalyze alpha/beta-amyrin oxidation in tobacco to form ursolic acid/oleanolic acid;

MdTYP 716C1 expression catalyzes the hydroxylation of ursolic acid/oleanolic acid to form corosolic acid/crataegolic acid in tobacco. The two genes can promote the synthesis of important triterpenic acid such as apple ursolic acid, corosolic acid and the like, and promote the biosynthesis of ursolic acid-corosolic acid, oleanolic acid-crataegolic acid and other ursane-type or oleanane-type triterpenic acid derived from the same in vitro. The invention lays a foundation for the application of the synthetic biological methods such as gene tandem expression, a biological generator and the like in apple metabolism research, and provides application support for fruit functional variety improvement and triterpenic acid component modification.

Drawings

FIG. 1 is a GC-MS chromatogram of the detection of major triterpenic acid components in apple leaves and fruits.

FIG. 2 is a P450 protein cluster tree of MdTYP 716A2, mdTYP 716C1 and other plants.

FIG. 3 shows the CYP716A protein conserved domains of MdTYP 716A2 and other plants.

FIG. 4 shows the CYP716C protein conserved domains of MdTYP 716C1 and other plants.

FIG. 5 is a GC-MS detection chromatogram of the tobacco transient expression product of MdTYP 716A2 and a synthetic route.

FIG. 6 is a GC-MS detection chromatogram of the tobacco transient expression product of MdTYP 716C1 and a synthetic route.

Detailed Description

The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1 comparison of the triterpenic acid content of apple leaves and fruits

1. Experimental method

1. Sample collection

The mature leaf and fruit samples of the 'golden crown' apples are collected from apple germplasm resource nursery of the institute of fruit tree research of the Shandong national academy of agricultural sciences. Fresh leaves, separated pulp and peel samples were quickly frozen in liquid nitrogen and stored in a-80 ℃ refrigerator.

2. Extraction and detection of triterpenic acid component

The apple tissue samples described above were lyophilized in a Frerzone 12L lyophilizer, and the lyophilized material was ground to a fine powder and sieved through a 100 mesh screen. Weighing 5.0mg of tissue dry powder, and adding 500 mu L of ethanol mixed solution (volume ratio is ethanol: water: KOH=9:1:1); violent shaking; water bath at 70 ℃ for 1 hour; evaporating ethanol in a rotary evaporator; adding 500 mu L of ethyl acetate, and shaking vigorously for 1min; adding 500 mu L of water, and continuing to shake vigorously for 1min; centrifuging at 16000g for 5min; taking an upper ethyl acetate extraction layer; placing the ethyl acetate extract into a nitrogen blowing instrument for blow-drying; silylation derivatization of the dried triterpenic acid extract with methoxyamine hydrochloride and N-methyl-N- (trimethylsilyl) trifluoroacetamide in a water bath at 37 ℃.

50 μl of the derivative was used to detect the triterpenic acid component in the extract by a 7890A/5975C GC-MS combined instrument under the following chromatographic conditions: HP-5M (30M. Times.0.25 mm. Times.0.25 μm) column, temperature programmed to 40deg.C for 1min, and 20 ℃/min to 320℃for 15min; sample injection is not carried out in a split way, the sample injection amount is 1 mu L, and the helium flow rate is 1mL/min; the mass spectrum conditions are as follows: interface temperature 250 ℃, ion source temperature 200 ℃, quaternary rod temperature 150 ℃, solvent delay 15min, voltage 1500V, and mass scanning range 60-800 m/s. Beta-amyrin (CAS number: 559-70-6), alpha-amyrin (CAS number: 508-04-3), oleanolic acid (CAS number: 508-02-1), ursolic acid (CAS number: 77-52-1), corosolic acid (4547-24-4), crataegolic acid (CAS number: 4373-41-5) and the like were purchased from Sigma-Aldrich company as standard substances, and were detected under the same conditions.

2. Experimental results

GC-MS analysis shows that the triterpenic acid in the leaf is richer in components and higher in total content; no other triterpene acids were detected in the pulp except for the unknown triterpene acid precursor; there were very few alpha-and beta-amyrin in both leaf and pericarp (figure 1). The content of ursolic acid and oleanolic acid in the leaves and the peel is equivalent, and the ursolic acid is taken as the main ingredient; but leaves also contain a significant amount of corosolic acid and crataegolic acid (especially corosolic acid). The method predicts that the triterpenic acid synthesis capacity of the leaf is stronger, and the unknown gene for specifically expressing the triterpenic acid in the leaf can provide new application for regulating the synthesis and distribution of natural active compounds of fruits.

EXAMPLE 2 apple MdTYP 716A2 and MdTYP 716C1 Gene cloning and vector construction

1. Experimental method

1. Extraction of RNA

Samples stored in example 1 were ground to a fine powder in liquid nitrogen using a plant grinder, 100mg of each sample was added to a 1.5mL centrifuge tube, 1.0mL RLT lysate was added, and RNA was extracted according to the EASYspin Plus plant RNA flash extraction kit (Edley) protocol, as follows: shaking the tube lysate with the sample on a shaker for 20s, and then cracking in a water bath at 55 ℃ for 20min; centrifuging the lysate at 13000rpm for 10min; transferring the supernatant into a new centrifuge tube, adding absolute ethyl alcohol with half of the volume of the supernatant, and gently sucking and beating to mix uniformly; adding the mixture into an RA adsorption column, standing for 30s, and centrifuging at 13000rpm for 2min; discarding the waste liquid, repeating the previous step, and filtering all the mixtures through an RA adsorption column; discarding the waste liquid, adding 700 μl deproteinized liquid RW1 into the adsorption column, standing for 1min, and centrifuging at 13000rpm for 30s; discarding the waste liquid, adding 500 μl of rinsing liquid RW into the adsorption column, standing for 1min, and centrifuging at 13000rpm for 30s; discarding the waste liquid, and adding the rinsing liquid RW for repeating the steps; discarding the waste liquid, putting the adsorption column back into the hollow tube, and centrifuging at 13000rpm for 2min; taking out the adsorption column, putting the adsorption column into a new RNase free centrifuge tube, adding 30 mu l RNase free water in the middle of the adsorption membrane, standing for 1min at room temperature, and centrifuging at 12000rpm for 1min; the eluted RNA was sucked back into the middle of the original adsorption film, left stand at room temperature for 2min, and centrifuged at 12000rpm for 1min to obtain RNA.

3. Reverse transcription synthesis of cDNA

After measuring the concentration of RNA extracted from different samples, water is added to adjust the concentration to be the same, and then PrimeScript is adopted ^TM RT reagent Kit with gDNA Eraser reverse transcription System Specification A first strand of cDNA was synthesized. Wherein, the reaction system for removing DNA is as follows: 2 mu L gDNA Eraser buffer +1 mu L gDNA Eraser+7 mu L RNA, and digesting gDNA at 42 ℃ after adding the system; after the reaction, the following components are added into each system: 1 mu L PrimeScript RT Enzyme Mix I +1 mu L RT Primer mix+4 mu L5X PrimeScript Buffer 2+4 mu L RNase Free dH ₂ O was subjected to a reverse transcription reaction. The reverse transcription procedure is: storing at 37 deg.C for 15min to 85 deg.C for 10s to 4 deg.C.

4. Cloning and vector construction of target Gene

Taking the predicted full length of coding region sequences of MdTYP 716A2 and MdTYP 716C1 genes from apple genome, designing amplification primers by using Primer Premier 5.0, adding homologous sequence joints on two sides of cleavage sites of pSAK277 vectors (EcoRI and XhoI) on two sides of the designed upstream and downstream primers respectively to prepare recombinant primers, and entrusting the recombinant primers to be synthesized by Shanghai bioengineering limited company, wherein the sequence information of the specific primers is as follows: mdTYP 716A2-F:5'-actagtggatccaaagaattcATGGAGCACTTCTATCTGACC-3', mdCYP716A2-R:5'-gactctagaagtactctcgagTTAAGCTGCGGCGGTCTTAG-3'; mdTYP 716C1-F:5'-actagtggatccaaagaattcATGGAGACCCTTTACCTTATATT-3', mdCYP716C 1-R5'-gactctagaagtactctcgagCTAGTGACGTCGAAGGCGAA-3'.

Blade cDNA is used as template, high-fidelity enzyme is usedHS DNA Polymerase the target gene is amplified. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min;95 ℃ for 30s;56 ℃ for 30s;72 ℃,90s; running for 33 cycles; extending at 72 deg.C for 10min; and (5) storing at 12 ℃.

The amplified product is recovered by gel cutting after gel electrophoresis detection and is connected to a linearized pSAK277 expression vector, and the specific connection steps are as follows: the purified cleavage product was ligated to the target vector using a ClonExpress one-step directed cloning seamless cloning kit, and a 10. Mu.l reaction system comprised of: 1 μl of purified and recovered PCR amplification product, 1 μl of linearization vector, 2 μl of 5 XCE II buffer,1 μl of Exnase ^TM II and 4. Mu.l of sterile water. The reaction system was allowed to react at 37℃for half an hour and then cooled on ice for 5 minutes. Thereafter, 5. Mu.l of the ligation product was transferred into 50. Mu.l of E.coli DH 5. Alpha. Competent cells. And (3) screening positive escherichia coli clones by kanamycin, performing PCR detection, and then delivering the positive escherichia coli clones to Shanghai bioengineering limited company for Sanger sequencing, wherein a plasmid with correct sequencing is the constructed expression vector.

2. Experimental results

The result shows that the designed primer can amplify MdTYP 716A2 gene with the length of 1455bp from leaves (the nucleotide sequence is shown as SEQ ID NO. 1), and the sequence codes 484 amino acid residues (the amino acid sequence is shown as SEQ ID NO. 2). Meanwhile, the single MdTYP 716C1 gene can be amplified, and the coding sequence of the gene is 1443bp (the nucleotide sequence is shown as SEQ ID NO. 3), and 480 amino acid residues are coded (the amino acid sequence is shown as SEQ ID NO. 4).

CYP716 family evolutionary trees are constructed by using CYP81, CYP93 and the like as outer groups and MEGA6 software, and the apple MdTYP 716A2 gene is found to be clustered with reported CYP716A branch P450 families such as MdTY 716A175 and the like. The MdTYP 716C1 gene was clustered with LsCYP716C55 and ObYP 716C50 gene from Lagerstroemia speciosa in another branch (FIG. 2). The MdTYP 716A2 and the apple MdTYP 716A175 were closest in relatedness, and the identity of the encoded amino acid was 98.14%. The sequence identity of MdTYP 716C1 to LsCYP716C55 and ObTYP 716C50 was 75.67% and 66.60%, respectively. Analysis of the conserved domains/motifs of these genes encoding amino acids using ClustalX software revealed that MdCTP 716A2 encoded amino acids, as reported for MdCTP 716A175 and other terpene synthase family genes, possessed conserved Membrane anchor hydrophic region, PPGXXGXP, A/GGXD/ET domains, EXXR motifs, PERF motifs and FXGXRXXG domains (FIG. 3). The conserved structure of CYP716C is significantly different from that of CYP716A, although there are also several conserved structures of PPGXXGXP domain, A/GGXD/ET domain, EXXR motif, PERF motif and FXGXRXXG domain; however, the a/GGXD/ET domain has a distinct conserved amino acid difference from CYP716A, while lacking a distinct conserved Membrane anchor hydrophic region (fig. 4).

Example 3 transient expression of Gene tobacco and detection of triterpenic acid products

1. Experimental method

Construction of vector referring to example 2, amplified full length was inserted into pSAK277 expression vector, and pSAK 277-MdTYP 716A2 recombinant plasmid containing the correct fragment was obtained by Sanger sequencing.

The constructed expression vector is transferred into GV3101 (pSoup) agrobacterium, meanwhile pSAK277 empty vector without inserted genes is used as a control, glycerol is added, and the mixture is placed in a refrigerator at the temperature of minus 80 ℃ for standby. Planting Nicotiana benthamiana in a greenhouse, sowing for about 1 month, completely expanding 5 th to 6 th true leaves of the tobacco with consistent growth vigor, and streaking on a plate containing antibiotics to activate the stored glycerinum. Meanwhile, pSAK277-EjOSC2 recombinant agrobacterium which is preserved in the subject group is activated (the nucleotide sequence of EjOSC2 is shown as SEQ ID NO. 5) so as to provide alpha/beta balsam pear catalytic substrate for oxidase coded by a target gene.

Inoculating 400 μl of the newly activated bacteria liquid into 10mL of YEP liquid culture medium containing 50 μl Kan and 100mL Rif, shaking for 24h to morning (if activated, the shaking time can be properly shortened) until the bacterial solution solubility is A ₆₀₀ =0.8 to 1.0. 30mL MES (100 mM), 30mL MgCl were taken ₂ (100 mM), 150. Mu.L of Acetosyringone (100 mM concentration of Acetosyringone), ultrapure water was added to a volume of 150mL, and the mixture was mixed to prepare an MMA working solution, which was stored in a refrigerator at 4 ℃. Taking 2mL of bacterial liquid, and centrifuging at 5000rpm for 10min; discarding the supernatant, adding 2mL MMA working solution into the precipitate, and suspending thalli; centrifuging at 5000rpm for 10min; discarding the supernatant, repeating suspension-centrifugation for 1 time, and adding into a 10mL test tube; adding working solution to dilute to bacterial solution A ₆₀₀ The value is about 0.2 (working solution is used as blank control); the diluted bacterial liquid is incubated for about 3 hours at the condition of light shielding and room temperature. The bacterial solution was injected into the back of the leaf blade using a 1mL syringe without needle, and the leaf blade injected with pSAK277 empty vector bacterial solution was used as a control. After injection, the tobacco is moved to a low light condition for 1 day at 25 ℃ for culture, and then is transferred to normal light culture. After 2 days, a small amount of leaf samples were collected, RNA was extracted by the method of reference example 2 and reverse transcribed into cDNA, and the expression of the foreign gene was examined.

After 7 days, tobacco leaves were collected, frozen in liquid nitrogen, and then transferred to a freeze dryer at-80℃under 0.1MPa to dry to a leaf balance, triterpenic acid components in the injection leaves were extracted by the method of reference example 1, and the gene transient expression products were tested by GC-MS under the same chromatographic conditions. The chromatographic conditions are as follows: HP-5M (30M. Times.0.25 mm. Times.0.25 μm) column, temperature programmed to 40deg.C for 1min, and 20 ℃/min to 320℃for 15min; sample injection is not carried out in a split way, the sample injection amount is 1 mu L, and the helium flow rate is 1mL/min; the mass spectrum conditions are as follows: interface temperature 250 ℃, ion source temperature 200 ℃, quaternary rod temperature 150 ℃, solvent delay 15min, voltage 1500V, and mass scanning range 60-800 m/s.

2. Experimental results

As a result, as shown in FIG. 5, after transient expression of EjoC 2, a large amount of alpha-amyrin and a small amount of beta-amyrin were detected in the leaf extract of Nicotiana benthamiana, which provided substrates for subsequent catalytic reactions, whereas no corresponding products were detected in the empty vector treated control leaf extract (FIG. 5A). When MdCTP 716A2 and EjOSC2 were expressed together, there were 2 new products, ursolic acid and oleanolic acid, in which the content of ursolic acid was much higher than that of oleanolic acid, than when EjOSC2 was expressed alone (FIGS. 5A and B). This suggests that the protein encoded by MdTYP 716A2 is a multifunctional C-28 oxidase that catalyzes the oxidation reaction at the C-28 position of alpha/beta-amyrin to synthesize ursolic acid and oleanolic acid.

Example 4 transient expression of MdTYP 716C1 Gene tobacco leaf and analysis of triterpenic acid product

1. Experimental method

The pSAK 277-MdTYP 716C1 recombinant plasmid was constructed as in example 2 or example 3.

The constructed expression vector is transferred into GV3101 (pSoup) agrobacterium, and glycerol is added and stored in a refrigerator at the temperature of minus 80 ℃ for standby. And (5) taking glycerinum to streak and activate on the antibiotic-containing flat plate after the 5 th to 6 th long true leaves of the tobacco are fully unfolded. Meanwhile, the agrobacterium glycerols of pSAK277-EjOSC2 and pSAK 277-MdCTYP 716A2 in example 3 are activated so as to provide alpha/beta amyrin, ursolic acid/oleanolic acid and other catalytic substrates for the hydroxylase coded by the target genes. Transient expression of tobacco, preparation of triterpene acid product and detection of triterpene acid were carried out as in example 3

2. Experimental results

As a result, as shown in FIG. 6, as in example 3, after transient expression of EjoC 2 and MdTYP 716A2, a large amount of alpha/beta-amyrin and ursolic acid/oleanolic acid were detected in the Nicotiana benthamiana leaf extract (FIG. 6A). Expression of MdCTP 716C1 on the basis of co-expression of EjoC 2-MdCTP 716A2 allows for almost complete consumption of ursolic acid/oleanolic acid, with the consequent appearance of another 2 new products, corosolic acid and crataegolic acid, and the content of corosolic acid is much higher than that of crataegolic acid (FIGS. 6A and B). This suggests that the MdTYP 716C1 encoded protein is a multifunctional C-2 hydroxylase, which can introduce hydroxyl group at the C-2 position of ursolic acid/oleanolic acid to catalyze the formation of corosolic acid and crataegolic acid.

Through the study of examples 3 and 4, the system reports the synthesis path of main triterpenic acid of apples: mdTYP 716A2 is an alpha/beta-amyrin C-28 oxidase gene, can catalyze alpha-amyrin to form ursolic acid and beta-amyrin to form oleanolic acid; mdTYP 716C1 is ursolic acid/oleanolic acid C-2 hydroxylase gene, and can catalyze ursolic acid to generate corosolic acid and oleanolic acid to form crataegolic acid.

Finally, it should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive of all the embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Sequence listing

<120> apple triterpenic acid synthesis key enzyme genes MdTYP 716A2 and MdTYP 716C1 and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1455

<212> DNA

<213> Malus × domestica apple

<400> 1

atggagcact tctatctgac cctcctttta gggtttgtct ccttcatcac tctttctctc 60

tccgtactct tttaccggca cagagcgcag ttcgtcggca ccaacctgcc gcctggcaaa 120

gttggctacc cggtgatcgg tgagacctac cagttcctgg ccacaggatg gaaaggtcac 180

ccagagaagt tcatcttcga ccgcatgacc aagtactctt ccgaagtgtt caagacctcc 240

ctcatgggcg 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

ttcgccgacg gctgggaaaa caagaaggaa gttgaagttt tccccctcgc caagaactac 540

accttttggc ttgctgcacg gttgtttgtg agcctggacg actcggttga aatcgccaag 600

ctaggcgacc cgttcgcagt tttggcctct ggaatcatat cgatgcctct ggatttcccg 660

ggaactcctt tttacaaagc gatcaaggca tccaacttca tcagggagga gctgacgaag 720

atcatcaagc agaggaagat agacttggcg gagggcaagg cgtcgccgac gcaagacata 780

ttgtcacaca tgttgttgtt gtgcgacgag cacggaagtc acatgaagga acacgatatc 840

gccgataaga ttttggggct gctgattggt gggcatgaca cagccagcgc tacctgcact 900

tttattgtca agtatcttgc cgaacttcct catatttatg acgaagtcta caaggagcaa 960

atggaggtcc taagcgcaaa agctccaggg gagttgttga attgggatga cctacagaag 1020

atgaaatact catggaacgt agctcaggaa gttctcagat tggctccacc tcttcaagga 1080

gctttcaggg aagccttgtc tgacttcgtc ttcaatggtt tcaccattcc aaagggctgg 1140

aagttatatt ggagcgcaaa ctcaacacac aagaacgcag cttacttccc ggaaccattc 1200

aaattcgacc cttcaagatt cgaaggaaaa ggtccagcac catacacgtt tgtgcccttt 1260

ggaggaggtc ctaggatgtg ccccggcaaa gagtacgccc gattggagat cctagtgttc 1320

atgcacaact tggtgaagag gttcaaatgg gagaaagtcc ttcccaacga gcagatcgta 1380

gttgacccac tccccatgcc cgccaaggga ctccctgtcc gcctttttcc tcaccctaag 1440

accgccgcag cttaa 1455

<210> 2

<211> 484

<212> PRT

<213> Malus × domestica apple

<400> 2

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 Ala 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 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 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> 3

<211> 1443

<212> DNA

<213> Malus × domestica apple

<400> 3

atggagaccc tttaccttat attttctgtg ggtgctgctt tcttagcctt taccatcttt 60

gcactcaagg gcaaatcaga tgatggaaaa aatcttccac cagggagcat ggggtggcct 120

attgtgggtg aatcaatcga gttcttgttc gggaagccgg aaaattttgt gttcaagagg 180

atgaggaagt actctcctga catcttcaag acctatattc ttggagagaa aactgcagtg 240

atctgtggtc ctagtggaca caaatttctg ttctccaatg aacaaaagta cttcacagct 300

tttcgaccac attcgatgca aaagatgttt cgatcataca aggctgccgc ccccactgct 360

tctgctgccc ccgctgtggc acaaccttct cgcgatgaag aatcgaaagt tataagatcg 420

ccggggtttt tgaagccgga agcattggtg aggtacttgg ggaaaatgga ctctatcact 480

caggagcaga tgaaggccta ttgggaaggc aaagatgagg tcaaggtcta ccctttggcc 540

aagacactca ctctaagtct tgcctgcaga ttcttcttgg ggatcgacga ttccgagcga 600

attgcaaggc tggtgagcaa ttttgatgat gttactgttg ggatgcattc acttattatc 660

aacttcccag gaacaacatt ctataaggca accaaagcag ccgacgcgct gcgaaaggag 720

ttgaggattg tgattcagga gaagaaggct gcaatggccg caggaggtcc catgcatgat 780

atattgtcac atatgatcgt ggctagtgat ccatccggta aacacatgcc cgaggctgag 840

gttgcagaca agatcatggg tttattgact gcaggatata gcactgtggc cactgctatg 900

actttcttca tgaaatatgt tggagagagg ccagacattt atgccaaggt cctagctgag 960

cacaaggaga ttgcagactc gaagaagcca ggagactttt tggagtggga agacatcaac 1020

aaaatgaagt actcgtggaa tgtgttgtat gaagtgatga gattcacacc gccacttcag 1080

gggacattta gagaggcctt gacagatttt acctacgcag gttacaccat cccgaagggc 1140

tggaaggtat attggacagt gagtacaaca aacatgaacc cacagtactt ccccaacccc 1200

gaaaagtttg atccatcaag atatgaggac ctaaacgcat tcccggcttt cacacttgtt 1260

ccatttggag gagggccaag aatgtgcccc gggaaagagt acgctcgtct agcgatactc 1320

actttcgttc acaacgtggt gaagaggttc gaatgggaag ttctgtttcc caaggagaag 1380

atcacaggtg atatgatgcc aacaccagag aaagggcttc cagttcgcct tcgacgtcac 1440

tag 1443

<210> 4

<211> 480

<212> PRT

<213> Malus × domestica apple

<400> 4

Met Glu Thr Leu Tyr Leu Ile Phe Ser Val Gly Ala Ala Phe Leu Ala

1 5 10 15

Phe 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 Lys Arg Met Arg Lys Tyr

50 55 60

Ser 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 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 Ala Pro Ala Val Ala 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 Arg His

465 470 475 480

<210> 5

<211> 2283

<212> DNA

<213> Eriobotrya japonica (Thunb.) Lindl

<400> 5

atgtggagga ttaagtttgg agaaggtgca aacgacccct tgttgttcag cacaaacaac 60

ttccacggaa ggcagacatg ggagttcgac ccagatgcag gtaccgagga ggaacgagca 120

gaagttgaag ctgctcgtga gcatttctac caaaatcgct tcaaggtcac gcctagcagt 180

gacctcctat ggcgttttca gattttaaga gaaaaaaact tcaaacaaga 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

aagaagatca gagacaatgc cattcaattc acaattgacc aaattcatta cgaagatgag 1080

aacagtcgct acattacaat cggatgtgtg gaaaagccat taatgatgct tgcttgctgg 1140

gccgaggatc ctagtggaga agctttcaag aagcatattc ctagagttac tgattatatc 1200

tggctcggag aggatggaat caagatgcag agttttggaa gccagtcatg ggattgtgct 1260

cttgtgattc aagctttgct tgctggcaat ctaaatactg aaatggcacc tacccttaag 1320

caagcacacg aatttctcaa gatatctcag gtgagggtga atacttctgg tgactaccta 1380

gctcatttcc gtcacgtttc taagggcgca tggacattct ctgaccgcga ccatggatgg 1440

caagtgtcgg attgtactgc agaagcactg aggtgttgct gcattttcgc aaatatgtcc 1500

ccagaacttg ttggtgagcc aatggatgct gagtgtatgt atgatgccgt caatgtcatc 1560

ttgacccttc agagtccaaa tggtggtgta tcagcctggg agccaacagg agcaccaaaa 1620

tggttggagt ggctcaaccc tgtggagttt cttgaggacc tagtcattga gtacgagtac 1680

atcgaatgca cttcatcttc gatccaggcc ttaactttgt ttaggaagtt gtaccctggc 1740

catagaagga aggagatcaa caatttcatc acgagagcag cagactacat tgaagacata 1800

cagtaccctg atggctcatg gtatggaaac tggggaatct gctttgtgta tggtacatgg 1860

ttcgcaatca aagggctgga ggccgctggt aggacgtaca acaactgcga ggcagtgcgc 1920

aaaggtgttg actttttgct caaaacacaa agggcagatg gtggctgggg agagcactac 1980

acctcatgca caaacaagaa atatacagct caagatagca caaatttggt ccaaactgca 2040

ctcggattaa tgggtctgat tcatggtcga caggctgaga gagatccaac tcctattcac 2100

cgtgctgctg cggtgttaat gaaaggtcag ttggatgatg gcgatttccc acaacaggaa 2160

ctgatgggag tttttatgag gaatgctatg ttgcactatg cagcatacag aaacatcttc 2220

ccattgtggg ctcttggaga ataccgcact ctggtttcgt tgcctataaa aaggattgct 2280

tga 2283

Claims (10)

1. The application of the gene with the nucleotide sequence shown as SEQ ID NO.1 and/or the protein with the amino acid sequence shown as SEQ ID NO.2 is characterized in that the application is for promoting the apple to generate triterpenic acid.

2. The use according to claim 1, wherein the triterpenic acid is an ursane-type and/or oleanane-type triterpenic acid.

3. The use according to claim 2, wherein the ursolic acid and/or corosolic acid is the oleanolic acid and/or crataegolic acid.

4. The application of the protein with the nucleotide sequence shown as SEQ ID NO.1 or the amino acid sequence shown as SEQ ID NO.2 is characterized in that the application is for promoting alpha-amyrin to generate ursolic acid, alpha-amyrin to generate corosolic acid, beta-amyrin to generate oleanolic acid and/or beta-amyrin to generate crataegolic acid.

5. A method for improving the yield of ursane-type and/or oleanane-type triterpene acid is characterized in that the gene with the nucleotide sequence shown as SEQ ID NO.1 is overexpressed and/or the expression level of the protein with the amino acid sequence shown as SEQ ID NO.2 is improved.

6. The method according to claim 5, wherein the ursolic acid and/or corosolic acid are ursolic acid and the oleanane-type triterpene acid and/or crataegolic acid.

7. The application of the gene with the nucleotide sequence shown as SEQ ID NO.3 and/or the protein with the amino acid sequence shown as SEQ ID NO.4 is characterized in that the application is for promoting apples to generate ursane type and/or oleanane type triterpenic acid.

8. The use according to claim 7, wherein said ursane-type triterpene acid is corosolic acid and said oleanane-type triterpene acid is crataegolic acid.

9. The application of the gene with the nucleotide sequence shown as SEQ ID NO.3 and/or the protein with the amino acid sequence shown as SEQ ID NO.4 is characterized in that the application is for promoting ursolic acid to generate corosolic acid and/or promoting oleanolic acid to generate crataegolic acid.

10. A method for improving the yield of corosolic acid and/or crataegolic acid is characterized in that the gene with the nucleotide sequence shown as SEQ ID NO.3 is over-expressed and/or the expression quantity of the protein with the amino acid sequence shown as SEQ ID NO.4 is improved.