Oxymethyltransferase as well as coding gene CsOMT5 and application thereof

Abstract

The invention relates to oxygen methyl transferase as well as a coding gene CsOMT5 and application thereof, and belongs to the technical field of gene engineering. The oxygen methyl transferase is obtained through prokaryotic expression of a CsOMT5 gene cloned from rock candy orange peel for the first time. In-vitro enzyme activity experiments show that the oxymethyltransferase can catalyze various flavonoid compounds, such as flavanones, flavonoids, flavanols and flavonols, and the catalytic site is 3 ' / 5' / 5 / 7. Citrus homologous transient overexpression and gene silencing experiments prove that the oxymethyltransferase participates in biosynthesis of citrus PMFs and promotes accumulation of the citrus PMFs, which further proves that the oxymethyltransferase has relatively high research prospects and application values in the aspects of improving the nutritional quality of citrus fruits and industrially producing PMFs.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an oxygen methyltransferase and its encoding gene. CsOMT5 and applications. Background Technology

[0002] PMFs (polymethoxyflavones) are a class of natural flavonoid compounds unique to citrus fruits. They not only play an important role in plant growth and development but also have antioxidant, anti-inflammatory, anti-cancer, and blood sugar-lowering functions in the human body. OMT ( O -methyltransferase, O β-methyltransferases (MTs) are the main methyltransferases in plants, catalyzing methylation of flavonoid substrates to improve their stability and transmembrane capacity, thus enabling the synthesis of PMFs. However, most reported methyltransferases (OMTs) currently favor methylation of flavonoid substrates, lacking OMTs that modify flavanones and flavonols. This undoubtedly limits the research progress on industrial-scale PMF production technology. Summary of the Invention

[0003] The purpose of this invention is to address the current situation where most existing oxygen methyltransferases (OMTs) prefer to methylate flavonoid substrates, and to provide an OMT capable of methylating flavanones and flavonols, along with its encoding gene. To this end, this invention provides an oxygen methyltransferase and its encoding gene. CsOMT5 and applications.

[0004] The present invention provides an oxymethyltransferase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] This invention also provides the encoding gene of the oxygen methyltransferase described in the above technical solution. CsOMT5 The encoding gene CsOMT5 The CDS nucleotide sequence is shown in SEQ ID NO.2.

[0006] The present invention also provides the oxygen methyltransferase described in the above-described technical solution or the encoding gene described in the above-described technical solution. CsOMT5 Application in improving the nutritional quality of citrus fruits.

[0007] Preferably, the improvement of the nutritional quality of citrus fruits includes positively regulating the content of polymethoxyflavones in citrus fruits.

[0008] The present invention also provides the oxygen methyltransferase described in the above-described technical solution or the encoding gene described in the above-described technical solution. CsOMT5 Application in the catalytic synthesis of polymethoxyflavonoids.

[0009] Preferably, the substrate for the synthesis of the polymethoxyflavonoids includes flavonoids; the oxymethyltransferase participates in catalyzing the synthesis of one or more hydroxyl sites in the flavonoids. O -Methylation reaction.

[0010] Preferably, the flavonoids include one or more of flavanones, flavones, dihydroflavonols, and flavonols; The hydroxyl sites include the 3', 5', 5', and 7' positions of the flavonoid substrate.

[0011] The present invention also provides a method for promoting the synthesis of polymethoxyflavonoids in citrus fruits, comprising the following steps: Increasing the content of oxymethyltransferase in citrus fruits and / or overexpressing the gene encoding oxymethyltransferase. CsOMT5 ; The amino acid sequence of the oxygen methyltransferase is shown in SEQ ID NO.1; The encoding gene CsOMT5 The CDS nucleotide sequence is shown in SEQ ID NO.2.

[0012] Preferably, the gene encoding oxymethyltransferase is overexpressed in citrus. CsOMT5 By introducing the gene into citrus fruits... CsOMT5 The overexpression vector is realized.

[0013] The present invention also provides a method for amplifying genes. CsOMT5 The primer pair, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; The gene CsOMT5 The CDS nucleotide sequence is shown in SEQ ID NO.2.

[0014] Beneficial effects: This invention provides an oxymethyltransferase, the amino acid sequence of which is shown in SEQ ID NO.1. The oxymethyltransferase of this invention was first cloned from the peel of a rock sugar orange. CsOMT5The gene was obtained through prokaryotic expression. In vitro enzyme activity experiments showed that the oxymethyltransferase described in this invention can catalyze various flavonoid compounds, such as flavanones, flavones, dihydroflavonols, and flavonols, with catalytic sites at 3' / 5' / 5 / 7. When catalyzing the 3' position, it is independent of the ortho-hydroxyl environment; when catalyzing the 5' position, it is dependent on the meta-hydroxyl environment. Furthermore, through citrus homologous transient overexpression and gene silencing experiments, it was demonstrated that the oxymethyltransferase participates in the biosynthesis of citrus PMFs and promotes their accumulation. This further demonstrates the high research prospects and application value of this invention in improving the nutritional quality of citrus fruits and the industrial production of PMFs. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 for CsOMT5 Expression profiles of sugar oranges during their developmental stages; among which, Figure 1 In the figure, A represents the results of the total flavonoid component analysis; Figure 1 Figure B in the graph represents the analysis results of total polymethoxyflavonoid components; Figure 1 C in the text is CsOMT5 Graph showing the results of gene relative expression level analysis; Figure 1 D in CsOMT5 Figure showing the results of the per-thousand-base fragment analysis of gene exons; Figure 2 The image shows the SDS-PAGE electrophoresis results of the CsOMT5 recombinant protein; in which, Figure 2 In this context, M represents the standard molecular weight of the protein. Figure 2 The EV in the text is the pET32a empty vector protein; Figure 3 Figure I shows the HPLC analysis of the in vitro enzyme activity of CsOMT5 recombinant protein with different substrates; where, Figure 3 The orange curve represents the reaction between the CsOMT5 recombinant protein and the substrate; the blue curve represents the reaction between the empty vector protein and the substrate; the green curve represents the flavonoid standard substance. Figure 3 In the figure, A represents the HPLC chromatogram of naringenin catalyzed by the CsOMT5 recombinant protein; Figure 3 B in the figure represents the HPLC chromatogram of isochordin catalyzed by the CsOMT5 recombinant protein. Figure 3 In the figure, C represents the HPLC chromatogram of CsOMT5 recombinant protein catalyzing sennaol; Figure 3 In the figure, D represents the HPLC chromatogram of hesperidin catalyzed by CsOMT5 recombinant protein; Figure 3 E in the figure represents the HPLC chromatogram of apigenin catalyzed by CsOMT5 recombinant protein; Figure 3F in the figure represents the HPLC chromatogram of luteolin catalyzed by CsOMT5 recombinant protein; Figure 3 In the figure, G represents the HPLC chromatogram of geraniol catalyzed by CsOMT5 recombinant protein; Figure 3 H in the figure represents the HPLC chromatogram of baicalin catalyzed by CsOMT5 recombinant protein; Figure 3 In the figure, I represents the AHPLC chromatogram of CsOMT5 recombinant protein catalyzing pectin; Figure 3 J in the figure represents the HPLC chromatogram of 5,7,3',4',5'-pentahydroxyflavone catalyzed by the CsOMT5 recombinant protein. Figure 3 K in the figure represents the HPLC chromatogram of demethoxycyanidin flavonoids catalyzed by CsOMT5 recombinant protein; Figure 4 Figure II shows the HPLC analysis of the in vitro enzyme activity of CsOMT5 recombinant protein with different substrates; in which, Figure 4 The orange curve represents the reaction between the CsOMT5 recombinant protein and the substrate; the blue curve represents the reaction between the empty vector protein and the substrate; the green curve represents the flavonoid standard substance. Figure 4 In the figure, L represents the HPLC chromatogram of hesperidin catalyzed by CsOMT5 recombinant protein; Figure 4 M in the figure represents the HPLC chromatogram of piperidine catalyzed by CsOMT5 recombinant protein; Figure 4 In the figure, N represents the HPLC chromatogram of kaempferol catalyzed by the CsOMT5 recombinant protein; Figure 4 O in the figure represents the HPLC chromatogram of quercetin catalyzed by the CsOMT5 recombinant protein; Figure 4 P in the figure represents the HPLC chromatogram of caffeic acid catalyzed by the CsOMT5 recombinant protein.

[0017] Figure 5 High-resolution mass spectra of the products generated by the in vitro enzyme activity of CsOMT5 recombinant protein with different substrates (I); where... Figure 5 A in the image represents the high-resolution mass spectrum of the product of naringenin catalyzed by the CsOMT5 recombinant protein. Figure 5 B in the image is a high-resolution mass spectrum of the product of isochorin catalyzed by the CsOMT5 recombinant protein. Figure 5 C and D in the image are high-resolution mass spectra of the product of CsOMT5 recombinant protein catalyzing sennaol; Figure 5 E in the image represents the high-resolution mass spectrum of the product of hesperidin catalyzed by the CsOMT5 recombinant protein. Figure 5 F in the image represents the high-resolution mass spectrum of the product of apigenin catalyzed by the CsOMT5 recombinant protein. Figure 5 G in the image represents the high-resolution mass spectrum of the product of luteolin catalyzed by the CsOMT5 recombinant protein. Figure 5 H in the image represents the high-resolution mass spectrum of the product of CsOMT5 recombinant protein catalyzing geraniol. Figure 5 In the image, I represents the high-resolution mass spectrum of the product of baicalin catalyzed by the CsOMT5 recombinant protein. Figure 5J in the image represents the high-resolution mass spectrum of the product of CsOMT5 recombinant protein catalyzing phloem A. Figure 5 K and L in the image are high-resolution mass spectra of the product of 5,7,3',4',5'-pentahydroxyflavone catalyzed by the CsOMT5 recombinant protein. Figure 5 M in the image represents the high-resolution mass spectrum of the product of demethoxycyanin flavonoids catalyzed by the CsOMT5 recombinant protein. Figure 6 High-resolution mass spectra II show the products generated by the in vitro enzyme activity of CsOMT5 recombinant protein with different substrates; among them... Figure 6 N in the image represents the high-resolution mass spectrum of the product of kaempferol catalyzed by the CsOMT5 recombinant protein. Figure 6 The O and P in the image represent the high-resolution mass spectra of the product of quercetin catalyzed by the CsOMT5 recombinant protein. Figure 6 Q in the image represents the high-resolution mass spectrum of the product of hesperidin catalyzed by the CsOMT5 recombinant protein. Figure 6 R in the image represents the high-resolution mass spectrum of the product of piperidine catalyzed by the CsOMT5 recombinant protein. Figure 6 S in the image represents the high-resolution mass spectrum of the product of caffeic acid catalyzed by the CsOMT5 recombinant protein.

[0018] Figure 7 for CsOMT5 Homologous transient overexpression of genes and VIGS results; among them, Figure 7 In this context, A represents homologous transient overexpression. CsOMT5 Image showing the results of detecting polymethoxyflavonoid content after gene sequencing; Figure 7 B in the equation represents homologous transient overexpression. CsOMT5 The results of relative expression level detection after gene expression; Figure 7 C in the text represents silence. CsOMT5 Image showing the results of detecting polymethoxyflavonoid content after gene sequencing; Figure 7 The D in the text represents silence. CsOMT5 The results of relative expression level detection after gene expression; Figure 8 This is a diagram showing the subcellular localization results of CsOMT5. Detailed Implementation

[0019] This invention provides an oxymethyltransferase, the amino acid sequence of which is shown in SEQ ID NO. 1. As one embodiment, the oxymethyltransferase of this invention is obtained for the first time from the peel of a rock sugar orange. CsOMT5 The gene is obtained through prokaryotic expression. As one embodiment, the oxymethyltransferase described in this invention can catalyze various flavonoid compounds, such as flavanones, flavones, dihydroflavonols, and flavonols, with catalytic sites at 3' / 5' / 5 / 7. Specifically, SEQ ID NO.1 of this invention is as follows: MANEGRDESFAYANQLVTASVLPMTMQAVIGLGVFEIIAKAGPGAKLSASEIAAQLPATKNKDAPMMLDRMLRLLASHSVVECSIDDADDYQRLYGLNDVSNYFVPNKDGVSFGPVLALIQDKVFMDSWSQLKEAITEGGVPFDRVHGTHAFEYPGLDPRFNEVFNIAMYNYTNLVIQKI LEAYKGFEHIEQLVDVGGCLGNTLKAITSKYPHIKGINFDLPHVIQHAPKYPGVEHVGGDMFQNVPKGDAIFMKWILHDWSDEHCLKLLKNCYKSIPEDGKVIVVEAILPELPDTSTHSKINSQGDVLMMTQNPGGKERTKHELTTLATEAGFSGIRFVCLFYNYWVMEFYK, length 352aa.

[0020] This invention also provides the encoding gene of the oxygen methyltransferase described in the above technical solution. CsOMT5 The encoding gene CsOMT5 The CDS nucleotide sequence is shown in SEQ ID NO.2. Specifically, SEQ ID NO.2 of this invention is: CsOMT5 The gene is mainly expressed in the oil cell layer of citrus peel, and almost not in the white peel layer.

[0021] The present invention also provides the oxygen methyltransferase described in the above-described technical solution or the encoding gene described in the above-described technical solution. CsOMT5 Application in improving the nutritional quality of citrus fruits. As one embodiment, the oxymethyltransferase or the encoding gene described in this invention... CsOMT5 It can participate in the biosynthesis of citrus PMFs and promote their accumulation. As one implementation method, the improvement of citrus fruit nutritional quality according to the present invention includes positively regulating the content of polymethoxyflavones in citrus.

[0022] The present invention also provides the oxygen methyltransferase described in the above-described technical solution or the encoding gene described in the above-described technical solution. CsOMT5 Application in the catalytic synthesis of polymethoxyflavones. As one embodiment, the substrate for the synthesis of polymethoxyflavones according to this invention includes flavonoids; the oxymethyltransferase participates in the catalytic synthesis of one or more hydroxyl sites in the flavonoids. O - Methylation reaction. As one embodiment, the flavonoids of this invention include one or more of flavanones, flavones, dihydroflavonols, and flavonols; the hydroxyl sites include the 3', 5', 5', and 7' positions of the flavonoid substrate. As one embodiment, the oxymethyltransferase or the encoding gene of this invention... CsOMT5 The catalytic activity at the 3' position of flavonoids is independent of the ortho-hydroxyl environment; however, it is dependent on the meta-hydroxyl environment when catalyzing the 5' position. As one embodiment, the oxymethyltransferase or the encoding gene described in this invention... CsOMT5 The new substance peak that catalyzes the formation of senna, demethoxycyanidin, hesperidin, kaempferol, and quercetin corresponds to the 5-methyl ester product. As one embodiment, the oxymethyltransferase or the encoding gene described in this invention... CsOMT5 It preferentially catalyzes the 3'-hydroxymethylation of sennaol, luteolin, 3'-hydroxyflavone, 5,7,3',4',5'-pentahydroxyflavone, and quercetin, generating the corresponding sennaol, luteolin, 3'-methylflavone, alfalfa extract, and isorhamnetin. As one embodiment, the oxygen methyltransferase or the encoding gene described in this invention... CsOMT5 It can catalyze the 7-hydroxymethylation of baicalin and 7,8-dihydroxyflavone to generate the corresponding baicalin-7-methyl ether and 7-methoxy-8-hydroxyflavone. As one embodiment, the oxymethyltransferase or the encoding gene described in this invention... CsOMT5 It can catalyze the formation of isoflavonic acid from caffeic acid.

[0023] The present invention also provides a method for promoting the synthesis of polymethoxyflavonoids in citrus fruits, comprising the following steps: Increasing the content of oxymethyltransferase in citrus fruits and / or overexpressing the gene encoding oxymethyltransferase. CsOMT5 ; The amino acid sequence of the oxygen methyltransferase is shown in SEQ ID NO.1; The encoding gene CsOMT5 The CDS nucleotide sequence is shown in SEQ ID NO.2.

[0024] This invention increases the content of oxymethyltransferase in citrus fruits and / or overexpresses the gene encoding oxymethyltransferase. CsOMT5 In one embodiment, the present invention overexpresses the gene encoding oxymethyltransferase in citrus fruits. CsOMT5 By introducing the gene into citrus fruits... CsOMT5 The overexpression vector is used for implementation. As one embodiment, the present invention will... CsOMT5 The CDS sequence after removing the terminator was loaded onto the pBI121 vector to construct... CsOMT5 -pBI121 recombinant expression vector.

[0025] The present invention also provides a method for amplifying genes. CsOMT5 The primer pair, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; the gene CsOMT5 The CDS nucleotide sequence is shown in SEQ ID NO.2. As one embodiment, SEQ ID NO.3 of the present invention specifically includes: 5'-TATCGGATCCGAATTCATGGCTAATGAAGGGAGAGACG-3'.

[0026] As one implementation method, SEQ ID NO.4 of the present invention specifically refers to: 5'-GGTGGTGGTGCTCGAGCTTGTAGAACTCCATAACCCAAT-3'.

[0027] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, describes an oxygen methyltransferase and its encoding gene provided by the present invention. CsOMT5 The invention is described in detail in terms of its application, but it should not be construed as limiting the scope of protection of this invention.

[0028] Example 1 CsOMT5 Gene cloning 1. Experimental Methods ①RNA extraction and reverse transcription Total RNA was extracted from the peel of the rock sugar orange according to the instructions of the RN38-EASYspin Plus Plant RNA Kit (Aidlab, Beijing). cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Vazyme, Nanjing).

[0029] ② Gene cloning Based on the cDNA sequence of OMT in the sweet orange genome, a design was created. CsOMT5 PCR primers for amplifying the coding region (CDS) of the gene were used, and homologous arms were constructed by adding partial sequences of the pET32a restriction sites BamHI and XhoI. Forward primer (SEQ ID NO.3): 5'-TATCGGATCC GAATTC ATGGCTAATGAAGGGAGAGACG-3', Reverse primer (SEQ ID NO.4): 5'-GGTGGTGGTG C TCGAG CTTGTAGAACTCCATAACCCAAT-3' (underlined is the restriction enzyme site). PCR amplification was performed using 2 × Phanta MaxMaster Mix (Vazyme, Nanjing). The PCR reaction system and procedure are as follows: The reaction mixture (30 μL) consisted of: 15 μL 2×Buffer, 0.6 μL dNTPs, 1.2 μL each of forward and reverse primers (10 mM), 0.6 μL enzyme, 1 μL cDNA, and 10.4 μL RNase-free water. The reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 58℃ annealing for 15 sec, 72℃ extension for 45 sec, for 35 cycles; and 72℃ final extension for 5 min.

[0030] Subsequently, the product was analyzed by agarose gel electrophoresis, and the product was recovered using a gel recovery kit (Vazyme, Nanjing). The product was then ligated into the pET32a vector using homologous recombination and transformed into DH5α Escherichia coli. Positive single clones were preserved after verification by colony PCR and sequencing.

[0031] 2. Experimental Results Successfully cloned from the peel of rock sugar orange CsOMT5 The full-length sequence is 1059 bp (as shown in SEQ ID NO.2), and a sequence containing... CsOMT5 Total length CsOMT5 -pET32a vector.

[0032] Example 2 CsOMT5 Expression profiles of the developmental stages of rock sugar orange 1. Experimental Methods ① Fruit material collection Using rock sugar oranges as material, a total of seven developmental stages of rock sugar oranges were collected: 30 days after full bloom (S1), 60 days (S2), 80 days (S3), 100 days (S4), 120 days (S5), 150 days (S6), and 180 days (S7). Three biological replicates were performed for each developmental stage, with three fruits per replicate. The peel was separated into oil cell layer and white peel layer, cut into small pieces, flash-frozen with liquid nitrogen, and stored at -80℃.

[0033] ② HPLC analysis of flavonoids The oil cell layer and the white skin layer were ground into powder. 0.05 g of the powder was weighed and 0.5 mL of 80% ethanol was added. The mixture was sonicated for 30 min and then centrifuged at 1,2000 rpm for 10 min. The supernatant was collected and the process was repeated once. The two supernatants were combined and centrifuged at high speed for 30 min to precipitate impurities for HPLC detection. HPLC detection system: The mobile phase consisted of 100% chromatographic acetonitrile (A) and water (B) containing 0.1% formic acid. The injection volume was 10 μL, the column temperature was 25℃, and the flow rate was 1 mL / min. Gradient elution was used: 0–5 min, 20% A; 5–10 min, 20%–27% A; 10–15 min, 27% A; 15–25 min, 27%–40% A; 25–35 min, 40%–60% A; 35–40 min, 60%–80% A; 40–42 min, 80%–100% A; 42–45 min, 100%–20% A; 45–52 min, 20% A. The detection wavelengths were 280 nm and 330 nm.

[0034] ③RNA extraction and reverse transcription The experimental method is the same as in Example 1.

[0035] ④ Gene expression analysis Citrus β -actin was used as an internal reference gene. Differential gene expression analysis was performed using ChamQ Blue Universal SYBR qPCR Master Mix (Vazyme, Nanjing). The specific system was described in the instruction manual. Citrus fruits were included. β -actin forward Q-PCR primer (SEQ ID NO.5): 5'-CATCCCTCAGCACCTTCC-3', reverse Q-PCR primer (SEQ ID NO.6): 5'-CCAACCTTAGCACTTCTCC-3'; CsOMT5Forward Q-PCR primer (SEQ ID NO.7): 5'-CTTCCAGAACTGCCTGACAC-3' Reverse Q-PCR primer (SEQ ID NO.8): 5'-GTTCGCTCCTTTCCCCCTG-3'.

[0036] The reaction program was as follows: pre-denaturation at 95℃ for 5 min; 95℃ for 10 s, 60℃ for 30 s, for 40 cycles; 95℃ for 10 s; melting curve from 65℃ to 95℃, increasing by 0.5℃ every 5 s. The detection instrument was a CFX96s real-time quantitative PCR (Bio-Rad, USA). Two... -ΔΔCt The method calculates the relative expression level of genes.

[0037] 2. Experimental Results Results of flavonoid component analysis were obtained. CsOMT5 Expression profiles of the developmental stages of sugar oranges (e.g.) Figure 1 As shown, the analysis results of flavonoid components indicate that the total flavonoids in the peel of the rock sugar orange generally show a trend of first increasing and then decreasing at different developmental stages; PMFs in the peel mainly accumulate in the oil cell layer and hardly accumulate in the white peel layer. CsOMT5 Gene expression trends were largely consistent with those of PMFs: expression levels were highest in the early stages, then gradually decreased, and expression was specific in the oil cell layer. Preliminary assessment... CsOMT5 It plays an important role in the synthesis of PMFs.

[0038] Example 3 CsOMT5 recombinant protein expression and purification 1. Experimental Methods ① Recombinant cell construction Correct sequencing CsOMT5 Using the pET32a recombinant expression vector as a template, the strain was transformed into the BL21(DE3)pLysS Escherichia coli expression strain (Weidi, Shanghai) via heat shock. Positive single clones were screened on LB solid plates with 100 ug / mL ampicillin and preserved with 20% glycerol at -80℃.

[0039] ② Prokaryotic expression and protein purification After the above bacterial culture was reactivated, 10 mL of the culture was added to 500 mL of LB liquid medium containing Amp and cultured at 37°C and 150 rpm for 2 h until OD was reached. 600≈0.6; Add IPTG to a final concentration of 1 mM, and induce protein expression at 16℃ and 150 rpm for 20 h. Then, centrifuge the bacterial suspension at 4℃ and 3000 rpm for 30 min; discard the supernatant, resuspend the cells in 1×PBS, and incubate at -80℃ overnight. The next day, freeze-thaw the bacterial suspension at 30℃, then disrupt it using an ultrasonic cell disruptor; centrifuge at 4℃ and 12000 rpm for 30 min, the supernatant being the crude protein. Subsequent protein purification was performed according to the His-tagged protein purification kit (reduction-resistant chelate type) instructions (Beyotime, Shanghai). Finally, 20 μL of purified protein was analyzed by SDS-PAGE electrophoresis, and the remaining protein was passed through a PD-10 desalting column and stored at -80℃.

[0040] 2. Experimental Results The SDS-PAGE electrophoresis results of the recombinant CsOMT5 protein are as follows: Figure 2 As shown, according to Figure 2 The SDS-PAGE electrophoresis results show that the molecular weight of the CsOMT5-pET32a recombinant protein is approximately 55 kDa, consistent with the theoretical prediction of 58.27 kDa, and the protein concentration is high, which can be used for subsequent experiments.

[0041] Example 4 CsOMT5 in vitro enzyme activity identification 1. Experimental Methods ① Substrate regional selectivity determination The following flavonoids were selected to verify the CsOMT5 protein: naringenin, isochorin, sennaol, and hesperidin; flavonoids: apigenin, luteolin, geraniol, baicalin, sennain A, 3'-hydroxyflavone, 7,8-dihydroxyflavone, demethoxycyanidin, and 5,7,3',4',5'-pentahydroxyflavone; dihydroflavonols: hesperidin and piperidin; flavonols: quercetin and kaempferol; and caffeic acid. O - Methylation catalysis function. The total in vitro enzyme activity reaction system was 100 μL: 200 μM substrate, 2 mM... S -Adenosine- L -Methionine ( S -adenosyl- L10 μL of recombinant protein and Tris-HCl buffer were added to the sample. After reacting at 37℃ for 1 h, 100 μL of ice-cold methanol was added to terminate the reaction. The sample was centrifuged at 13000 rpm for 30 min, and the supernatant was collected for HPLC analysis. The HPLC system was as follows: 100% chromatographic acetonitrile (A) and water containing 0.1% formic acid (B) were used as the mobile phase. The injection volume was 10 μL, the column temperature was 25℃, the flow rate was 1 mL / min, and gradient elution was used: 0~2 min, 20% A; 2~4 min, 20%~30% A; 4~12 min, 30%~50% A; 12~19 min, 50%~80% A; 19~21 min, 80%~100% A; 21~22 min, 100% A; 22~23 min, 100%~20% A; 23~26 min, 20% A. The detection wavelengths were 280 nm and 330 nm. Enzyme reaction products were analyzed by mass spectrometry using an AB TripleTOF 5600 time-of-flight LC-MS / MS system (AB SCIEX, USA), in negative ion scanning mode: ion source temperature (TEM) 550℃, voltage (IS) -4500V; scan range (m / z) 100–1500 Da; nebulizer gas (GS1), nebulizer gas (GSI2), and curtain gas (CUR) pressures were 55 psi, 55 psi, and 35 psi, respectively.

[0042] ② Determination of enzyme kinetic parameters Refer to MTase-Glo TM The Methyltransferase Assay kit (Promaga, USA) was used to determine the affinity and catalytic efficiency of recombinant CsOMT5 protein for different substrates. The reaction system consisted of 20 μL: 2 mM SAM, reaction buffer, substrates of varying concentrations (0–400 μM), and 10 μL of diluted CsOMT5 recombinant protein. After incubation at 37°C for 30 min, the reaction was terminated with 0.5% TFA (trifluoroacetic acid). The luminescent reagent was then added according to the manufacturer's instructions, and the RLU values ​​were measured using a microplate reader (BioTek, USA). The Michaelis-Menten equation in GraphPad Prism 8.0 was used for calculation. K m and k cat value.

[0043] 2. Experimental Results ① Results of in vitro enzyme activity The reaction products were determined by combining the results of HPLC analysis of the standard and mass spectrometry analysis. The in vitro enzyme activity HPLC chromatograms of CsOMT5 recombinant protein with different substrates were obtained as follows: Figure 3 and Figure 4 As shown, and the high-resolution mass spectra of the products generated by the in vitro enzyme activity of CsOMT5 recombinant protein with different substrates are as follows. Figure 5 and Figure 6 As shown. According to Figures 3-6 The results show that CsOMT5 generates new peaks after reacting with the above substrates. In vitro, the recombinant CsOMT5 protein can catalyze the 5-hydroxymethylation of naringenin, isochorin, and apigenin to generate the corresponding 5-methoxynaringenin, 7-hydroxy-5,4'-dimethoxyflavanone, and oleander flavonoids. Furthermore, based on the characteristics of the 5-methyl ester product peaks (the 5-methylation product peak precedes the substrate peak) and previous reports on the catalytic formation of 5-methyl esters in some flavonoids (Peng Zhaoxin. Evaluation of polymethoxyflavones in different citrus germplasms, mining and functional analysis of genes related to biosynthesis. [Doctoral Dissertation]. Huazhong Agricultural University, 2021.), it is determined that the new peaks generated by CsOMT5 in the formation of senna, demethoxycyanidin, hesperidin, kaempferol, and quercetin are also the corresponding 5-methyl ester products. In addition, CsOMT5 preferentially catalyzes the 3'-hydroxymethylation of senna, luteolin, 3'-hydroxyflavone, 5,7,3',4',5'-pentahydroxyflavone, and quercetin, generating the corresponding senna, luteolin, 3'-methylflavone, alfalfa, and isorhamnetin. Furthermore, based on CsOMT's preference for 3'-position catalysis, it is determined that it also catalyzes the 3'-position methylation of hesperidin, geraniol, piperidin, and quercetin, generating the corresponding 3'-methyl ester products. CsOMT5 can also catalyze the 7-position hydroxymethylation of baicalin and 7,8-dihydroxyflavone, generating the corresponding baicalin-7-methyl ether and 7-methoxy-8-hydroxyflavone. Based on CsOMT5's characteristic catalysis of the 7-position, it is determined that it catalyzes the 7-position methylation of phloroglucin A, generating the corresponding 7-methyl ester product. Furthermore, CsOMT5 can catalyze the formation of isoflavonic acid from caffeic acid.

[0044] ② Enzyme kinetic results The relative enzyme activities of four different types of flavonoids (flavanones, flavones, dihydroflavonols, and flavonols) and caffeic acid were determined, and the enzyme kinetic evaluation results are shown in Table 1. Relatively speaking, CsOMT5 exhibited the highest catalytic efficiency for sennaol (…). k cat / K m =257.89 M -1 s -1 ), followed by hesperidin ( k cat / K m =166.07 M -1 s -1 ), baicalin ( k cat / K m =123.32 M -1 s -1 ), apigenin ( k cat / K m =122.55 M -1 s -1 ), Douglas fir extract ( k cat / K m =121.37 M -1 s -1 CsOMT5 showed the lowest catalytic efficiency for naringenin. k cat / K m =32.25 M -1 s -1 ), but is quite friendly to it ( K m =5.19±0.75 μM), while the catalytic efficiency and affinity for caffeic acid were relatively low ( k cat / K m =76.43 M -1 s -1 , K m =20.82±3.56 μM). Overall, the CsOMT5 recombinant protein prefers to catalyze flavonoid substrates containing 3' hydroxyl groups and is independent of the ortho-hydroxyl environment; the enzyme also prefers to catalyze the methylation of substrates containing meta-dihydroxyl groups in the A ring of flavonoids at the 5 and 7 positions.

[0045] Table 1. Results of enzyme kinetic evaluation

[0046] Example 5 Homologous transient overexpression 1. Experimental Methods ① Construction of recombinant expression vectors Will CsOMT5 The CDS sequence after removing the terminator was loaded onto the pBI121 vector to construct... CsOMT5 The pBI121 recombinant expression vector was used, with the empty pBI121 vector as a control. CsOMT5 Forward primer (SEQ ID NO.9): 5'-GGACTCTAGA G GATCCATGGCTAATGAAGGGAGAGACG-3', Reverse primer (SEQ ID NO.10): 5'-GACCACCCGG GGATCC CTTGTAGAACTCCATAACCCAAT-3' (underlined is the restriction enzyme site).

[0047] Using chemical transformation method to CsOMT5 The recombinant vector pBI121 and the empty vector pBI121 were transformed into Agrobacterium EHA105 and cultured on LB solid medium containing 50 μg / mL Kanamycin and 25 μg / mL Rif. Positive single clones were screened and the bacteria were preserved at -80°C with 20% glycerol.

[0048] ② Homologous transient overexpression The bacterial culture was activated twice, the bacterial cells were collected, and resuspended in osmotic buffer (10 mM MES, 10 mM MgCl2, 200 μM acetylsuccine) until OD500 was reached. 600 ≈0.75. Select healthy, uniformly sized, and uniformly colored rock sugar oranges (160 days after full bloom), with each fruit representing one biological replicate. Inject bacterial solution into the peels of opposite sides of the equatorial plane of the same fruit, with one side injected containing ≈0.75. CsOMT5 One side was injected with Agrobacterium tumefaciens pBI121, and the other side was injected with an empty pBI121 vector as a control. Approximately 5 mL was injected into each site, and the samples were protected from light for 12 hours after injection. Samples were collected 5 days later, flash-frozen in liquid nitrogen, and stored at -80°C. ③ HPLC analysis of flavonoids The same experimental method as in Example 2.

[0049] ④RNA extraction and reverse transcription The experimental method is the same as in Example 1.

[0050] ⑤ Gene expression analysis The same experimental method as in Example 2.

[0051] 2. Experimental Results Obtaining homologous transient overexpression CsOMT5 The results of the genes are as follows Figure 7 A and Figure 7 As shown in B in the figure. Homologous transient overexpression CsOMT5 Gene analysis results showed that, compared to the control group pBI121 empty vector, overexpression... CsOMT5 The relative expression levels of the gene were significantly upregulated, and the contents of the four major PMFs in the pericarp were also significantly increased, indicating that... CsOMT5 It promotes the biosynthesis of PMFs in citrus peel.

[0052] Example 6 TRV-mediated viral-induced gene silencing (VIGS) 1. Experimental Methods ① Construction of recombinant expression vectors Select CsOMT5 A 300bp fragment of the gene's CDS region was inserted into the TRV2 vector. CsOMT5 Forward primer (SEQ ID NO.11): 5'-GCCTCCATGG GGATCC AATCAATTGGTGACGGCTTCAGTG-3', Reverse primer (SEQ ID NO. 12): 5'-ATGCCCGGGC CTCGAG GTCTCTGATAATCATCAGCATCATC-3' (underlined are restriction enzyme sites BamHI and XhoI). The constructed [product / process] was generated using a chemical transformation method. CsOMT5 The TRV2 recombinant expression vector was transformed into Agrobacterium EHA105 and cultured on LB solid medium containing 50 μg / mL Kanamycin and 25 μg / mL Rifampicin. Positive single clones were screened.

[0053] ② Homologous transient overexpression Includes TRV1 (unloaded), TRV2 (unloaded), CsOMT5 Agrobacterium with the -TRV2 plasmid was cultured in liquid LB medium until OD. 600 =1.0, centrifuged to collect bacteria, and resuspended in osmotic buffer. The resuspended TRV1 and TRV2 bacterial solutions were mixed at a 1:1 ratio and incubated for 3 h. Then, newly germinated orange seeds were immersed in the bacterial solution and kept under a vacuum of -100 kPa for 1 min. After infection, the germinated shoots were placed on MT (Murashige & Tucker medium) and cultured in the dark for 2 days, then transferred to a 25°C artificial climate chamber for about 2 weeks, and then transplanted into the soil to continue growing for about 4-7 weeks. The aboveground parts of the seedlings were then harvested, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent analysis.

[0054] ③ HPLC analysis of flavonoids The same experimental method as in Example 2.

[0055] ④RNA extraction and reverse transcription The experimental method is the same as in Example 1.

[0056] ⑤ Gene expression analysis The same experimental method as in Example 2.

[0057] 2. Experimental Results The VIGS experimental results were obtained as follows: Figure 7 C and Figure 7As shown in D in the figure. The VIGS experimental results indicate that, compared to the control group, CsOMT5 The relative expression levels of VIGS-positive plants were significantly decreased; simultaneously, the contents of the four major PMFs were also significantly reduced. The VIGS results further confirm... CsOMT5 It participated in the biosynthesis of citrus PMFs.

[0058] Example 7 Subcellular localization 1. Experimental Methods ① Construction of recombinant expression vectors After removing the terminator CsOMT5 The full-length CDS was loaded into the 35S-eGFP vector, with the forward primer (SEQ ID NO.13) being 5'-CGGTACCCGG. GGATCC ATGGCTAATGAAGGGAGAGACG-3', Reverse primer (SEQ ID NO.14): 5'-CGACTCTAGA GGATCC CTTGTAGAACTCCATAACCCAAT-3' (underlined is the restriction enzyme site BamHI).

[0059] And CsOMT5 The -35s-eGFP recombinant vector was transformed into Agrobacterium GV3101::pSoup by chemical transformation. Positive single clones were screened on LB solid medium containing 50 μg / mL Kan and 25 μg / mL Get (gentamicin), and the bacteria were preserved at -80℃ with 20% glycerol.

[0060] ② Subcellular localization The above bacterial culture was activated twice and resuspended in permeate to OD. 600 =1.0. Select 4-week-old, well-grown Nicotiana benthamiana, and include... CsOMT5 Agrobacterium tumefaciens carrying -35S-eGFP and 35S-eGFP empty vectors was injected into the lower epidermis of tobacco leaves. After being kept in the dark for 12 h, the leaves were cultured in a climate chamber at 25°C for 2 days. The fluorescence distribution of GFP (green fluorescent protein) was observed using a Zeiss LSM 880 laser confocal microscope.

[0061] 2. Experimental Results Obtaining subcellular localization results as follows Figure 8 As shown. The results indicated that after injection of Agrobacterium containing the 35S-eGFP empty vector, GFP fluorescence was observed in both the nucleus and cytoplasm of tobacco leaf cells, demonstrating that the vector localization results were reliable and specific; after injection... CsOMT5 In tobacco leaves of Agrobacterium -35S-eGFP, its GFP fluorescence is distributed in both the cytoplasm and the nucleus, exhibiting non-targeting characteristics.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An oxygen methyltransferase, characterized in that, The amino acid sequence of the oxymethyltransferase is shown in SEQ ID NO.

1.

2. The gene encoding the oxymethyltransferase of claim 1 CsOMT5 Its characteristics are, The encoding gene CsOMT5 The CDS nucleotide sequence is shown in SEQ ID NO.

2.

3. The oxymethyltransferase of claim 1 or the encoding gene of claim 2 CsOMT5 Application in improving the nutritional quality of citrus fruits.

4. The application according to claim 3, characterized in that, The improvement of the nutritional quality of citrus fruits includes positively regulating the content of polymethoxyflavonoids in citrus fruits.

5. The oxymethyltransferase of claim 1 or the encoding gene of claim 2 CsOMT5 Application in the catalytic synthesis of polymethoxyflavonoids.

6. The application according to claim 5, characterized in that, The substrates for the synthesis of the polymethoxyflavonoids include flavonoids; the oxymethyltransferase participates in the catalysis of one or more hydroxyl sites in the flavonoids. O -Methylation reaction.

7. The application according to claim 6, characterized in that, The flavonoids include one or more of flavanones, flavones, dihydroflavonols, and flavonols; The hydroxyl sites include the 3', 5', 5', and 7' positions of the flavonoid substrate.

8. A method for promoting the synthesis of polymethoxyflavonoids in citrus fruits, characterized in that, Includes the following steps: Increasing the content of oxymethyltransferase in citrus fruits and / or overexpressing the gene encoding oxymethyltransferase. CsOMT5 ; The amino acid sequence of the oxygen methyltransferase is shown in SEQ ID NO.1; The encoding gene CsOMT5 The CDS nucleotide sequence is shown in SEQ ID NO.

2.

9. The method according to claim 8, characterized in that, Overexpression of the gene encoding oxymethyltransferase in citrus CsOMT5 By introducing the gene into citrus fruits... CsOMT5 The overexpression vector is realized.

10. A method for amplifying genes CsOMT5 The primer pair is characterized in that, The nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; The gene CsOMT5 The CDS nucleotide sequence is shown in SEQ ID NO.2.

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