O-methyltransferase participating in citrus peel flavone synthesis and coding gene and application thereof

An oxygen methyltransferase, citrus peel technology, applied in transferase, application, genetic engineering and other directions, to achieve high application value and research prospects, the effect of simple enzymatic reaction conditions

Active Publication Date: 2019-02-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the isolation and identification of oxymethyltransferases that can simultaneously catalyze the 3', 4', 6, and 8 positions of flavones from plants has not been reported.

Method used

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  • O-methyltransferase participating in citrus peel flavone synthesis and coding gene and application thereof
  • O-methyltransferase participating in citrus peel flavone synthesis and coding gene and application thereof
  • O-methyltransferase participating in citrus peel flavone synthesis and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Cloning of the CitOMT1 gene

[0026] (1) Experimental method

[0027] Gene cloning: CitOMT1 was obtained by performing BLAST analysis on the reported OMT gene of Arabidopsis thaliana and the citrus genome database (http: / / www.citrusgenomedb.org / ), the nucleotide sequence of which is SEQ: No.2. Combining the primer pair SEQ:No.3 and SEQ:No.4, using the cDNA of the oil cell layer of Oumandarin fruit as a template, and referring to the FastStart High FidelityPCR System (Roche) for PCR amplification, the PCR system is: 10×Buffer 3μL, dNTP 2.4 μL, 1.2 μL each of upstream and downstream primers (10 μM), 0.3 μL enzyme, 21.3 μL DEPC-treated water, 0.6 μL cDNA; PCR program: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 sec, 58 °C annealing for 30 sec, 72 °C Extend 1min, 35 thermal cycles; 72 extend 1min, store at 4°C. The PCR product was recovered by 1% agarose gel electrophoresis and connected to pGEM-T Easy vector (Promega) for sequencing analysis. ...

Embodiment 2

[0030] Example 2: Analysis of the expression pattern of CitOMT1 in the developmental stage of Ou mandarin

[0031] (1) Experimental method

[0032] 1. Fruit material collection

[0033] Ou mandarin fruit of different developmental stages was used as material. Picked at 30(S1), 60(S2), 80(S3), 100(S4), 120(S5), 140(S6), 170(S7), 200(S8) days after full flowering, divided into 3 Biological replicates, 8 fruits were picked for each replicate; the peel of Ou mandarin was divided into two parts, the oil cell layer and the white cortex, cut into small pieces, put them in liquid nitrogen quickly, and then store them at -80°C.

[0034] 2. High performance liquid chromatography (HPLC) analysis of flavonoids

[0035] Weigh 0.5g of the sample powder of Ou mandarin pericarp oil cell layer and white cortex layer, sonicate with 3mL of 80% ethanol for 30min, centrifuge the extract (4000rpm, 10min), repeat three times, combine the supernatant (about 9mL), and use it for the flavonoid group...

Embodiment 3

[0044] Example 3: Identification of CitOMT1 Enzyme Activity in Vitro

[0045] (1) Experimental method

[0046] 1. Recombinant vector and recombinant cell construction

[0047] The verified primer pair SEQ:No.3 and SEQ:No.4, plus pET32a restriction site BamHI, XhoI and protection base constitute a new primer pair SEQ:No.10 and SEQ:No.11. Using the sequence-verified CitOMT1-T vector as a template, combined with PCR technology to clone the open reading frame of CitOMT1, and put it on the pET32a vector to construct a CitOMT1-pET recombinant expression vector, and introduce it into the E. coli expression strain by heat shock method In BL21(DE3)pLysS (Promega), spread the bacterial solution evenly on LB plates containing 100 μg / mL ampicillin (Amp) to screen positive single colonies, and store them in 20% glycerol at -80°C for later use.

[0048] 2. Induction and purification of CitOMT1 protein

[0049] After inoculating and activating the glycerol bacteria stored at -80°C, pick a...

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Abstract

The invention provides O-methyltransferase participating in citrus peel flavone synthesis and a coding gene and application thereof. The expression level of CitOMT1 is positively correlated with the accumulation of polymethoxylated flavones in a fruit development process of Citrus reticulata cv. Suavissima; in vitro enzyme activity assays indicate that the O-methyltransferase can be applied to catalyze specific site methylation of flavones, in particular to catalyze quercetin to produce quercetin 3',4' dimethyl ester, catalyze luteolin to produce chrysoeriol, catalyze eriodictyol to produce hesperidin and homoeriodictyol, catalyze baicalein to produce oroxylin A, and catalyze 7,8-dihydroxyflavone to produce 7-hydroxy-8-methoxy flavone. The enzyme activity assays have simple reaction conditions, do not require additional metal ions, and have high application value and research prospects.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biotechnology and genetic engineering, and specifically relates to an oxygen methyltransferase CitOMT1 involved in the synthesis of citrus peel flavonoids, its coding gene and its application. Background technique [0002] Flavonoids are one of the important secondary metabolites in plants, which play an important role in plant growth, development and stress resistance. Existing literature has shown that oxymethylated flavones have stronger activities in terms of anti-oxidation, anti-tumor, hypoglycemic and cardiovascular disease prevention, and their activities are different due to changes in the position and number of methoxy groups. However, the process of isolating and purifying or chemically synthesizing oxymethylated flavones from plants is relatively complicated. Oxymethyltransferase (OMT) in plants can catalyze the methylation of hydroxylated flavonoids at specific sites, so the s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P17/06
CPCC12N9/1007C12P17/06
Inventor 孙崇德刘晓娟赵晨拧王岳曹锦萍李鲜陈昆松
Owner ZHEJIANG UNIV
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