Oxymethyltransferases involved in the synthesis of flavonoids in citrus peel and their coding genes and applications

A technology of oxymethyltransferase and methylation, applied in the field of oxymethyltransferase CitOMT1 and its coding gene and application, to achieve high application value and research prospects, and the effect of simple enzyme activity reaction conditions

Active Publication Date: 2021-07-13
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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  • Oxymethyltransferases involved in the synthesis of flavonoids in citrus peel and their coding genes and applications
  • Oxymethyltransferases involved in the synthesis of flavonoids in citrus peel and their coding genes and applications
  • Oxymethyltransferases involved in the synthesis of flavonoids in citrus peel and their coding genes and applications

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 BLAST analysis of the reported Arabidopsis thaliana OMT gene and the Citrus Genome Database (http: / / www.citrusgenomedb.org / ), and the nucleotide sequence is SEQ: No. 2. Combining the primer pairs of SEQ: No.3 and SEQ: No.4, using the cDNA of Ou mandarin fruit oil cell layer as a template, PCR amplification was carried out with reference to FastStart High Fidelity PCR System (Roche), wherein the PCR system was: 10×Buffer 3μL, dNTP 2.4 μL, 1.2 μL of upstream and downstream primers (10 μM), 0.3 μL of enzyme, 21.3 μL of DEPC-treated water, 0.6 μL of cDNA; PCR program: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 sec, annealing at 58 °C for 30 sec, 72 °C Extension for 1 min, 35 thermal cycles; extension for 1 min at 72, and storage at 4°C. The PCR products were recovered by 1% agarose gel electrophoresis and then connected to pGEM-T Ea...

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 fruits at different developmental stages were used as materials. Picked at 30(S1), 60(S2), 80(S3), 100(S4), 120(S5), 140(S6), 170(S7), 200(S8) days after full bloom, divided into 3 Biological repetitions, 8 fruits were picked for each repetition; the pericarp of Ou mandarin was divided into two parts: oil cell layer and white cortex, cut into small pieces, quickly placed in liquid nitrogen, and then stored at -80°C.

[0034] 2. Analysis of flavonoids by high performance liquid chromatography (HPLC)

[0035] Weigh 0.5 g of the sample powders of the oil cell layer and white cortex of the pericarp of Ou citrus peel, ultrasonically use 3 mL of 80% ethanol for 30 min, centrifuge the extract (4000 rpm, 10 min), repeat three times, and combine the supernatant (about 9 mL) for the flavon...

Embodiment 3

[0044] Example 3: In vitro functional identification of CitOMT1 enzyme activity

[0045] (1) Experimental method

[0046] 1. Construction of recombinant vectors and recombinant cells

[0047] Using the verified primer pairs of SEQ:No.3 and SEQ:No.4, plus the restriction sites BamHI, XhoI and protective bases of pET32a, new primer pairs SEQ:No.10 and SEQ:No.11 were constructed. Using the sequence-verified CitOMT1-T vector as a template, combined with PCR technology, the open reading frame of CitOMT1 was cloned and loaded into the pET32a vector to construct a CitOMT1-pET recombinant expression vector, which was introduced into E. coli expression strains by heat shock method In BL21 (DE3) pLysS (Promega), the bacterial solution was evenly spread on LB plates containing 100 μg / mL ampicillin (Amp) to screen positive single colonies, and stored at -80°C with 20% glycerol for later use.

[0048] 2. Induction and purification of CitOMT1 protein

[0049] After inoculation and activa...

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Abstract

The invention provides an oxymethyltransferase involved in the synthesis of citrus peel flavonoids and its coding gene and application. The expression level of CitOMT1 is positively correlated with the accumulation of polymethoxylated flavonoids during the fruit development of Citrus reticulata cv. Suavissima; The in vitro enzyme activity test shows that the oxygen methyltransferase can be used in catalyzing the specific site methylation of flavonoids, especially can catalyze quercetin to generate quercetin 3', 4' dimethyl ester; catalyze eriodiction to generate hesperetin and homo eriodictyol, catalyze baicalein to generate saflavone A; catalyze 7,8-dihydroxyflavone to generate 7-hydroxy-8-methoxyflavone . The enzyme activity reaction conditions are simple, and no additional metal ions need to be added, so it has high application value and research prospect.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biotechnology and genetic engineering, and in particular relates to an oxygen methyltransferase CitOMT1 involved in the synthesis of citrus peel flavonoids and its encoding gene and application. Background technique [0002] Flavonoids are one of the important secondary metabolites in plants and play an important role in plant growth, development and stress resistance. Existing literature shows that oxymethylated flavonoids have stronger activities in antioxidant, anti-tumor, hypoglycemic and prevention of cardiovascular diseases, and their activities vary depending on the site and number of methoxy groups. However, the process of separating and purifying flavonoids from plants or chemically synthesizing oxymethylated flavonoids is complicated. The presence of O-methyltransferase (OMT) in plants can catalyze the methylation of hydroxylated flavonoids at specific sites, so the OMT enzymati...

Claims

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Application Information

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