Hawthorn fruit cyanidin-3-hydroxyl glycosyl transferase and encoding genes and application thereof

A cyanidin, encoding technology, applied in the field of genetic engineering, can solve the problems of low content, unstable anthocyanins, and high price

Active Publication Date: 2016-06-15
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the instability of anthocyanins, it has caused difficulties in chemical separation, resulting in extremely high market prices for s...

Method used

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  • Hawthorn fruit cyanidin-3-hydroxyl glycosyl transferase and encoding genes and application thereof
  • Hawthorn fruit cyanidin-3-hydroxyl glycosyl transferase and encoding genes and application thereof
  • Hawthorn fruit cyanidin-3-hydroxyl glycosyl transferase and encoding genes and application thereof

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1, cloning of cyanidin-3-O-galactosidyl transferase gene Cp3OGT

[0035] The total RNA of hawthorn fruit was extracted by the improved CTAB method, and the cDNA obtained by reverse transcription was used as a template. The forward primer P1: 5'ATGGCACCGCCGCAGCCCACCG3' and the reverse primer P2: 5'CTATGCTTTATTGGATCCTGAT3' were used for PCR amplification to obtain a 1.4kb PCR product .

[0036] The above amplification system (10 μL) was as follows: 2 μL of 5×PSGXLBuffer, 0.8 μL of dNTPs (2.5 mmol L-1), 0.2 μL of each of primers P1 and P2, 0.2 μL of PrimeSTARGXL, 1 μL of template, and the rest was supplemented with sterile double-distilled water. Reaction conditions: pre-denaturation at 98°C for 3 min; 40 cycles at 98°C for 10 s, annealing at 55°C for 15 s, extension at 68°C for 1 min and 30 s; extension at 68°C for 5 min, and storage at 4°C.

[0037] After sequencing, the 1.4kb PCR product has the nucleotides shown in sequence 1 in the sequence table, the open...

Embodiment 2

[0038] Example 2. Functional research of cyanidin-3-O-galactosidyl transferase gene Cp3OGT

[0039] 1. Preparation of recombinant vector

[0040] Extract the total RNA of hawthorn fruit, reverse transcribe to obtain cDNA as a template, and use the following primers:

[0041] Upstream primer P3: 5'GTGGACAGCAAATGGGTCGCGGATCCATGGCACCGCCGCAGCCCACCG3' (SEQ ID NO: 3) and downstream primer P4: 5' TGTCGACGGAGCTCGAATTCCTATGCTTTATTGGATCCTGAT3' (SEQ ID NO: 4) were used for PCR amplification.

[0042] The PCR reaction system (100 μL) was: 20 μL of 5×PSGXLBuffer, 8 μL of dNTPs (2.5 mmol L-1), 2 μL of primers P1 and P2, 2 μL of PrimeSTARGXL, 2 μL of template, and the rest were supplemented with sterile double-distilled water. PCR reaction conditions: pre-denaturation at 98°C for 3min; 40 cycles at 98°C for 10s, annealing at 55°C for 15s, extension at 68°C for 1min and 30s; extension at 68°C for 5min, and storage at 4°C.

[0043] The obtained 1.4kb PCR product is the Cp3OGT gene.

[0044]...

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Abstract

The invention discloses hawthorn fruit cyanidin-3-hydroxyl glycosyl transferase and encoding genes and application thereof. The invention provides protein. The protein is protein as shown in sequence 2 in a sequence table or protein derived from the sequence 2. The protein derived from the sequence 2 has the same function of the protein as shown in the sequence 2 and is obtained by subjecting the amino acid sequence as shown in the sequence 2 in the sequence table to one or more amino acid residue substitution and/or loss and/or adding. Hawthorn fruit cyanidin-3-O-galactoside transferase (Cp3OGT) genes are cloned from hawthorn fruit for the first time. In-vitro experiments show that Cp3OGT has the activity for catalyzing cyanidin and UDP-D-galactose to generate cyanidin-3-O-galactoside.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to hawthorn fruit cyanidin-3-hydroxyglycosyltransferase and its coding gene and application. Background technique [0002] Anthocyanins belong to flavonoids, which are water-soluble plant pigments widely distributed in nature. They have various biological activities such as anti-oxidation, anti-mutation, anti-tumor, and prevention and treatment of cardiovascular diseases. Previous studies have shown that the anthocyanins in hawthorn fruit are mainly two kinds of anthocyanins, cyanidin 3-O-β-galactoside and cyanidin-3-O-α-arabinoside, which were separated by chemical Means of isolation to obtain its monomeric compounds. Due to the instability of anthocyanins, chemical separation is difficult, resulting in extremely high market prices of anthocyanin standard products; some types of anthocyanins cannot be obtained by chemical separation due to their extremely l...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/11C12P19/60
CPCC12N9/1051C12P19/60C12Y204/01254
Inventor 黄璐琦袁媛唐金富杨滨闫述模
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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