Artemisia annua flavanonol oxidase gene AaDHFO2 as well as encoding protein and application thereof

A dihydroflavone and alcohol oxidase technology, applied in the field of genetic engineering, can solve problems such as unclear regulation mechanism, and achieve the effect of reducing costs

Inactive Publication Date: 2016-02-03
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the biosynthesis and regulation mechanism of Artemisia annua flavonoids are not clear at present, and there is no report on the cloning of related key enzyme genes

Method used

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  • Artemisia annua flavanonol oxidase gene AaDHFO2 as well as encoding protein and application thereof
  • Artemisia annua flavanonol oxidase gene AaDHFO2 as well as encoding protein and application thereof
  • Artemisia annua flavanonol oxidase gene AaDHFO2 as well as encoding protein and application thereof

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Experimental program
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Embodiment 1

[0028] The acquisition of embodiment 1AaDHFO2 gene fragment

[0029] Total RNA was extracted from young leaves of Artemisia annua (HJ2013020) and Artemisia meridiana (NJ201007), respectively, and the DNA in the total RNA was digested with Qiagen’s FreeDNaseSet kit to prevent genomic DNA contamination. Take 0.5 μL of the RNA sample digested with DNA, and use a ThermoScientific company Nanodrop2000c spectrophotometer to measure the sample RNA concentration and OD260 / OD280 value. 1 μL of RNA was taken and detected by 1% Agarosegel electrophoresis to determine the integrity and purity of the RNA sample. Dilute the RNA whose integrity and purity meet the requirements to 200ng / μL for use. Take about 2 μL (2 μg) poly(A) RNA of Artemisia annua and Artemisia annua, add 1 μL of cDNAsythesisPrimer (10 mmol / L) to each, add sterile water to a total volume of 5 μL, mix well, centrifuge briefly, and perform PCR Incubate at 70°C for 2 minutes, cool on ice for 2 minutes, and centrifuge brief...

Embodiment 2

[0030] Cloning of embodiment 2AaDHFO2 gene full-length cDNA sequence

[0031] 5'-RACE primer (DHFO5) was designed according to the sequence of the obtained conserved region fragment: 5'-AGCTCAGGGCATGGACATGGTGGGTAGT-3' (SEQ ID No.3); 3'-RACE primer (DHFO3): 5'-TTGGGTTGTTGTCAAAAAGGTCTTGGACTG-3' (SEQ ID No. 4). The products of primers DHFO5 and UPM have a bright band around 1160bp (see figure 2 ), and the products of primers DHFO3 and UPM have a bright band around 1300bp (see figure 2 ). The PCR products were recovered, ligated and transformed, and among the obtained large number of clones, positive clones were selected for sequencing. By sequencing and splicing in the conserved region sequence, removing the overlapping part, the full-length deoxyribonucleotide sequence was 1978bp, and the analysis showed that the sequence contained a complete coding region of 1008bp, 5'untranslated region (UTR) of 547bp, 3' The untranslated region is 388bp and the polyA tail is 35bp, encod...

Embodiment 3 2

[0033] Example 3 Heterologous expression of dihydroflavonol oxidase gene AaDHFO2

[0034] 1. Construction of recombinant expression vector carrying Artemisia annua dihydroflavonol oxidase gene AaDHFO2

[0035] Using Artemisia annua cDNA as a template, primers were designed according to the sequence SEQIDNo.1, and the deoxyribonucleotide sequence of the coding region of the dihydroflavonol oxidase gene of Artemisia annua was amplified by PCR. EcoRI and XhoI restriction sites were introduced at both ends of the primers respectively, and the primer sequences are as follows:

[0036] Upstream: 5'-CCGGAATTCATGGAGGTGGAAAGAGTTCAAGA-3' (SEQ ID No.5), introducing an EcoRI restriction site, downstream: 5'-CCGCTCGAGTCACTGTGGAAGCTTATTTAGCTTG-3' (SEQ ID No.6), introducing an XhoI restriction site;

[0037] Perform agarose gel electrophoresis on the PCR amplification product, cut out the target band, purify and recover, connect the recovered DNA fragment to the pGEM-Teasy vector, transform...

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Abstract

The invention provides an artemisia annua flavanonol oxidase gene AaDHFO2. A nucleotide sequence of the artemisia annua flavanonol oxidase gene AaDHFO2 is shown in SEQ ID No.1, and an amino acid sequence of the encoding protein is shown in SEQ ID No.2. The artemisia annua flavanonol oxidase gene AaDHFO2 can catalyze flavanonol to form flavonol and provides a new optional approach for generation of flavonol through in-vitro catalysis of flavanonol and regulation of artemisia annua component in artemisia annua; besides, recombinant AaDHFO2 is prepared through prokaryotic expression, the difficulty of directly separating the protein from the artemisia annua is overcome, and the cost is reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an Artemisia annua dihydroflavonol oxidase gene AaDHFO2 and its encoded protein and application. Background technique [0002] Flavonoids (flavonoid) are a class of yellow pigments derived from flavone (2-phenylchromone), including isomers of flavone and their hydrogenated reduction products, that is, C6-C3 -C6 is a series of compounds of the basic carbon frame. Flavonoids are widely distributed in the plant kingdom, and most of them are combined with sugars to form glycosides or carbon sugar groups in plants, and some exist in free forms. The core of natural flavonoids often contains substituents such as hydroxyl, methoxy, alkoxy, isopentenyloxy and the like. Due to the existence of these auxiliary chromophores, the compounds are mostly yellow. And because the oxygen atom on the γ-pyrrole ring in the molecule can form a salt with a strong acid, it is weakly alkali...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/82A01H5/00C12P17/06
Inventor 刘硕谦唐雨薇田娜梁恒郑泽华熊硕龙金花
Owner HUNAN AGRICULTURAL UNIV
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