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Artemisia annua flavanone 3-hydroxylase gene AaF3HY as well as encoded protein and application thereof

A technology of Artemisia annua and flavanones, applied in the field of genetic engineering, can solve problems such as unclear regulation mechanism, and achieve the effect of reducing cost

Inactive Publication Date: 2015-04-29
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 flavanone 3-hydroxylase gene AaF3HY as well as encoded protein and application thereof
  • Artemisia annua flavanone 3-hydroxylase gene AaF3HY as well as encoded protein and application thereof
  • Artemisia annua flavanone 3-hydroxylase gene AaF3HY as well as encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Obtaining of AaF3HY Gene Fragment

[0028]Total RNA was extracted from young leaves of Artemisia annua with high flavonoid content (HF2010001) and Artemisia annua with low flavonoid content (LF2009001), and the DNA in the total RNA was digested with Qiagen’s Free DNase Set kit to prevent genomic DNA contamination. Take 0.5 μL of RNA sample digested with DNA, and use a Thermo Scientific Nanodrop 2000c spectrophotometer to measure the sample RNA concentration and OD260 / OD280 value. 1 μL of RNA was taken and detected by 1% Agarose gel 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. Double-strand cDNA was digested with EcoRI and MseI, and the EcoRI adapter sequences used for the ligation reaction were 5'-CTCGTAGACTGCGTACC-3'(SEQ ID No.3) and 3'-CTGACGCATGGTTAA-5'(SEQ ID No.4 ), the MseI linker sequences are 5'-GACGATGAGTCCTGAG-3' (SEQ ID No.5) and 3'-TAC...

Embodiment 2

[0029] Example 2 Cloning of the full-length cDNA sequence of AaF3HY gene

[0030] Design the 5'-RACE primer (F3HY5) according to the sequence of the obtained conserved region fragment:

[0031] 5'-TCATCTTCTTTCTTATACATGTCCGTG-3'(SEQ ID No.9); 3'-RACE primer (F3HY3):

[0032] 5'-TATCAATAGCAACCTTTCAAAACCCT-3' (SEQ ID No. 10).

[0033] The product of primers F3HY5 and UPM has a bright band around 1400bp (see image 3 ), and the product of primers F3HY3 and UPM has a bright band around 1600bp (see image 3 ). 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 2136bp, and the analysis showed that the sequence contained a complete coding region of 1095bp, 5'untranslated region (UTR) of 563bp, 3' The untranslated region is 441bp and the polyA ...

Embodiment 3

[0035] Example 3 Heterologous expression of flavanone 3-hydroxylase gene AaF3HY

[0036] 1. Construction of a recombinant expression vector carrying the flavanone 3-hydroxylase gene AaF3HY of Artemisia annua

[0037] Using Artemisia annua cDNA as a template, primers were designed according to the sequence SEQ ID No.1, and the deoxyribonucleotide sequence of the coding region of Artemisia annua flavanone 3-hydroxylase gene was amplified by PCR. BamHI and Xho I restriction sites were introduced at both ends of the primers respectively, and the primer sequences are as follows:

[0038] Upstream: 5'-CGCCATATG GCACCTATATCCTTAAAATGGGAC-3' (SEQ ID No.11), introducing an NdeI restriction site, downstream: 5'-TGGGAGCTCCTAAGCAAAGATACTTTCAATGGGC-3' (SEQ ID No.12), introducing a SacI restriction site;

[0039] 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-T easy vector, tra...

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Abstract

The invention provides an artemisia annua flavanone 3-hydroxylase gene AaF3HY, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO.1, and the amino acid sequence of encoded protein of the gene is as shown in SEQ ID NO.2. The artemisia annua flavanone 3-hydroxylase gene AaF3HY can be used for catalyzing flavanone so as to form 3-hydroxyl flavanone, therefore a new available way is offered for generating the 3-hydroxyl flavanone through in vitro catalysis of the flavanone and for the in vivo regulation of artemisia annua components of artemisia annua. In addition, the recombinant AaF3HY is prepared by virtue of prokaryotic expression, so that the difficulty of isolating the protein directly from the artemisia annua is solved and cost is reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an Artemisia annua flavanone 3-hydroxylase gene AaF3HY and its encoded protein and application. Background technique [0002] Artemisia annua L., also known as Artemisia annua L., is a plant of the genus Artemisia in the family Asteraceae. The leaves, flowers and stems of Artemisia annua L. are rich in flavonoids. Flavonoids are a class of compounds that exist in nature and have a 2-phenylchromone (flavone) structure, including isomers of flavones and their hydrogenated reduction products, that is, C6-C3 -C6 is a series of compounds of the basic carbon frame. 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. There is a ketone carbonyl group in their molecules, and the oxygen atom at the first position is bas...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/82C12P17/12
Inventor 田娜曹娟易庆峰熊硕杨威田冬铭
Owner HUNAN AGRICULTURAL UNIV
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