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Artemisia carvifolia WRKY type transcription factor coding sequence, cloning method and application

A coding sequence and transcription factor technology, applied in the field of genetic engineering, can solve problems such as the difficulty of single gene modification and the complexity of synthetic pathways

Active Publication Date: 2016-11-09
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Considering that the synthetic pathway of plant secondary metabolites is complex, the number of genes involved in the reaction is large, and it is affected by various factors such as development and environment, it is sometimes difficult to modify a single gene in the pathway.

Method used

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  • Artemisia carvifolia WRKY type transcription factor coding sequence, cloning method and application
  • Artemisia carvifolia WRKY type transcription factor coding sequence, cloning method and application
  • Artemisia carvifolia WRKY type transcription factor coding sequence, cloning method and application

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1, the cloning of embodiment 1 Artemisia annua AaWRKY2 gene

[0045] 1. Artemisia annua was cultivated in an artificial climate chamber under the photoperiod of 18h / 6h (light / dark), 25°C;

[0046] 2. Extraction of total RNA from leaves of Artemisia annua. Take about 100 mg of tender young Artemisia annua leaf tissue material, put it in liquid nitrogen and grind it into powder, and extract the total RNA of the leaf according to the method of the plant total RNA extraction kit (Tiangen Biochemical, Beijing). 3 μL of the obtained plant total RNA was subjected to agarose gel electrophoresis to identify the quality of the total RNA, and then the concentration of the total RNA was measured on a NanoDrop (Thermo Fisher, USA) spectrophotometer.

[0047] 3. Gene cloning. Using the extracted total RNA as a template (500ng), according to the reverse transcription kit PrimeScript1st Strand cDNA Synthesis Kit (TaKaRa, Dalian) instructions, reverse transcription to pro...

Embodiment 2

[0054] Embodiment 2, the construction of the plant expression vector comprising AaWRKY2 gene

[0055] 1. Construction of the intermediate vector pENTR-TOPO-AaWRKY2.

[0056] Design and amplify the AaWRKY2 gene open reading frame sequence according to the sequence information of SEQ ID NO.1, and the amplification primers are as follows:

[0057] Forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

[0058] Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3'

[0059] The pENTR / D-TOPO vector was purchased from Invitrogen Company, and it is the entry vector of the company's Gateway cloning technology. According to the requirements of the product specification, four bases of CACC were added before the ATG base of the forward primer. Using the pLB-AaWRKY2 plasmid as a template, PCR amplification was performed with the blunt end high-fidelity enzyme KOD. After the PCR product was recovered and purified, it was connected to the pENTR-TOPO vector by Gateway cloning technology. The specific ...

Embodiment 3

[0061] Example 3. Construction of dual fluorescein reporter vectors for artemisinin synthesis pathway-specific gene promoters

[0062] 1. PCR amplification of the promoter of the specific gene of the artemisinin synthesis pathway. According to the sequence information of the ADS gene promoter (GenBank: DQ448297.1) in the NCBI database, design the ADS gene promoter amplification specific primers, the forward and reverse specific primers contain Kpn I and Pst I restriction sites respectively, and the primer sequences are as follows :

[0063] ProADS F 5'-ggtaccACCGGGGACCTCTAGAGATC-3',

[0064] ProADS R 5'-ctgcagGATTTTACAAACTTTGAA-3'.

[0065] Similarly, according to the sequence information of the CYP71AV1 gene promoter (GenBank: FJ870128.1) in the NCBI database, CYP71AV1 promoter-specific primers containing Kpn I and Pst I restriction sites were designed, and the primer sequences were as follows:

[0066] ProCYP F 5'-ggtaccATGGGTCAATTTCGGGTTG-3',

[0067] ProCYP R 5'-ctgc...

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Abstract

The invention discloses cloning of an artemisia carvifolia WRKY gene coding sequence and application of cloning. Cloning of gene AaWRKY2, construction of a plant expression vector containing the gene and an activation effect of the gene on a specific gene promoter for biosynthesis of artemisinin are specifically comprised. A nucleotide sequence of the artemisia carvifolia gene AaWRKY2 is shown as SEQ ID NO. 1, and a coded amino acid sequence is shown as SEQ ID NO. 2. The invention further discloses the characteristic of the gene AaWRKY2 that expression of two specific gene promoters ADS and DBR2 for biosynthesis of the artemisinin can be activated. The gene AaWRKY2 can be applied to quality improvement of artemisia carvifolia by excessive expression and the like, and the content of the artemisinin in the artemisia carvifolia can be increased.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an artemisia annua WRKY transcription factor coding sequence AaWRKY2 and its application. Background technique [0002] Metabolism in plants is divided into primary metabolism and secondary metabolism. Primary metabolites (such as sugars, lipids and nucleic acids) exist in all plants and are necessary for plants to maintain cell life activities. Plant secondary metabolites refer to A large class of small molecule organic compounds not necessary for plant growth and development in plants, whose synthesis and distribution are specific to species, tissues and organs, and production and development. For example, artemisinin, a specific medicinal ingredient for treating malaria, is only synthesized and stored in secretory glandular hairs on the plant surface of the plant Artemisia annua L. In recent years, with the gradual deepening of the research on the components of Ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/10C12N15/82A01H5/00
CPCC07K14/415C12N15/8243
Inventor 唐克轩沈乾王蕾江伟民郝小龙黎凌
Owner SHANGHAI JIAO TONG UNIV
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