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Artemisia apiacea bZIP-type transcription factor encoding sequence, cloning method and application

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

Active Publication Date: 2016-10-26
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 apiacea bZIP-type transcription factor encoding sequence, cloning method and application
  • Artemisia apiacea bZIP-type transcription factor encoding sequence, cloning method and application
  • Artemisia apiacea bZIP-type transcription factor encoding sequence, cloning method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the cloning of embodiment 1 Artemisia annua AabZIP6 gene

[0047] 1. Artemisia annua was cultivated in an artificial climate chamber under the photoperiod of 18h / 6h (light / dark), 25°C;

[0048] 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.

[0049] 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

[0056] Embodiment 2, the construction of the plant expression vector comprising AabZIP6 gene

[0057] 1. Construction of the intermediate vector pENTR-TOPO-AabZIP6.

[0058] Design and amplify the AabZIP6 gene open reading frame sequence according to the sequence information of SEQ ID NO.1, and the amplification primers are as follows:

[0059] Forward primer P3: 5'-CACCATGTCGAGGCCACCTCGACTTCC-3'

[0060] Reverse primer P4: 5'-GTTAATATGAAGCTTGCCCATGTC-3'

[0061] 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-AabZIP6 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 the method of Gateway cloning tech...

Embodiment 3

[0063] Example 3. Construction of dual fluorescein reporter vectors for artemisinin synthesis pathway-specific gene promoters

[0064] 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 :

[0065] ProADS F 5'-ggtaccACCGGGGACCTCTAGAGATC-3',

[0066] ProADS R 5'-ctgcagGATTTTACAAACTTTGAA-3'.

[0067] 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:

[0068] ProCYP F 5'-ggtaccATGGGTCAATTTCGGGTTG-3',

[0069] ProCYP R 5'-ctgc...

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Abstract

The invention discloses clone of an artemisia apiacea AabZIP6 gene encoding sequence and an application thereof, and particularly includes clone of the AabZIP6 gene, establishment of a plant expression vector containing the gene, and an activation effect of the gene on a gene promoter being specific in a bio-synthetic route of artemisinin. The nucleic acid sequence of the artemisia apiacea AabZIP6 gene is represented as the SEQ ID No.1, and an amino acid sequence encoded by the gene is represented as the SEQ ID No.2. The invention also discloses a character that the AabZIP6 gene can activate expression of the gene promoters, comprising ADS and ALDH1, which are specific in the bio-synthetic route of artemisinin. The AabZIP6 gene is applied in quality improvement of the artemisia apiacea and can increase the content of the artemisinin in the artemisia apiacea.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an Artemisia annua bZIP-like transcription factor coding sequence AabZIP6 and its cloning method and 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...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N15/10C12N15/66C12Q1/66C07K14/415A01H5/00
CPCC07K14/415C12N15/66C12N15/8205C12N15/8243C12Q1/686C12Q2521/107
Inventor 唐克轩沈乾马亚男吕宗友潘琪芳唐岳立
Owner SHANGHAI JIAO TONG UNIV
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