Artemisia annua L. bZIP class transcription factor coding sequence and 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 annua L. bZIP class transcription factor coding sequence and cloning method and application
  • Artemisia annua L. bZIP class transcription factor coding sequence and cloning method and application
  • Artemisia annua L. bZIP class transcription factor coding sequence and cloning method and application

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1, the cloning of embodiment 1 Artemisia annua AabZIP9 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 AabZIP9 gene

[0057] 1. Construction of the intermediate vector pENTR-TOPO-AabZIP9.

[0058] Design and amplify the AabZIP9 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'-CACCATGGCCGCAAACCCTGTCTGG-3'

[0060] Reverse primer P4: 5'-AACGTTACGATGAGAATCCAAAGG-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-AabZIP9 plasmid as a template, the blunt end high-fidelity enzyme KOD was used for PCR amplification. After the PCR product was recovered and purified, it was connected to the pENTR-TOPO vector by the method of Gateway cloning technology. ...

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 cloning and application of an artemisia annua L. bZIP class transcription factor coding sequence. Cloning particularly comprises cloning of a gene AabZIP9, construction of a plant expression vector containing the gene and activation of the gene on a gene promoter special for an artemisinin biosynthetic pathway. A nucleotide sequence of the artemisia annua L. AabZIP9 gene is as shown in SEQ ID NO.1, and a coded nucleotide sequence of the artemisia annua L. AabZIP9 gene is as shown in SEQ ID NO.2. The invention further discloses an AabZIP9 gene which has characteristics of being capable of activating artemisinin biosynthetic pathway specific gene ADS and ALDH1 expression. The AabZIP9 gene can be used for improving the artemisia annua L. quality and can improve the content of artemisinin in artemisia annua L..

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an Artemisia annua bZIP-type transcription factor coding sequence AabZIP9 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/10C12N15/82A01H5/00
CPCC07K14/415C12N15/8243
Inventor 唐克轩沈乾黄华仪颜廷祥陈明慧何倩
Owner SHANGHAI JIAO TONG UNIV
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