Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Artemisia-apiacea bHLH transcription factor coded sequence and clone method and application thereof

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: 2016-12-14
SHANGHAI JIAO TONG UNIV
View PDF3 Cites 4 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Artemisia-apiacea bHLH transcription factor coded sequence and clone method and application thereof
  • Artemisia-apiacea bHLH transcription factor coded sequence and clone method and application thereof
  • Artemisia-apiacea bHLH transcription factor coded sequence and clone method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the cloning of embodiment 1 Artemisia annua AaMYC3 gene

[0046] 1. Artemisia annua was cultivated in an artificial climate chamber under the photoperiod of 18h / 6h (light / dark), 25°C;

[0047] 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.

[0048] 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 prod...

Embodiment 2

[0055] Embodiment 2, the construction of the plant expression vector comprising AaMYC3 gene

[0056] 1. Construction of the intermediate vector pENTR-TOPO-AaMYC3.

[0057] Design and amplify the AaMYC3 gene open reading frame sequence according to the sequence information of SEQ ID NO.1, and the amplification primers are as follows:

[0058] Forward primer P3: 5'-CACCATGGATGATGATTTCCTAATTC-3'

[0059] Reverse primer P4: 5'-CTGATTTAATCTAGCTAGAAGATG-3'

[0060] 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-AaMYC3 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 specifi...

Embodiment 3

[0062] Example 3. Construction of dual fluorescein reporter vectors for artemisinin synthesis pathway-specific gene promoters

[0063] 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 :

[0064] ProADS F 5'-ggtaccACCGGGGACCTCTAGAGATC-3',

[0065] ProADS R 5'-ctgcagGATTTTACAAACTTTGAA-3'.

[0066] 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:

[0067] ProCYP F 5'-ggtaccATGGGTCAATTTCGGGTTG-3',

[0068] ProCYP R 5'-ctgc...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a cloning method and application of an artemisia-apiacea AaMYC3 gene coded sequence. The cloning method includes the specific steps of cloning of a gene AaMYC3; constructing of a plant expression vector with the gene; the activation effect of the gene on a specific gene promoter of an artemisinin biosynthetic pathway. The nucleotide sequence of the artemisia-apiacea AaMYC3 gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO.2. The invention further discloses the application of the AaMYC3 gene to activating expression of the four specific gene promoters including ADS, CYP71AV1, DBR2 and ALDH1 of the artemisinin biosynthetic pathway. According to the cloning method an application, the AaMYC3 gene can be applied to artemisia apiacea with the RNAi method, the antisense inhibiting method and the like, quality is improved, and the content of artemisinin in artemisia apiacea can be increased.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an Artemisia annua bHLH transcription factor coding sequence AaMYC3 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 Chi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/415C12N15/82C12N15/66C12N1/21A01H5/00
CPCC07K14/415C12N15/8205C12N15/8243
Inventor 唐克轩沈乾赵彧张婷婷孙小芬
Owner SHANGHAI JIAO TONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com