Artemisia annua aagtd1 gene and its encoded protein and application

A kind of gene coding, Artemisia annua technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem that there is no report on the AaGTD1 gene protein of Artemisia annua

Active Publication Date: 2018-02-09
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] After searching the literature of the prior art, it is found that there is no report on the AaGTD1 gene and its encoded protein of Artemisia annua

Method used

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  • Artemisia annua aagtd1 gene and its encoded protein and application
  • Artemisia annua aagtd1 gene and its encoded protein and application
  • Artemisia annua aagtd1 gene and its encoded protein and application

Examples

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Effect test

Embodiment 1

[0043] Example 1: Cloning of Artemisia annua AaGTD1 gene

[0044] 1. Extraction of Total RNA from Artemisia annua Genome

[0045] Take an appropriate amount of fresh Artemisia annua leaves and quickly grind them into powder in liquid nitrogen, then take about 100mg of the powder and add it to a 1.5ml EP tube pre-filled with plant tissue lysate, shake and mix well, and then extract total plant RNA according to TIANGEN RNAprep Pure The kit's instructions were used to extract total RNA from Artemisia annua. The quality of RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA concentration was determined on a spectrophotometer.

[0046] 2. Cloning of the AaGTD1 gene of Artemisia annua

[0047] Using the extracted total RNA as a template, cDNA of Artemisia annua was synthesized using the Full-Form Gold TansScript First-Strand cDNASynthesis Supermix kit.

[0048] Gene-specific primers were designed according to the sequence of the AaGTD1 gene:

[00...

Embodiment 2

[0053] Example 2: Spatiotemporal expression analysis of Artemisia annua AaGTD1 gene

[0054] 1. Material preparation

[0055] According to the extraction method of total RNA used in Example 1, different parts of Artemisia annua were extracted, including: roots, stems, old leaves, young leaves, early flower buds, flower buds before flowering, total RNA of flowers in full flowering stage, and reversed cDNA to obtain materials for spatial expression analysis; at the same time, according to the above method, obtain RNA and cDNA from leaves of different parts of Artemisia annua growing to a height of 45-55 cm, and obtain materials for AaGTD1 gene expression analysis in leaves of different developmental stages (such as figure 2 shown).

[0056] 2. Real-time fluorescent quantitative PCR analysis

[0057] Quantitative PCR primers (Table 1) across introns of AaGTD1 gene and Actin internal reference gene were designed by primer 5, and real-time fluorescent quantitative PCR analysis w...

Embodiment 3

[0061] Example 3: Analysis of subcellular localization of Artemisia annua AaGTD1 gene

[0062] According to the content of the bioinformatics analysis in Example 1, the AaGTD1 gene is an AP2 / ERF type transcription factor with an AP2 domain. In order to further verify the nature of AaGTD1 gene transcription factor, we constructed the subcellular localization vector of AaGTD1 gene, and confirmed that AaGTD1 is localized in the nucleus by transforming rice protoplasts, which conforms to the characteristics of AaGTD1 gene transcription factor.

[0063] 1. Construction of subcellular localization vector

[0064] In this example, the forward primer was designed as subGTD-F:aaCCATGGGA atgggtcaaaagaagtttag (SEQ ID NO:9), containing the NCO I restriction site, and the reverse primer was subGTD-R:aaACTAGTATTCGTATTAAGCAATTCTT (SEQ ID NO:10), Contains Spe I restriction site. Perform PCR, enzyme digestion and ligation to finally obtain the AaGTD1 gene subcellular localization vector with...

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Abstract

The invention relates to the field of biotechnology, in particular to an AaGTD1 gene and its encoded protein in Artemisia annua controlling the synthesis of artemisinin and the development of glandular hairs and its application in the preparation of artemisinin. The present invention provides an Artemisia annua AaGTD1 gene, the nucleotide sequence of which is shown in SEQ ID NO:1; the amino acid sequence of the protein AP2 / ERF transcription factor encoded by the gene is shown in SEQ ID NO:2. The application described in the present invention refers to a method for increasing the content of artemisinin in Artemisia annua through AaGTD1 involved in the present invention, comprising the following steps: transforming the plant expression vector comprising the gene shown in SEQ ID NO:1 into Artemisia annua cells: cultivating transformed Artemisia annua cells to obtain Artemisia annua plants with increased artemisinin content. The protein encoded by the AaGTD1 gene of the present invention can be used to increase the yield of artemisinin, and it is of great significance to provide high-yield and stable plant materials for the large-scale production of artemisinin.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an AaGTD1 gene and its encoded protein in Artemisia annua controlling the synthesis of artemisinin and the development of glandular hairs and its application in the preparation of artemisinin. Background technique [0002] Malaria is a global disease that threatens the health of approximately half of the world's population. In 2010 there were approximately 219 million malaria cases and 660 000 deaths. According to the WHO report in 2013, 99 countries and regions have persistent malaria transmission. Non-immune travelers from malaria-free areas become particularly ill after infection. The best available treatment for malaria, especially P. falciparum, is an artemisinin-based combination therapy. Artemisinin is a sesquiterpene lactone peroxide synthesized from the glandular hairs of the medicinal plant Artemisia annua in my country, and Artemisia annua is the only natural source of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
Inventor 谭何新张磊肖玲
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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