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Artemisia apiacea WRKY type transcription factor coding sequence and application

A technology of transcription factor and coding sequence, which is applied to the coding sequence and application field of Qinghao WRKY transcription factor, can solve the problems of low feasibility of artemisinin, low content of artemisinin, and no detection of artemisinin.

Active Publication Date: 2016-11-23
唐克轩 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the main source of artemisinin is extracted from the aerial parts of Artemisia annua plants. However, the content of artemisinin in Artemisia annua is very low. The average content of artemisinin in different planting environments and planting varieties is 0.01-1% of the dry weight of leaves of Artemisia annua. , making the large-scale commercial production of this drug limited
Due to the complex structure of artemisinin, the artificial synthesis is difficult, the yield is low, and the cost is high
Although some people have successfully used yeast fermentation to produce artemisinin precursor artemisinin acid, artemisinin still needs to be artificially chemically semi-synthesized to artemisinin, and this research is still in the initial stage of laboratory research and development
There are also attempts to produce artemisinin by tissue culture and cell engineering. However, the content of artemisinin in the callus is less than 0.1% of the dry weight, and the highest in the bud is only 0.16% of the dry weight. Study detects no artemisinin in roots
Therefore, the feasibility of using tissue culture and cell engineering to produce artemisinin is not high.

Method used

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  • Artemisia apiacea WRKY type transcription factor coding sequence and application
  • Artemisia apiacea WRKY type transcription factor coding sequence and application
  • Artemisia apiacea WRKY type transcription factor coding sequence and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the cloning of Artemisia annua AaGSW2 gene

[0035] 1. Extraction of Total RNA from Artemisia annua Genome

[0036] Take the leaf tissue of Artemisia annua, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, shake it fully, and extract total RNA according to the instructions of the TIANGEN kit. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0037] 2. Cloning of Artemisia annua AaGSW2 gene

[0038] Using the extracted total RNA as a template, cDNA was synthesized under the action of PowerScript reverse transcriptase; gene-specific primers (SEQ ID NO:3 and SEQ ID NO:4) were designed according to the sequence of the AaGSW2 gene, and the total cDNA was obtained by PCR. The AaGSW2 gene was amplified and sequenced.

[0039] Through the above steps, the full-length coding sequence (SEQ ID NO: 1) of the tran...

Embodiment 2

[0040] Example 2, construction of plant binary interference expression vector containing AaGSW2 gene

[0041] In order to study the effect of AaGSW2 gene on the development of secretory glandular hairs in Artemisia annua, the overexpression vector PHB-AaGSW2 overexpressing AaGSW2 was constructed. In order to facilitate the construction of the expression vector, the restriction site of BamH1 was introduced into the forward primer, and the restriction site of Sac1 was introduced into the reverse primer. The primers are shown in Table 1;

[0042] Table 1 PCR primers constructed by PHB-AaGSW2 vector

[0043]

[0044] In this example, the Artemisia annua AaGSW2 gene is operably linked to the expression control sequence, and the vector can be used to regulate the content of artemisinin in Artemisia annua through a developmental regulation strategy.

Embodiment 3

[0045] Example 3. Agrobacterium tumefaciens-mediated AaGSW2 interference vector genetically transforms Artemisia annua to obtain transgenic Artemisia annua plants

[0046] 1. Acquisition of Agrobacterium tumefaciens Engineering Bacteria Containing AaGSW2 Overexpression Vector

[0047] The plant binary overexpression vector containing AaGSW2 in Example 2 was transformed into Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material in the market, which can be purchased from CAMBIA Company in Australia, and the strain number is Gambar 1 ), and validated by PCR. The results showed that the plant binary interference expression vector containing AaGSW2 had been successfully constructed into the Agrobacterium tumefaciens strain.

[0048] 2. Agrobacterium tumefaciens mediated AaGSW2 gene transformation of Artemisia annua

[0049] 2.1. Preculture of explants

[0050] Artemisia annua seeds were soaked in 75% ethanol for 1 min, then soaked in 20% Na...

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Abstract

The invention discloses an artemisia apiacea WRKY type transcription factor coding sequence. The coding sequence is marked as AaGSW2, the nucleotide sequence is shown by SEQ ID NO:1, and the amino acid sequence is shown by SEQ ID NO:2. According to the coded WRKY type transcription factor AaGSW2 disclosed by the invention, the artemisinin content is increased by controlling the expression of artemisinin synthesis key enzyme gene. The artemisinin content in the blades of non-transgenic common artemisia apiacea is 9mg / g DW, and the blade artemisinin content of the transgenic artemisia apiacea expressed by the over-expression AaGSW2 gene is increased to 21mg / g DW. The invention is of great significance in providing a high-yield and stable new drug resource for large-scale production of artemisinin.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a coding sequence and application of an Artemisia annua WRKY transcription factor. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin (artemisinin) is a sesquiterpene lactone compound containing a peroxide bridge structure isolated from its aerial part. It is currently the most effective drug for treating malaria in the world, especially for cerebral malaria and anti-malaria Chloroquine malaria has the characteristics of quick action and low toxicity. Currently, the most effective treatment for malaria recommended by the World Health Organization is artemisinin combination therapy (ACTs). In addition, with the deepening of pharmacological research on artemisinin, scientists have discovered that artemisinin and its derivatives also have anti-inflammatory, anti-schistosome, anti-tumor, and immune-regulati...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84C07K14/415A01H5/00
CPCC07K14/415C12N15/8205C12N15/8243
Inventor 唐克轩陈明慧颜廷祥黄悠然马亚男郝小龙
Owner 唐克轩
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