Method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene

A kind of artemisinin and transgenic technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problem of low artemisinin content, large-scale commercial production restrictions, and low feasibility of artemisinin problem, to achieve the effect of increasing the content of artemisinin

Inactive Publication Date: 2012-11-14
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] At present, the main source of artemisinin is extracted from the above-ground 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, so it is not feasible
Some people try 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. Artemisinin was not detected in roots
Therefore, the feasibility of using tissue culture and cell engineering to produce artemisinin is not high.

Method used

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  • Method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene
  • Method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Cloning of AaWRKY1 Gene of Artemisia annua

[0027] 1.1 Extraction of total RNA from Artemisia annua genome

[0028] Take 100-200mg young leaves of Artemisia annua, quick-freeze them with liquid nitrogen, grind them quickly with a mortar, add them into a 1.5mL Eppendorf tube filled with 1mL TRlzol (TRlzol Reagents, GIBCOBRL, USA), shake them fully, and place them at room temperature Stand for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 sec, place at room temperature for 2-3 minutes, then centrifuge at 12,000 rpm for 15 minutes at 4°C; suck the supernatant (about 600 μL) into a clean 1.5 mL Eppendorf tube, add an equal volume of isopropyl Alcohol, mix it upside down, place it at room temperature for 10 minutes, then centrifuge at 12,000rmp for 10min at 4°C; discard the supernatant, add 1mL of 75% ethanol to wash, shake, and centrifuge at 7,500rmp for 5min at 4°C; dry at room temperature for 10-15min and dissolve In an appropriate amount (30-40 μL) of RN...

Embodiment 2

[0033] Construction of Plant Binary Expression Vector Containing AaWRKY1 Gene

[0034] 2.1 Construction of intermediate vector pMD18T-AaWRKY1

[0035]The pMD18T-simple vector (Takara, Dalian) was selected as the basic element to construct the intermediate vector pMD18T-AaWRKY1. Specifically, the full length of the gene with KpnI and SacI restriction sites introduced before and after the AaWRKY1 gene was amplified by high-fidelity enzymes, connected to the pMD18T vector by ligase, and the correctness of the gene was confirmed by Shanghai Invitrogen Company sequencing.

[0036] 2.2 Construction of plant expression vector FSN::p35S-AaWRKY1-NOS

[0037] Using the FSN (FSN expression vector transformed from the pCAMBIA2300 expression vector) as the expression vector, the AaWRKY1 gene in Example 1 was connected to its corresponding restriction site. Specifically, KpnI and SacI double-digested the intermediate vector pMD18T-AaWRKY1 and the expression vector FSN. The AaWRKY1 gene f...

Embodiment 3

[0040] Agrobacterium tumefaciens mediated AaWRKY1 gene genetic transformation of Artemisia annua to obtain transgenic Artemisia annua plants

[0041] 3.1 Obtaining the AaWRKY1 gene-containing binary plant expression vector Agrobacterium tumefaciens engineering bacteria

[0042] The plant binary expression vector containing the AaWRKY1 gene 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 Gambar1), and PCR was carried out verify.

[0043] 3.2 Agrobacterium tumefaciens mediated AaWRKY1 gene transformation of Artemisia annua

[0044] (1) Pre-cultivation of explants

[0045] Artemisia annua seeds were soaked in 75% ethanol for 1 min, then soaked in 20% NaClO for 20 min, rinsed with sterile water for 3 to 4 times, blotted the surface water with sterile absorbent paper, and inoculated in hormone-free MS (Mura...

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Abstract

The invention discloses a method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene. The method includes the steps of cloning WRKY type transcription factor AaWRKY1 gene in sweet wormwood; constructing a plant expression vector containing the AaWRKY1; introducing the AaWRKY1 gene into the sweet wormwood to regrow a plant under mediation of Agrobacterium Tumefaciens; performing PCR (polymerase chain reaction) to detect integration status of the foreign target gene AaWRKY1; measuring the content of artemisinin in the transgenic sweet wormwood by efficient liquid chromatography and an evaporative light scattering detector, and screening to obtain the plants of sweet wormwood with artemisinin content increased. The content of artemisinin in the transgenic sweet wormwood with the AaWRKY1 gene is increased to 24.59mg / g DW by the method and is 4.44 times of that of the non-transgenic sweet wormwood. The method provides new high-yield and stable drug sources for large-scale production of artemisinin.

Description

technical field [0001] The invention relates to a method for increasing the content of artemisinin in Artemisia annua by using transgenic technology, in particular to a method for increasing the content of artemisinin in Artemisia annua by transgenic AaWRKY1 gene. 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29C12Q1/68G01N30/02A01H5/00
Inventor 唐克轩江伟民陆续邱波王国丰
Owner SHANGHAI JIAO TONG UNIV
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