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Production method of high-artemisinin-content transgene sweet wormwood plants

A production method and artemisinin technology, applied in the fields of botanical equipment and methods, plant products, measuring devices, etc., can solve the problems of difficulty in artificial synthesis, restrictions on large-scale commercial production, and no detection of artemisinin, etc., To achieve the effect of stabilizing the new drug source

Inactive Publication Date: 2012-11-14
SHANGHAI JIAO TONG UNIV
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  • 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, which limits the large-scale commercial production of this drug.
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 callus is less than 0.1% of dry weight, and the highest in shoot is only 0.16% of 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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  • Production method of high-artemisinin-content transgene sweet wormwood plants
  • Production method of high-artemisinin-content transgene sweet wormwood plants
  • Production method of high-artemisinin-content transgene sweet wormwood plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1, Cloning of Artemisia annua ADS, CYP71AV1 and CPR Three Genes

[0022] 1.1 Extraction of total RNA from Artemisia annua genome

[0023] 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.

[0024] 1.2 Cloning of three genes of Artemisia annua ADS, CYP71AV1 and CPR

[0025] The obtained Artemisia annua genome total RNA was reverse-transcribed by reverse transcriptase XL (AMV) to obtain the first-strand cDNA, according to the coding sequences of the three genes of Artemisia annua ADS, CYP71AV1 and CPR (respectively as SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3), design the upstream and downstream primers to amplify the complete coding frame,...

Embodiment 2

[0027] Embodiment 2, containing ADS, the construction of the plant binary expression vector of CYP71AV1 and CPR three genes

[0028] 2.1 Construction of intermediate vector pMD18-p35S-gfp-gus-nos

[0029] The intermediate vector pMD18-p35S-gfp-gus-nos was constructed by selecting pMD18-T and pCAMBIA1304 as basic components. Specifically, a pair of primers were designed according to the sequence of p35S-gfp-gus-nos on pCAMBIA1304, and restriction endonuclease sites were introduced on the upstream and downstream primers respectively, so as to construct the expression vector. Using the pCAMBIA1304 plasmid as a template, the expression cassette of the gfp-gus fusion gene was amplified by PCR, connected to the pMD18-T vector, transformed and screened, and the single clone was picked and sequenced to confirm that it was correct.

[0030] 2.2 Construction of intermediate vector

[0031] Construct pMD18-p35S-ads-nos, pMD18-p35S-cyp71av1-nos, pMD18-p35S-cpr-nos, based on the pMD18-...

Embodiment 3

[0039] Example 3, Agrobacterium tumefaciens mediated genetic transformation of ADS, CYP71AV1 and CPR three genes to obtain transgenes in Artemisia annua Artemisia annua plant

[0040] 3.1 Acquisition of Agrobacterium tumefaciens Engineering Bacteria Containing ADS, CYP71AV1 and CPR Triple-gene Binary Plant Expression Vector

[0041] In Example 2, the plant binary expression vector containing ADS, CYP71AV1 and CPR three genes is transformed into Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material in the market, which can be purchased from Australia's CAMBIA company, and the strain number is Gambar 1), and perform PCR verification. The results showed that the plant binary expression vector containing ADS, CYP71AV1 and CPR three genes has been successfully constructed into the Agrobacterium tumefaciens strain.

[0042] 3.2 Agrobacterium tumefaciens mediates ADS, CYP71AV1 and CPR three gene vectors to transform Artemisia annua

[0043...

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Abstract

The invention relates to a production method of high-artemisinin-content transgene sweet wormwood plants in the biological technical field. The production method comprises the following steps that three genes including ADS, CYP71AV1 and CPR are cloned from sweet wormwoods, plant expression carriers containing the ADS, CYP71AV1 and CPR genes are built, agrobacterium tumefaciens are used for mediating, the ADS, CYP71AV1 and CPR genes are transferred into the sweet wormwoods, in addition, the plants are regenerated, the screening is carried out, and the transgene sweet wormwood plants with the improved artemisinin content are obtained. When the uninverted ordinary artemisinin content is 6.43mg / g DW, the artemisinin content in the obtained transgene sweet wormwood plant system is as high as 15.09 mg / g DW, and is 2.35 times of the content in the uninverted ordinary artemisinin. The method has the important significance on providing high-yield and stable new medicine sources for the large-scale production of the artemisinin.

Description

technical field [0001] The invention relates to a method for increasing the content of artemisinin in the field of biotechnology, in particular to a method for producing transgenic Artemisia annua plants with high artemisinin content. 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 gradual deepening of pharmacological research on artemisinin, scientists have found that artemisinin and its derivatives also have anti-inflammatory, ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/82C12Q1/68A01H5/00G01N30/02
Inventor 唐克轩陆续张凌沈乾高尔娣江伟民张芳源吕宗友
Owner SHANGHAI JIAO TONG UNIV
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