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Method for improving the content of artemisia annua patchouli calcohol by utilizing pts gene and RNA interferon ads gene

A technology of RNA interference and patchouli alcohol, which is applied in the field of increasing the content of patchouli alcohol in Artemisia annua, can solve the problems of increasing the content of patchouli alcohol and not yet discovering the ads gene

Inactive Publication Date: 2011-07-27
FIRMENICH AROMATICS (CHINA0 CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching the literature of the prior art, it is found that there is no report related to the use of the pts gene and the ads gene of RNA interference to increase the content of patchouli alcohol

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Step 1, cloning of Artemisia annua adsi gene, pts gene, tp gene

[0023] ① Extraction of total RNA from Artemisia annua genome

[0024] Take a small amount of young leaves of Artemisia annua, quick-freeze them with liquid nitrogen, grind them quickly with a mortar, add 1 mL of TRIzol Reagents produced by GIBCOBRL Company in the United States to a 1.5 mL Eppendorf tube, shake them fully, and place them at room temperature for 5 min. Add 200 μL of chloroform, shake vigorously for 15 sec, place at room temperature for 2min-3min, then centrifuge at 12,000g at 4°C for 15min; pipette about 600μL of the supernatant into a clean 1.5mL Eppendorf tube, add an equal volume of isopropanol, and mix by inversion After standing at room temperature for 10 minutes, centrifuge at 4°C and 12,000g for 10 minutes; discard the supernatant, add 1mL of 75% (v / v) ethanol to wash, shake, and centrifuge at 4°C and 7,500g for 5 minutes; dry at room temperature for 15min-20min Dissolve in 30μL-50μ...

Embodiment 2

[0067] In this embodiment, step 1, step 3 and step 5 are all the same as in embodiment 1, and the difference between this embodiment and embodiment 1 is that ①, ② and ③ in step 2 are all the same as in embodiment 1, and the difference is that

[0068] ④ Construction of intermediate vector pCAMBIA2300::cyp71av1 promoter-gus-nos

[0069] Using pCAMBIA2300::p35S-gus-nos as the expression vector, replace the P35S gene with the cyp71av1 promoter gene. Use PstI / BamHI to double digest pGEM T-easy+cyp71av1 promoter and pCAMBIA2300::p35S-gus-nos, recover cyp71av1 promoter and pCAMBIA2300::p35S-gus-nos large fragments, ligate and transform, pick single clones, and extract plasmids to make PCR detection and enzyme digestion verification.

[0070] ⑤Construction of plant expression vector pCAMBIA2300::cyp71av1 promoter-tp-pts-nos

[0071] Using pCAMBIA2300::cyp71av1 promoter-gus-nos as the expression vector, replace the gus gene with the tp-pts gene. Digest pGEM T-easy+tp-pts and pCAMBI...

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Abstract

The invention discloses a method for improving the content of artemisia annua patchouli calcohol by utilizing pts gene and RNA interferon ads gene, including the following specific steps: obtaining the partial sequence of gene ads and all reading frame sequence of gene pts and tp segment of plasmid positioning signal titanium in arabidopsis; connecting the partial sequence of gene ads and the plasmid positioned pts gene building the hairpin structure to the expression regulating sequence respectively, forming the plant expressing carrier containing the adsi and plasmid positioned pts gene; utilizing the built plate expressing carrier to transform the agrobactrium tumefaciens; utilizing the bacterial strain of built agrobactrium tumefaciens to transform the artemisia annua explant; measuring the content of patchouli calcohol in the transgene artemisia annua by HPLS-ELSD. The invention establishes a method for improving the content of patchouli calcohol, thereby laying a strong foundation for producing the patchouli calcohol in large scale by utilizing the transgene artemisia annua and the other terpene secondary metabolites except for the artemisinin.

Description

technical field [0001] The invention relates to a method for increasing the content of patchouli alcohol in Artemisia annua in the field of biotechnology, in particular to a method utilizing pts (patchoulol synthase, patchoulol synthase) gene and RNA interference ads (Amorpha-4, 11-dienesynthase, purple Sophoradiene synthase) gene to increase the content of patchouli alcohol in Artemisia annua. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin is a sesquiterpene lactone compound containing a peroxide bridge structure isolated from the aerial part of Artemisia annua. It is currently recognized as the most effective drug for treating malaria in the world, especially for cerebral malaria and chloroquine-resistant malaria. better effect. Ads is a key enzyme in the synthetic pathway of artemisinin and an important target of artemisinin metabolic engineering. Using genetic engineering means, using antisense RNA to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12Q1/68G01N30/02
Inventor 唐克轩王玥月唐岳立
Owner FIRMENICH AROMATICS (CHINA0 CO LTD
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