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Method for improving patchouli alcohol content in southernwood by pts gene and antonymous ads gene
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A technology of patchouli alcohol and gene, which is applied in the field of increasing the content of patchouli alcohol in Artemisia annua, and can solve the problem that the content of patchouli alcohol has not been found yet
Inactive Publication Date: 2011-01-12
FIRMENICH AROMATICS (CHINA0 CO LTD
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[0005] After searching the literature of the prior art, it is found that there is no report related to the use of pts gene and antisense ads gene to increase the content of patchouli alcohol in Artemisia annua
Method used
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[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 for 15min at 4°C; pipette about 600μL of the supernatant into a clean 1.5mL Eppendorf tube, add an equal volume of isopropanol, and invert to mix Evenly, place at room temperature for 10 minutes, centrifuge at 4°C, 12,000g for 10min; discard the supernatant, add 1mL 75% (v / v) ethanol to wash, shake, then centrifuge at 4°C, 7,500g for 5min; dry at room temperature for 15min-20min Dissolve in 30μL-50μL RNAase-...
Embodiment 2
[0065] In this embodiment, step one, step three and step five are all the same as embodiment 1, and the difference between this embodiment and embodiment 1 is:
[0066] In step 2, ① and ② are the same as in Example 1, the difference is that,
[0067]③ Construction of the intermediate vector pCAMBIA2300::cyp71av1 promoter-gus-nos
[0068] 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 enzymedigestionverification.
[0069] ④Construction of plant expression vector pCAMBIA2300::cyp71av1 promoter-tp-pts-nos
[0070] 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 pCA...
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Abstract
A method for improving patchoulialcohol content in southernwood by pts gene and antonymous ads gene specifically comprises the steps of obtaining all reading frame sequences of genes ads and pts, andtp segments of plastid located signalpeptide in Arabidopsis; connecting the ads gene of the southernwood reversely and the plastid located pts gene positively to an expression regulatory sequence toform a plantexpression vector including antisense ads and plastid located pts gene; transforming Agrobactrium tumefaciens by the plantexpression vector including antisense ads and plastid located pts gene; transforming a southernwood explant by the constructed Agrobactrium tumefaciens strain to obtain a transgenic southernwood plant; and measuring the content of patchoulialcohol in the obtained transgenic southernwood plant. The invention establishes a method for stably improving the content of patchoulialcohol and lays a firm foundation for mass production of patchouli alcohol and othersecondary metabolites such as terpene except artemisinine by the transgenic southernwood.
Description
technical field [0001] The present invention relates to a method for increasing the content of patchouli alcohol in Artemisia annua in the field of biotechnology, specifically a method utilizing pts (patchoulol synthase, patchouli alcohol synthase) gene and antisense ads (Amorpha-4, 11-dienesynthase, Amorphadiene 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 sesquiterpenelactone 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 artemisininmetabolic engineering. Using genetic engineering means, using antisense RNA to in...
Claims
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