CN101560519B - Method for improving patchouli alcohol content in southernwood by pts gene and antonymous ads gene - Google Patents
Method for improving patchouli alcohol content in southernwood by pts gene and antonymous ads gene Download PDFInfo
- Publication number
- CN101560519B CN101560519B CN2009100507852A CN200910050785A CN101560519B CN 101560519 B CN101560519 B CN 101560519B CN 2009100507852 A CN2009100507852 A CN 2009100507852A CN 200910050785 A CN200910050785 A CN 200910050785A CN 101560519 B CN101560519 B CN 101560519B
- Authority
- CN
- China
- Prior art keywords
- gene
- ads
- pts
- plastid
- sweet wormwood
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
A method for improving patchouli alcohol 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 signal peptide in Arabidopsis; connecting the ads gene of the southernwood reversely and the plastid located pts gene positively to an expression regulatory sequence toform a plant expression vector including antisense ads and plastid located pts gene; transforming Agrobactrium tumefaciens by the plant expression 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 patchouli alcohol in the obtained transgenic southernwood plant. The invention establishes a method for stably improving the content of patchouli alcohol 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
The present invention relates to the method for patchouli alcohol content in a kind of raising sweet wormwood of biological technical field, specifically be a kind of pts of utilization (patchoulol synthase, the Patchoulicalcohol synthetic enzyme) gene and antonymous ads (Amorpha-4,11-dienesynthase, Amorpha-4,11-diene synthase) gene improves the method for patchouli alcohol content in the sweet wormwood.
Background technology
Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.Artemisinin is from the isolated a kind of sesquiterpene lactones compound that contains the peroxide bridge structure of sweet wormwood over-ground part, is the most effective medicine of treatment malaria of generally acknowledging in the world at present, especially has better effect for encephalic malaria and anti-chloroquine malaria.Ads is a key enzyme in the Artemisinin route of synthesis, is the important target spot of Artemisinin metabolic engineering.Adopt genetic engineering means, method with sense-rna suppresses the ads gene, to make plant not produce Artemisinin or produce Artemisinin less, and the metabolism flow point be flow on secondary meta-bolites such as other terpenes synthetic, thereby obtain producing the sweet wormwood plant of other secondary metabolites.
Patchoulicalcohol, English name Patchouli Alcohol; Be commonly called as: Patehouli aleohal; Molecular weight: 222; Molecular formula: C15H26O; Fusing point: 55 ℃-59 ℃; Solubleness: water insoluble, be soluble in organic solvents such as ethanol, acetone, sherwood oil.Patchoulicalcohol is a kind of natural monomeric compound with special aromatic odour that extracts from Wrinkled Gianthyssop Herb, be used for essence or perfumery, it is the significant composition in the definite essence and flavoring agent of the normes Francaises council, can be used for treating dermatomycosis, also can be used for the synthetic basic material of other compound, improve Patchoulicalcohol and have great commercial value and vast market prospect.Pts is a key enzyme in the Patchoulicalcohol route of synthesis in the Wrinkled Gianthyssop Herb, is the important target spot of Patchoulicalcohol metabolic engineering.Adopt genetic engineering means, transform sweet wormwood, will break the biosynthetic speed limit bottleneck of Patchoulicalcohol, obtain the sweet wormwood plant of Patchoulicalcohol high yield, for the large-scale production Patchoulicalcohol provides a new way with ground, key gene pts plastid location.
Joe Chappell etc. have delivered the paper that is entitled as " Redirection of cytosolic or plastidic isoprenoid precursorselevates terpene production in plants " (plastid or kytoplasm by the isoprene precursor in plant are shunted the output that improves terpenoid) at " Nature Biotechnology " (Nature Biotechnol) 2006 24 phase 1441-1447 page or leaf, comment in the literary composition, can make the tobacco that does not produce Patchoulicalcohol originally produce Patchoulicalcohol by overexpression Patchoulicalcohol synthetic enzyme in transgene tobacco, patchouli alcohol content reaches 0.5 microgram in every gram weight in wet base material, as seen can make the phytosynthesis Patchoulicalcohol that does not produce Patchoulicalcohol originally by metabolic regulation Patchoulicalcohol route of synthesis committed step.
Find through literature search, do not find as yet at present to improve the relevant report of patchouli alcohol content in the sweet wormwood with utilizing pts gene and antonymous ads gene to prior art.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of pts of utilization gene and antonymous ads gene to improve the method for patchouli alcohol content in the sweet wormwood.The present invention has set up the method for patchouli alcohol content in a kind of stable raising sweet wormwood, establishes solid basis for utilizing transgene abrotanum scale operation Patchoulicalcohol and the secondary metabolites such as other terpenes except that Artemisinin.
The present invention is achieved by the following technical solutions, and it is as follows to the present invention includes step:
Step 1 obtains the whole reading frame sequences of Artemisinin biosynthetic pathway key gene ads, the tp fragment of plastid localization signal peptide in whole reading frame sequences of Patchoulicalcohol biosynthetic pathway key gene pts and the Arabidopis thaliana;
Step 2, with the ads gene that obtains oppositely and the plastid located pts gene forward be connected in respectively on the expression regulation sequence, formation contains the plant expression vector of antisense ads and plastid located pts gene;
Step 3 transforms agrobacterium tumefaciens with the plant expression vector that contains antisense ads and plastid located pts gene, obtains to be used to transform the agrobacterium tumefaciens bacterial strain of sweet wormwood;
Step 4 utilizes the agrobacterium tumefaciens bacterial strain that makes up to transform the sweet wormwood explant, and PCR detects, and obtains the transgene abrotanum plant;
Step 5 is measured the patchouli alcohol content in the transgene abrotanum that obtains.
In the step 2, described plastid located pts gene is started by P35S or cyp71av1 gene promoter.
In the step 4, described PCR detects and is specially: the primer that detects antisense ads and detect plastid located pts gene is synthesized in design respectively, carries out DNA cloning, observes the positive plant of purpose band under the ultraviolet ray and is the transgene abrotanum plant.
In the step 5, described mensuration is specially: the HPLC-ELSD method is measured, and chromatographic column is a C-18 reverse phase silica gel post, and moving phase is methyl alcohol and water, 30 ℃ of column temperatures, flow velocity 1.0mL/min, sample size 10 μ L, 40 ℃ of light scattering detector drift tube temperatures, scale-up factor are 7, nebulizer gas pressure 5bar.
In the step 5, the volume ratio of methyl alcohol and water is 70: 30 in the described moving phase.
The present invention clones the entire reading frame pts sequence of Patchoulicalcohol synthase gene from Wrinkled Gianthyssop Herb, make up the plant expression vector that contains plastid located pts gene; The entire reading frame sequence of clone's Amorpha-4,11-diene synthase gene oppositely is built into plant expression vector from sweet wormwood; Utilize and Agrobacterium tumefaciens mediated plastid located pts and antisense ads gene are imported the plant and the plant that regenerates simultaneously; PCR detects the integration situation of external source goal gene plastid located pts and antisense ads, and the HPLC-ELSD method is measured patchouli alcohol content in the plant.
The present invention imports the key enzyme ads inverted defined gene and the localized Patchoulicalcohol route of synthesis of the plastid key gene pts of Artemisinin route of synthesis in the sweet wormwood plant, has obtained the transgene abrotanum plant that patchouli alcohol content significantly improves.The content of Patchoulicalcohol reaches 0.85% of weight in wet base in the transgene abrotanum plant; be 850 times of the common sweet wormwood of non-conversion, the present invention is to the Patchoulicalcohol large-scale production and utilize transgene abrotanum scale operation other terpene secondary metabolites except that Artemisinin significant.
The agrobacterium tumefaciens EHA105 that the present invention relates to is at " Huang Yali, Jiang Xiliang, Yunlong, field, Guo Ping, Zhu Changxiong; The research of Agrobacterium tumefaciens mediated trichoderma harzianum genetic transformation, Chinese biological engineering magazine, 2008,28 (3): 38-43 " open in the document.Agrobacterium tumefaciens EHA105 can obtain by disclosing commercially available commercial channel, and as buying from Australian CAMBIA company, strain number is Gambar1.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Be interpreted as: these embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, Sambrook equimolecular clone for example: laboratory manual is seen the condition described in the New York Cold Spring HarborLaboratory press version in 1989, or the condition of advising according to manufacturer.
Embodiment 1
Step 1, the clone of sweet wormwood antisense ads gene, pts gene and tp gene
1. the extraction of the total RNA of sweet wormwood genome
The sweet wormwood young leaflet tablet takes a morsel, behind liquid nitrogen flash freezer, grind with mortar rapidly, add and fill TRIzol Reagents that 1mL produces by U.S. GIBCOBRL company in the Eppendorf of 1.5mL pipe, fully after the vibration, under room temperature, place 5min, add 200 μ L chloroforms, use forced oscillation 15sec, after room temperature is placed 2min-3min, in 4 ℃, 12, the centrifugal 15min of 000g; About 600 μ L supernatant liquors are sucked in the clean 1.5mL Eppendorf pipe, add isopyknic Virahol, put upside down mixing, place 10min under the room temperature after, in 4 ℃, 12, the centrifugal 10min of 000g; Abandon supernatant, add 1mL 75% (v/v) ethanol and clean, after the vibration, in 4 ℃, 7, the centrifugal 5min of 500g; Be dissolved in behind the drying at room temperature 15min-20min in the 30 μ L-50 μ L RNAase-free water; Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
2. the extraction of the total RNA of Wrinkled Gianthyssop Herb genome
The Wrinkled Gianthyssop Herb young leaflet tablet takes a morsel, behind liquid nitrogen flash freezer, grind with mortar rapidly, add and fill TRIzol Reagents that 1mL produces by U.S. GIBCO BRL company in 1.5mL Eppendorf pipe, fully after the vibration, under room temperature, place 5min, add 200 μ L chloroforms, use forced oscillation 15sec, after room temperature is placed 2min-3min, in 4 ℃, 12, the centrifugal 15min of 000g; About 600 μ L supernatant liquors are sucked in the clean 1.5mL Eppendorf pipe, add isopyknic Virahol, put upside down mixing, place 10min under the room temperature after, in 4 ℃, 12, the centrifugal 10min of 000g; Abandon supernatant, add 1mL 75% (v/v) ethanol and clean, after the vibration, in 4 ℃, 7, the centrifugal 5min of 500g; Be dissolved in behind the drying at room temperature 15min-20min in the 30 μ L-50 μ L RNAase-free water; Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
3. the extraction of the total RNA of arabidopsis gene group
The Arabidopis thaliana young leaflet tablet takes a morsel, behind liquid nitrogen flash freezer, grind with mortar rapidly, add and fill TRIzol Reagents that 1mL produces by U.S. GIBCO BRL company in 1.5mL Eppendorf pipe, fully after the vibration, under room temperature, place 5min, add 200 μ L chloroforms, use forced oscillation 15sec, after room temperature is placed 2min-3min, in 4 ℃, 12, the centrifugal 15min of 000g; About 600 μ L supernatant liquors are sucked in the clean 1.5mL Eppendorf pipe, add isopyknic Virahol, put upside down mixing, place 10min under the room temperature after, in 4 ℃, 12, the centrifugal 10min of 000g; Abandon supernatant, add 1mL 75% (v/v) ethanol and clean, after the vibration, in 4 ℃, 7, the centrifugal 5min of 500g; Be dissolved in behind the drying at room temperature 15min-20min in the 30 μ L-50 μ L RNAase-free water; Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
4. the clone of sweet wormwood ads gene
The total RNA of sweet wormwood genome is obtained the first chain cDNA by ThermoScript II XL (AMV) reverse transcription, amplify the upstream and downstream primer of complete encoder block according to encoding sequence (the SEQ ID NO.1) design of sweet wormwood ads gene, and on the upstream and downstream primer, introduce restriction endonuclease sites SacI and BamH respectively, so that construction of expression vector.With the first chain cDNA is template, checks order behind pcr amplification.Determined dna sequence adopts 3730 automatic sequencers to finish by Shanghai English fine horse biotechnology Services Co., Ltd.Sequencing result shows, the encoding sequence (SEQ ID NO.1) of the sweet wormwood ads gene of being reported among the sequence of being cloned and the GenBank is consistent.
5. the clone of Wrinkled Gianthyssop Herb pts gene
The total RNA of Wrinkled Gianthyssop Herb genome is obtained the first chain cDNA by ThermoScript II XL (AMV) reverse transcription, amplify the upstream and downstream primer of complete encoder block according to encoding sequence (the SEQ ID NO.2) design of Wrinkled Gianthyssop Herb pts gene, and on the upstream and downstream primer, introduce restriction endonuclease sites NcoI and SacI respectively, so that construction of expression vector.With the first chain cDNA is template, checks order behind pcr amplification.Determined dna sequence adopts 3730 automatic sequencers to finish by Shanghai English fine horse biotechnology Services Co., Ltd.Sequencing result shows, the encoding sequence (SEQ ID NO.2) of the Wrinkled Gianthyssop Herb pts gene of being reported among the sequence of being cloned and the GenBank is consistent.
6. the clone of Arabidopis thaliana tp gene
The total RNA of arabidopsis gene group is obtained the first chain cDNA by ThermoScript II XL (AMV) reverse transcription, amplify the upstream and downstream primer of complete function according to encoding sequence (the SEQ ID NO.3) design of Arabidopis thaliana tp gene, and on the upstream and downstream primer, introduce restriction endonuclease sites SacI and BamHI respectively, so that construction of expression vector.With the first chain cDNA is template, checks order behind pcr amplification.Determined dna sequence adopts 3730 automatic sequencers to finish by Shanghai English fine horse biotechnology Services Co., Ltd.Sequencing result shows, the encoding sequence (SEQ ID NO.3) of the Arabidopis thaliana tp gene of being reported among the sequence of being cloned and the GenBank is consistent.
Step 2 contains the structure of the plant binary expression vector of plastid located pts and antisense ads gene
1. the structure of intermediate carrier pCAMBIA2300::p35S-gus-nos
Selecting pBI121 and pCAMBIA2300 for use is primary element, makes up double base plant expression vector pCAMBIA2300::p35S-gus-nos.Utilize HindIII and EcoRI double digestion pBI121 and pCAMBIA2300 plasmid; Reclaim GUS expression cassette and the big fragment of pCAMBIA2300 of pBI121; Connect and reclaim product, transformation and selection is taken out the plasmid enzyme restriction checking.
2. the structure of plant expression vector pCAMBIA2300::p35S-antisense ads-nos
With pCAMBIA2300::p35S-gus-nos is expression vector, oppositely replaces gus gene on it with the ads gene.Utilize BamHI/SacI double digestion pGEM T-easy+antisense ads and pCAMBIA2300::p35S-gus-nos, reclaim antisense ads and the big fragment of pCAMBIA2300::p35S-gus-nos, connect conversion, the picking mono-clonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
3. the structure of plant expression vector pCAMBIA2300::p35S-tp-pts-nos
With pCAMBIA2300::p35S-gus-nos is expression vector, replaces gus gene on it with the tp-pts gene.Utilize BamHI/SacI double digestion pGEM T-easy+tp-pts and pCAMBIA2300::p35S-gus-nos, reclaim tp-pts and the big fragment of pCAMBIA2300::p35S-gus-nos, connect conversion, the picking mono-clonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
Step 3 contains the preparation of antisense ads and plastid located pts gene double base plant expression vector agrobacterium tumefaciens engineering bacteria
The plant binary expression vector that contains antisense ads and plastid located pts gene in the step 2 is changed among the agrobacterium tumefaciens EHA105.The plasmid DNA of getting 1 μ g joins in the 200ml EHA105 competent cell, and behind the mixing, ice bath 30min places 10min for-70 ℃.At 37 ℃ of water-bath 5min or 42 ℃ of water-bath 1min, then ice bath 2min adds 28 ℃ of 800ml LB liquid nutrient mediums again, and 175rpm is coated on the LB flat board that contains 50 μ g/ml Kanamycin after shaking training 3hr, cultivates for 28 ℃ and forms single bacterium colony.
Step 4 utilizes the agrobacterium tumefaciens bacterial strain that makes up to transform the sweet wormwood explant
1. the pre-cultivation of explant
Seeds of southernwood is with 75% (v/v) alcohol immersion 1min; Use 20% (w/v) NaClO to soak 20min again; Aseptic water washing 3 times-4 times; Blot surface-moisture with aseptic thieving paper, be inoculated in the MS solid medium of no hormone, MS is invented in 1962 by Murashige and Skoog, can obtain by disclosed commercial channel; 25 ℃, cultivate the night on 16 hour daytime and 8 hours, cultivated 3 days, can obtain the sweet wormwood aseptic seedling, treats that seedling grows to 5cm, and clip aseptic seedling leaf explant is used for transforming.
2. the common cultivation of Agrobacterium and explant
Leaf explant is forwarded in the common culture medium of 1/2MS and 100 μ mol/L AS composition, dropping contains the 1/2MS suspension of the agrobacterium tumefaciens engineering bacteria of the good described antisense of containing ads of activation and pts gene plant binary expression vector, explant is fully contacted with bacterium liquid, 28 ℃ of dark 3d that cultivate are contrast with the leaf explant that drips at the 1/2MS of the agrobacterium tumefaciens that does not have goal gene liquid nutrient medium suspension.
3. the screening of resistance regeneration plant
The sweet wormwood explant of cultivating 3d altogether is transferred on the germination screening culture medium of MS, 0.5mg/L 6-BA, 0.05mg/L NAA, 50mg/LKan and 500mg/L Cb composition, in 25 ℃, the 16h/8h illumination condition is cultivated down, per two all succeeding transfer culture once, through obtaining the Kan resistance bud of growing thickly behind 2 times-3 times subcultures.Well-grown resistance bud of growing thickly is cut and changed 1/2MSO and 125mg/L Cb over to and form to be cultured on the root media and take root, thereby obtain Kan resistance regeneration sweet wormwood plant.
4. the PCR of transgene abrotanum plant detects
Design the forward primer design respectively and reverse primer detects goal gene according to goal gene place expression cassette p35s-antisense ads-nos sequence p35s and antisense ads.The result shows, utilizes designed PCR special primer, can amplify the specific DNA fragment of 1882bp, and when being template with non-conversion sweet wormwood genomic dna, does not amplify any fragment.
Design the forward primer design respectively and reverse primer detects goal gene according to goal gene place expression cassette p35s-tp-pts-nos sequence p35s and tp-pts.The result shows, utilizes designed PCR special primer, can amplify the specific DNA fragment of 2966bp, and when being template with non-conversion sweet wormwood genomic dna, does not amplify any fragment.
Step 5, the HPLC-ELSD method is measured patchouli alcohol content in the transgene abrotanum
1. the preparation of HPLC-ELSD condition and system suitability and standardized solution
HPLC: adopt water alliance 2695 systems, chromatographic column is a C-18 reverse phase silica gel post, and this silicagel column is the SymmetryShieldTM C18 that is produced by Waters company, and specification is specially: thickness 5 μ m, and length * internal diameter is 250mm * 4.6mm; Moving phase is methyl alcohol and water, and wherein the volume ratio of methyl alcohol and water is 70: 30, and this moment, the retention time of Patchoulicalcohol was 5.1min, and the peak type is good; 30 ℃ of column temperatures; Flow velocity 1.0mL/min; Sample size 10 μ L; Sensitivity AUFS=1.0; Theoretical plate number is calculated by the Patchoulicalcohol peak and is not less than 2000.
ELSD: adopt water alliance 2420 systems, 40 ℃ of light scattering detector drift tube temperatures, scale-up factor are 7, nebulizer gas pressure 5bar;
Accurately take by weighing the Patchoulicalcohol standard substance that 2.0mg Sigma company produces and dissolve fully, obtain 2mg/mL Patchoulicalcohol standard solution with 1mL methyl alcohol, be stored in-20 ℃ standby.
2. the making of typical curve
With reference substance solution difference sample introduction 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l under corresponding chromatographic condition, record collection of illustrative plates and chromatographic parameter carry out regression analysis with peak area (Y) to standard substance content (X, μ g) respectively.The result indicates that Patchoulicalcohol presents good log-log linear relationship in 4 μ g-20 μ g scopes, and the log-log equation of linear regression of Patchoulicalcohol reference substance is: Y=1.28e+000X+4.71e+000, R=0.979546.
3. the mensuration of the preparation of sample and patchouli alcohol content
The leaching process of Patchoulicalcohol is based on rolling up the 2nd phase 180-187 page or leaf " Journal of Chromatography A " (chromatogram magazine A) 23 days the 1118th June in 2006 by people's reported method such as Van Nieuwerburgh: get the fresh sweet wormwood blade of 1g-2g fresh weight, in the 50ml test tube, it is immersed in and swayed in the 10ml chloroform 1 minute, leach liquor poured into make chloroform volatilization fully in the new test tube, get the 3ml dehydrated alcohol and fully dissolve extract, be used for HPLC and detect.Adopt high performance liquid chromatography and light scattering detector (HPLC-ELSD) to measure patchouli alcohol content, the sample feeding volume is 20 μ l, go out patchouli alcohol content (mg) in the sample according to the linear regression equation calculation of peak area substitution, again divided by the artemisia leaf weight in wet base (g) of sample, thereby calculate the content of Patchoulicalcohol in the sweet wormwood plant.
The result shows that the content of Patchoulicalcohol reaches 0.8% of sample weight in wet base in the present embodiment sweet wormwood, is 800 times of the common sweet wormwood of non-conversion.
Embodiment 2
Step 1, step 3 and step 5 are all with embodiment 1 in the present embodiment, and 1 difference of present embodiment and embodiment is:
1. with 2. with embodiment 1, difference is in the step 2,
3. the structure of intermediate carrier pCAMBIA2300::cyp71av1 promoter-gus-nos
With pCAMBIA2300::p35S-gus-nos is expression vector, replaces P35S gene on it with cyp71av1 promoter gene.Utilize PstI/BamHI double digestion pGEM T-easy+cyp71av1 promoter and pCAMBIA2300::p35S-gus-nos, reclaim cyp71av1 promoter and the big fragment of pCAMBIA2300::p35S-gus-nos, connect and transform, the picking mono-clonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
4. the structure of plant expression vector pCAMBIA2300::cyp71av1 promoter-tp-pts-nos
With pCAMBIA2300::cyp71av1 promoter-gus-nos is expression vector, replaces gus gene on it with the tp-pts gene.Utilize BamHI/SacI double digestion pGEM T-easy+tp-pts and pCAMBIA2300::cyp71av1promoter-gus-nos, reclaim tp-pts and the big fragment of pCAMBIA2300::cyp71av1 promoter-gus-nos, connect and transform, the picking mono-clonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
1., 2. and 3. with embodiment 1, difference is in the step 4,
4. the PCR of transgene abrotanum plant detects
Design the forward primer design respectively and reverse primer detects goal gene according to goal gene place expression cassette cyp71av1 promoter-antisense ads-nos sequence cyp71av1promoter and antisense ads.The result shows, utilizes designed PCR special primer, can amplify the specific DNA fragment of 1914bp, and when being template with non-conversion sweet wormwood genomic dna, does not amplify any fragment.
Design the forward primer design respectively and reverse primer detects goal gene according to goal gene place expression cassette cyp71av1 promoter-tp-pts-nos sequence cyp71av1 promoter and tp-pts.The result shows, utilizes designed PCR special primer, can amplify the specific DNA fragment of 2998bp, and when being template with non-conversion sweet wormwood genomic dna, does not amplify any fragment.
The result shows that the content of Patchoulicalcohol reaches 0.85% of sample weight in wet base in the present embodiment sweet wormwood, is 850 times of the common sweet wormwood of non-conversion.
Sequence table
＜110〉Shanghai Communications University
＜120〉utilize pts gene and antonymous ads gene to improve the method for patchouli alcohol content in the sweet wormwood
<160>3
<170>PatentIn?version?3.1
<210>1
<211>1641
<212>DNA
＜213〉sweet wormwood (Artemisia annua L.)
<400>1
atgtcactta?cagaagaaaa?acctattcgc?cccattgcca?actttcctcc?aagcatttgg 60
ggagatcagt?ttctcatcta?tgaaaagcaa?gtagagcaag?gggtggaaca?gatagtgaat?120
gatttaaaaa?aagaagtgcg?gcaactacta?aaagaagctt?tggatattcc?tatgaaacat?180
gccaatttat?tgaagctgat?tgatgaaatc?caacgccttg?gaataccgta?tcactttgaa?240
cgggagattg?atcatgcatt?gcaatgtatt?tatgaaacat?atggtgataa?ctggaatggt?300
gaccgctctt?ccttatggtt?ccgtcttatg?cgaaagcaag?gatattatgt?tacatgtgat?360
gttttcaata?actataaaga?caaaaatgga?gcgttcaagc?aatcgttagc?taatgatgtt?420
gaaggtttgc?ttgagttgta?cgaagcaact?tctatgaggg?tacctgggga?gattatatta?480
gaagatgctc?ttggttttac?acgatctcgt?cttagcatta?tgacaaaaga?tgctttttct?540
acaaaccccg?ctctttttac?cgaaatacaa?cgggcactaa?agcaacccct?ttggaaaagg?600
ttgccaagaa?tagaggcggc?gcagtacatt?cctttctatc?aacaacaaga?ttctcataac?660
aagactttac?ttaaacttgc?taagttagag?ttcaatttgc?ttcagtcatt?gcacaaggaa?720
gagctcagcc?atgtgtgcaa?atggtggaaa?gctttcgata?tcaagaagaa?cgcaccttgt?780
ttaagagata?gaattgttga?atgctacttt?tggggactag?gttcaggcta?tgagccacag?840
tattcccggg?ctagagtttt?cttcacaaaa?gctgttgctg?ttataactct?tatagatgac?900
acttatgatg?cgtatggtac?ttatgaagaa?cttaagatct?ttactgaagc?tgttgaaagg?960
tggtcaatta?catgcttaga?cacacttcca?gaatacatga?aaccgatata?caaattattc?1020
atggatacat?acacagaaat?ggaagaattt?cttgcaaagg?agggaagaac?agatctattt?1080
aactgcggca?aagaatttgt?gaaagagttt?gttagaaacc?tgatggttga?agcaaaatgg?1140
gcaaatgagg?gacacatacc?aaccactgaa?gagcatgatc?cagttgtaat?cattactggc?1200
ggtgctaacc?tgcttacaac?aacttgttat?cttggcatga?gtgatatatt?cacaaaagag?1260
tctgtcgaat?gggctgtctc?tgcacctcct?ctttttagat?actcaggtat?acttggtcga?1320
cgcctaaatg?atctcatgac?ccacaaggcc?gagcaagaaa?gaaaacatag?ttcatcgagc?1380
cttgaaagtt?atatgaagga?atataatgtc?aatgaggagt?atgcccaaac?cttgatttac?1440
aaggaagtag?aagatgtgtg?gaaagatata?aaccgagagt?acctcacaac?taaaaacatt?1500
ccaaggccgt?tattgatggc?tgtgatctat?ttgtgccagt?ttcttgaagt?tcaatatgca?1560
ggaaaggata?acttcacacg?tatgggagac?gaatacaaac?atctcataaa?gtctctactc?1620
gtttatccta?tgagtatatg?a 1641
＜110〉Shanghai Communications University
＜120〉utilize pts gene and antonymous ads gene to improve the method for patchouli alcohol content in the sweet wormwood
<160>3
<170>PatentIn?version?3.1
<210>2
<211>1689
<212>DNA
＜213〉Wrinkled Gianthyssop Herb (Pogostemon cablin)
<400>2
gggggaagat?ctagctgcat?gaaggagctc?atggagttgt?atgcccaaag?tgttggagtg 60
ggtgctgctt?ctcgtcctct?tgcgaatttt?catccatgtg?tgtggggaga?caaattcatt?120
gtctacaacc?cacaatcatg?ccaggctgga?gagagagaag?aggctgagga?gctgaaagtg?180
gagctgaaaa?gagagctgaa?ggaagcatca?gacaactaca?tgcggcaact?gaaaatggtg?240
gatgcaatac?aacgattagg?cattgactat?ctttttgtgg?aagatgttga?tgaagctttg?300
aagaatctgt?ttgaaatgtt?tgatgctttc?tgcaagaata?atcatgacat?gcacgccact?360
gctctcagct?ttcgccttct?cagacaacat?ggatacagag?tttcatgtga?agtttttgaa?420
aagtttaagg?atggcaaaga?tggatttaag?gttccaaatg?aggatggagc?ggttgcagtc?480
cttgaattct?tcgaagccac?gcatctcaga?gtccatggag?aagacgtcct?tgataatgct?540
tttgacttca?ctaggaacta?cttggaatca?gtctatgcaa?ctttgaacga?tccaaccgcg?600
aaacaagtcc?acaacgcatt?gaatgagttc?tcttttcgaa?gaggattgcc?acgcgtggaa?660
gcaaggaagt?acatatcaat?ctacgagcaa?tacgcatctc?atcacaaagg?cttgctcaaa?720
cttgctaagc?tggatttcaa?cttggtacaa?gctttgcaca?gaagggagct?gagtgaagat?780
tctaggtggt?ggaagacttt?acaagtgccc?acaaagctat?cattcgttag?agatcgattg?840
gtggagtcct?acttctgggc?ttcgggatct?tatttcgaac?cgaattattc?ggtagctagg?900
atgattttag?caaaagggct?ggctgtatta?tctcttatgg?atgatgtgta?tgatgcatat?960
ggtacttttg?aggaattaca?aatgttcaca?gatgcaatcg?aaaggtggga?tgcttcatgt?1020
ttagataaac?ttccagatta?catgaaaata?gtatacaagg?cccttttgga?tgtgtttgag?1080
gaagttgacg?aggagttgat?caagctaggc?gcaccatatc?gagcctacta?tggaaaagaa?1140
gccatgaaat?acgccgcgag?agcttacatg?gaagaggccc?aatggaggga?gcaaaagcac?1200
aaacccacaa?ccaaggagta?tatgaagctg?gcaaccaaga?catgtggcta?cataactcta?1260
ataatattat?catgtcttgg?agtggaagag?ggcattgtga?ccaaagaagc?cttcgattgg?1320
gtgttctccc?gacctccttt?catcgaggct?acattaatca?ttgccaggct?cgtcaatgat?1380
attacaggac?acgagtttga?gaaaaaacga?gagcacgttc?gcactgcagt?agaatgctac?1440
atggaagagc?acaaagtggg?gaagcaagag?gtggtgtctg?aattctacaa?ccaaatggag?1500
tcagcatgga?aggacattaa?tgaggggttc?ctcagaccag?ttgaatttcc?aatccctcta?1560
ctttatctta?ttctcaattc?agtccgaaca?cttgaggtta?tttacaaaga?gggcgattcg?1620
tatacacacg?tgggtcctgc?aatgcaaaac?atcatcaagc?agttgtacct?tcaccctgtt?1680
ccatattaa 1689
＜110〉Shanghai Communications University
＜120〉utilize pts gene and antonymous ads gene to improve the method for patchouli alcohol content in the sweet wormwood
<160>3
<170>PatentIn?version?3.1
<210>3
<211>177
<212>DNA
＜213〉Arabidopis thaliana (Aribdopsis)
<400>3
atggcttcct?ctatgctctc?ctccgccgct?gtggttacat?ccccggctca?ggccaccatg 60
gtcgctccat?tcaccggctt?gaagtcatcc?gctgcattcc?cggtcacccg?caagaccaac?120
aaggacatca?cttccatcgc?aagcaacggg?ggaagatcta?gctgcatgaa?ggagctc 177
Claims (5)
1. a method of utilizing pts gene and antonymous ads gene to improve patchouli alcohol content in the sweet wormwood is characterized in that, comprises that step is as follows:
Step 1 obtains the whole reading frame sequences of Artemisinin biosynthetic pathway key gene ads, the tp fragment of plastid localization signal peptide in whole reading frame sequences of Patchoulicalcohol biosynthetic pathway key gene pts and the Arabidopis thaliana;
The tp fragment of plastid localization signal peptide is shown in Seq ID No.3 in the described Arabidopis thaliana;
Step 2, with the ads gene that obtains oppositely and the plastid located pts gene forward be connected in respectively on the expression regulation sequence, formation contains the plant expression vector of antisense ads and plastid located pts gene;
Step 3 transforms agrobacterium tumefaciens with the plant expression vector that contains antisense ads and plastid located pts gene, obtains to be used to transform the agrobacterium tumefaciens bacterial strain of sweet wormwood;
Step 4 utilizes the agrobacterium tumefaciens bacterial strain that makes up to transform the sweet wormwood explant, and PCR detects, and obtains the transgene abrotanum plant;
Step 5 is measured the patchouli alcohol content in the transgene abrotanum that obtains.
2. the method for utilizing pts gene and antonymous ads gene to improve patchouli alcohol content in the sweet wormwood according to claim 1 is characterized in that, in the step 2, described plastid located pts gene is started by P35S or cyp71av1 gene promoter.
3. the method for utilizing pts gene and antonymous ads gene to improve patchouli alcohol content in the sweet wormwood according to claim 1, it is characterized in that, in the step 4, described PCR detects and is specially: the primer that detects antisense ads and detect plastid located pts gene is synthesized in design respectively, carry out DNA cloning, observing the positive plant of purpose band is the transgene abrotanum plant down in ultraviolet ray.
4. the method for utilizing pts gene and antonymous ads gene to improve patchouli alcohol content in the sweet wormwood according to claim 1, it is characterized in that in the step 5, described mensuration is specially: the HPLC-ELSD method is measured, chromatographic column is a C-18 reverse phase silica gel post, moving phase is methyl alcohol and water, 30 ℃ of column temperatures, flow velocity 1.0mL/min, sample size 10 μ L, 40 ℃ of light scattering detector drift tube temperatures, scale-up factor are 7, nebulizer gas pressure 5bar.
5. the method for utilizing pts gene and antonymous ads gene to improve patchouli alcohol content in the sweet wormwood according to claim 4 is characterized in that the volume ratio of methyl alcohol and water is 70: 30 in the described moving phase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100507852A CN101560519B (en) | 2009-05-07 | 2009-05-07 | Method for improving patchouli alcohol content in southernwood by pts gene and antonymous ads gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100507852A CN101560519B (en) | 2009-05-07 | 2009-05-07 | Method for improving patchouli alcohol content in southernwood by pts gene and antonymous ads gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101560519A CN101560519A (en) | 2009-10-21 |
| CN101560519B true CN101560519B (en) | 2011-01-12 |
Family
ID=41219495
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100507852A Expired - Fee Related CN101560519B (en) | 2009-05-07 | 2009-05-07 | Method for improving patchouli alcohol content in southernwood by pts gene and antonymous ads gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101560519B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925441B (en) * | 2012-10-11 | 2014-06-18 | 上海交通大学 | AaORA gene promoter and application thereof |
| CN107475265A (en) * | 2017-09-26 | 2017-12-15 | 上海交通大学 | Nucleotide sequence, carrier and the method for improving content of artemisinin in sweet wormwood |
| CN116267609B (en) * | 2023-03-06 | 2023-10-13 | 广东药科大学 | Seedling cultivation method for improving yield of patchouli and application thereof |
-
2009
- 2009-05-07 CN CN2009100507852A patent/CN101560519B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101560519A (en) | 2009-10-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101182544B (en) | Method for enhancing arteannuin content in southernwood by transforming ads gene | |
| CN103958673B (en) | Isopropylmalate synthase from nicotiana tabacum, and methods and uses thereof | |
| CN104152463B (en) | Coding sequence of AaMYBL1 protein of artemisia apiacea and application thereof | |
| CN102676578B (en) | Method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer | |
| CN102776225A (en) | Method for increasing artemisinin content of sweet wormwood by transferring AaWRKY1 gene | |
| CN102242145A (en) | Method for improving artemisinin content in artemisia annua L through transferring allene oxide cyclase (AOC) gene | |
| US8476492B2 (en) | Method of utilizing the pts gene and anti-sense ads to increase patchouli alcohol content in Artemisia annua L | |
| CN101597620B (en) | Method for improving the content of artemisia annua patchouli calcohol by utilizing pts gene and RNA interferon ads gene | |
| Argôlo et al. | The regulation of solasodine production by Agrobacterium rhizogenes-transformed roots of Solanum aviculare | |
| US8476491B2 (en) | Method of utilizing the PTS gene and RNA interference of the ads gene to increase patchouli alcohol content in Artemisia annua L. | |
| CN101560519B (en) | Method for improving patchouli alcohol content in southernwood by pts gene and antonymous ads gene | |
| CN101182543B (en) | Method for enhancing arteannuin content in southernwood using gene cyp71av1 and cpr co-transformation | |
| CN102643838A (en) | Method for improving content of artemisinin in artemisia apiacea by tran-ALDH1 gene | |
| CN101182545B (en) | Method for enhancing arteannuin content in southernwood using gene hmgr and fps co-transformation | |
| CN102776212A (en) | Production method of high-artemisinin-content transgene sweet wormwood plants | |
| CN105646686A (en) | Gene for regulating and controlling synthesis of plant flavonol and application | |
| CN102604987A (en) | Method for improving artemisinin content in Artemisia annua L. by DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene transfer | |
| CN104017797A (en) | Mutant and application of momordica grosvenori SgCAS gene | |
| CN106480088A (en) | A kind of method for improving content of artemisinin in sweet wormwood | |
| CN102558325B (en) | The preparation method of sweet wormwood AaORA albumen and encoding gene, transgene abrotanum plant | |
| CN102533804B (en) | Artemisia sphaerocephala krasch delta 12 fatty acid dehydrogenase (As flavin adenine dinucleotide 2 (FAD2)) gene and application | |
| CN105936914B (en) | The albumen and application of asparagus chalcone synthase genes and its coding | |
| Hosseini et al. | Transient Expression of cor Gene in Papaver somniferum | |
| ES2358643T3 (en) | POPULATION OF TRANSGENIC PLANTS, MATERIAL DERIVED FROM THE SAME, COLLECTION OF CORRESPONDING PLASID AND POPULATION OF TRANSFORMED GUESTS ORGANISMS, AS WELL AS THEIR USE AND METHODS FOR THEIR GENERATION. | |
| CN105713921A (en) | Method for increasing content of artemisinin in Artemisia annua L. by genetic modification with CHI (chalcone Isomerase) genes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: FIRMENICH FLAVOR(CHINA) CO., LTD. Free format text: FORMER OWNER: SHANGHAI JIAO TONG UNIVERSITY Effective date: 20100809 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 200240 NO.800, DONGCHUAN ROAD, MINXING DISTRICT, SHANGHAI CITY TO: 201108 NO.3901, JINDU ROAD, SHENZHUANG INDUSTRY PARK, MINXING DISTRICT, SHANGHAI CITY |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20100809 Address after: 201108, No. 3901, Jin Du Road, Xhenzhuang Industrial Park, Shanghai, Minhang District Applicant after: Firmenich Aromatics (China) Co .,Ltd. Address before: 200240 Dongchuan Road, Shanghai, No. 800, No. Applicant before: Shanghai Jiao Tong University |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110112 Termination date: 20180507 |