CN110438148A - A method of content of artemisinin in sweet wormwood is improved using AaSPL2 gene - Google Patents
A method of content of artemisinin in sweet wormwood is improved using AaSPL2 gene Download PDFInfo
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Abstract
A method of content of artemisinin in sweet wormwood is improved using AaSPL2 gene, include the following steps: to clone salicylic acid signal pathway AaSPL2 gene from sweet wormwood, construct the plant expression vector of the gene containing AaSPL2, with Agrobacterium tumefaciens mediated, AaSPL2 gene is transferred to sweet wormwood and regenerates plant, PCR detects the integration of external source target gene AaSPL2, then measures the content of qinghaosu in transgene abrotanum, and screening obtains the transgene abrotanum plant that artemislnin content improves.The content of qinghaosu significantly improves in the transgene abrotanum that the present invention obtains, up to the 1.8 of nontransformed control plant times, lays the foundation for qinghaosu is mass produced using transgene abrotanum.
Description
Technical field
The invention belongs to field of biotechnology.A kind of method for improving artemislnin content is particularly related to, in particular to
It is a kind of method for improving content of artemisinin in sweet wormwood using AaSPL2 gene.
Background technique
Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.Qinghaosu
(artemisinin) it is a kind of sesquiterpene lactone containing peroxide bridge structure separated from its aerial part, is current
The drug of generally acknowledged most effective treatment malaria in the world has quick-acting and low especially for encephalic malaria and anti-chlorine quinoline malaria
The feature of poison.Currently, the method for the most effective treatment malaria of world health organisation recommendations is exactly qinghaosu conjoint therapy
(ACTs).In addition, it is anti-that scientist has found that qinghaosu and its derivative also have with gradually going deep into qinghaosu pharmacological research
Inflammation, anti-schistosome, antitumor and immunoregulatory function.It can be seen that qinghaosu is a kind of natural drug of great potential.
The main source of qinghaosu is extracted from the aerial part of sweet wormwood plant at present, however the content of Artemisinin in Artemisia annuna
Very low (0.01%-1%), so that the large-scale commercial production of this drug is restricted.Since qinghaosu structure is multiple
Miscellaneous, artificial synthesized difficulty is big, and low output is at high cost, does not have feasibility.Have tried to the side with tissue cultures and cell engineering
Method produces qinghaosu, however qinghaosu content in callus is lower than the 0.1% of dry weight, and highest also only has dry weight in bud
0.16%, and most of research does not detect qinghaosu in root.Therefore it is produced using tissue cultures and cell engineering
The feasibility of qinghaosu is not also high.
Through to existing technical literature retrieve find, Qian Shen etc. " New Phytologist " (" new plant scholar ",
1269-1281 pages of 210 phase in 2016) deliver entitled " The jasmonate-responsive
AaMYC2transcription factor positively regulates artemisinin biosynthesis in
Artemisia annua " (" is closed by the biology that the AaMYC2 transcription factor that methyl jasmonic acid responds just is regulating and controlling Artemisinin in Artemisia annuna
At ") paper, report mentions artemislnin content by being overexpressed the AaMYC2 that is responded by hormone methyl jasmonic acid in sweet wormwood
It is 1.6 times high.Therefore, it is provided by being overexpressed the gene responded by methyl jasmonic acid to improve the content of Artemisinin in Artemisia annuna
One feasible method.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for improving content of artemisinin in sweet wormwood using AaSPL2 gene, will be green
The AaSPL2 gene cloned in wormwood artemisia, which is transferred in sweet wormwood, obtains transgenosis AaSPL2 sweet wormwood, sweet wormwood in transgenosis AaSPL2 sweet wormwood
Cellulose content significantly improves, and qinghaosu is mass produced using sweet wormwood to realize.
The present invention is achieved by the following technical solutions: the present invention clones AaSPL2 gene from sweet wormwood, and building contains
The plant expression vector of AaSPL2 gene, use is Agrobacterium tumefaciens mediated, by AaSPL2 channel genes sweet wormwood and regenerates plant；
PCR detects the integration of external source target gene AaSPL2, HPLC ELSD detector (HPLC-ELSD)
Content of artemisinin in sweet wormwood is measured, screening obtains the transgene abrotanum plant that artemislnin content improves.
The method provided by the invention for improving content of artemisinin in sweet wormwood using AaSPL2 gene, includes the following steps:
S1: sweet wormwood key gene AaSPL2 is obtained using gene clone method；
S2: AaSPL2 gene is operatively connectable to expression regulation sequence, constructs the plant table of the gene containing AaSPL2
Up to carrier；
S3: the plant expression vector of the gene containing AaSPL2 is converted into Agrobacterium tumefaciems, is obtained for converting containing for sweet wormwood
The Agrobacterium tumefaciens strain of AaSPL2 gene plant expression vector；
S4: sweet wormwood is converted using constructed Agrobacterium tumefaciens strain, obtains the transgene abrotanum through PCR test positive
Plant；
S5: HPLC-ELSD measurement is carried out to artemislnin content in the transgene abrotanum of acquisition, screening obtains artemislnin content
The transgene abrotanum plant significantly improved.
Preferably, step S1: sweet wormwood genome total serum IgE is extracted；Sweet wormwood genome total serum IgE is obtained first by reverse transcription
Chain cDNA；According to the design of the coded sequence of the sweet wormwood AaSPL2 gene as shown in SEQ ID NO.1 amplifies complete encoder block
Upstream and downstream primer, and restriction endonuclease sites are introduced respectively on upstream and downstream primer, so as to construction of expression vector；With institute
Stating the first chain cDNA is template, is sequenced after PCR amplification, obtains and correct sweet wormwood AaSPL2 gene is sequenced, and wherein PCR expands
The nucleotide sequence for increasing upstream and downstream primer used is respectively as follows: AaSPL2-PF:atggagtggaattgggacaa, AaSPL2-
PR:ttaatttgaacaaaagtagtcaaaa.
Preferably, in step S2, the plant expression vector of building gene containing AaSPL2 includes the following steps: to select
PBI121 and pCAMBIA2300 is primary element, constructs binary plant expression vector pCAMBIA2300::p35S-gus-nos；
BamHI and SacI double digestion carrier pCAMBIA2300::p35S-gus-nos is used again, recycles pCAMBIA2300::p35S-nos
Large fragment will be formed by seamless Cloning Kit in AaSPL2 gene swapping to pCAMBIA2300::p35S-nos
PCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos, conversion, picking monoclonal extract plasmid and do
PCR detection and digestion verification.
It is furthermore preferred that the building binary plant expression vector pCAMBIA2300::p35S-gus-nos includes following step
It is rapid: to use HindIII and EcoRI double digestion pBI121 and pCAMBIA2300 plasmid；Recycle pBI121 gus expression cassette and
PCAMBIA2300 large fragment；Recovery product is connected, transformation and selection takes out plasmid enzyme restriction verifying.
Preferably, described to convert the preculture for including the following steps: explant in step S4；Agrobacterium and explant
It co-cultures；The screening of resistance regeneration plant.
It is furthermore preferred that the preculture of the explant includes the following steps: that 75% ethyl alcohol of seeds of southernwood impregnates 1min, then
20min is impregnated with 20%NaClO, aseptic water washing 3-4 times blots surface moisture with sterile blotting paper, is inoculated in no hormone
In MS solid medium, illumination cultivation 16h, dark culture 8h, can be obtained sweet wormwood aseptic seedling at 25 DEG C, long to 5~8cm to seedling
Afterwards, clip tests for sterility explant is for converting.
It is furthermore preferred that the co-cultivation of the Agrobacterium and explant includes the following steps: the tests for sterility explant
Body is gone in co-culture medium, is added dropwise containing the activated binary expression vector of gene plant containing AaSPL2
The 1/2MS of the Agrobacterium tumefaciems engineering bacteria of pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos is outstanding
Liquid comes into full contact with explant with bacterium solution, 28 DEG C of dark culture 3d.
It is furthermore preferred that include the following steps: will be outside the sweet wormwood of the described co-cultivation 3d for the screening of the resistance regeneration plant
Implant is transferred on germination screening and culturing medium illumination cultivation 16h, dark culture 8h at 25 DEG C, and squamous subculture is primary every two weeks, warp
It can be obtained Kan resistance Multiple Buds after crossing 2-3 subculture, well-grown resistance Multiple Buds cut and are transferred on root media
Culture obtains Kan resistance and regenerates sweet wormwood plant to taking root.
Preferably, in step S4, the method for the PCR detection are as follows: separately design synthesis expression cassette p35s-AaSPL2-nos
The specific primer of AaSPL2 gene carries out PCR amplification in sequence, observes purpose in the UV lamp after agarose gel electrophoresis
The positive strain of band is transgene abrotanum plant；The specific primer includes upstream primer, downstream primer, particular sequence
It is respectively as follows: PF:gagagactggtgatttcagcg, PR:ttaatttgaacaaaagtagtcaaaa.
In step S5, the HPLC-ELSD measures content of artemisinin in sweet wormwood, method are as follows: chromatographic column C-18 reverse phase silicon
Rubber column gel column, mobile phase are methanol: water, methanol: the volume ratio of water be 70:30,30 DEG C of column temperature, flow velocity 1.0mL/min, 10 μ of sample volume
L, 40 DEG C of evaporative light scattering detector drift tube temperature, amplification coefficient (gain) is 7, nebulizer gas pressure 5bar.
Agrobacterium tumefaciems used is the biomaterial that there is public offering in market in step S3 of the present invention, can be from more companies
As Australian CAMBIA company buys.
The present invention also provides sweet wormwood AaSPL2 genes to improve the application in content of artemisinin in sweet wormwood.
This invention demonstrates that AaSPL2 gene is the important transcription factor of salicylic acid signal pathway and qinghaosu metabolic engineering
Important target spot.The present invention has broken the limit of qinghaosu biosynthesis with AaSPL2 genetic transformation sweet wormwood using genetic engineering means
Fast bottleneck obtains the sweet wormwood plant of qinghaosu high yield, establishes the method stablized and improve content of artemisinin in sweet wormwood, is scale
Production qinghaosu provides a new way.
Beneficial effects of the present invention:
The method provided by the invention for improving content of artemisinin in sweet wormwood using AaSPL2 gene, using genetic engineering side
Key gene AaSPL2 is imported in sweet wormwood plant, obtains the transgene abrotanum strain that artemislnin content significantly improves by method,
The content for turning AaSPL2 gene Artemisinin in Artemisia annuna can reach 20.6mg/g DW, be non-transformed common sweet wormwood (10.9mg/g
DW) 1.8 times, content of artemisinin in sweet wormwood is significantly improved, provides high yield, stable new drug for the large-scale production of qinghaosu
Source is of great significance.
Detailed description of the invention
Fig. 1 is AaSPL2 gene in the embodiment of the present invention 1 by MeJA induced map.
Fig. 2 be the embodiment of the present invention 1 in AaSPL2 gene different tissues express spectra.
Fig. 3 is 3 plant expression vector of the embodiment of the present invention: the building signal of pCAMBIA2300::p35S-AaSPL2-nos
Figure.
Fig. 4 is the content detection result figure of 5 transgenic Artemisinin in Artemisia annuna of the embodiment of the present invention.
Specific embodiment
Elaborate below to the embodiment of the present invention: the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sambrook etc.
Molecular cloning: described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989)
Condition, or according to the normal condition proposed by manufacturer.
Embodiment 1
Hormone MeJA plays a significant role in induction secondary metabolite.MeJA can induce a variety of secondary metabolites
Synthesis, such as danshinolic acid, qinghaosu in Radix Salviae Miltiorrhizae.SBP class transcription factor is a kind of important transcription factor, raw in regulation plant
It plays a crucial role in long development, secondary metabolism.The research that SPL regulates and controls qinghaosu in sweet wormwood at present not yet has been reported that.
1.MeJA handles sweet wormwood
It (uses by the sweet wormwood plant that the hormone MeJA of 100mM handles 30 days sizes and originates in Chongqing tenth of the twelve Earthly Branches sun artemislnin content
Higher sweet wormwood kind), screen the SBP class transcription factor induced by MeJA.Respectively hormone MeJA handle 0h, 1h, 3h, 6h,
9h, 12h and sweet wormwood blade is taken for 24 hours.It is placed in liquid nitrogen, the extraction for RNA.
2. the extraction of sweet wormwood genome total serum IgE
A small amount of sweet wormwood young leaflet tablet is taken to be ground with mortar rapidly after liquid nitrogen flash freezer, addition fills 1mL TRIzol
In the 1.5mL Eppendorf pipe of (TRIzol Reagents, GIBCO BRL, USA), sufficiently after oscillation, place at room temperature
5min adds 200 μ L chloroforms, with forced oscillation 15sec, after being placed at room temperature for 2-3min, is centrifuged 15min in 4 DEG C, 12,000g；By supernatant
Liquid (about 600 μ L) sucks in clean 1.5mL Eppendorf pipe, and isometric isopropanol is added, is mixed by inversion, room temperature decentralization
After setting 10min, 10min is centrifuged in 4 DEG C, 12,000g；Supernatant is abandoned, 75% ethyl alcohol of 1mL is added to clean, after oscillation, in 4 DEG C, 7,
500g is centrifuged 5min；It is dissolved in after drying at room temperature 15-20min in appropriate (30-50 μ L) RNAase-free water；With denaturing formaldehyde glue
Electroresis appraisal total serum IgE quality, then measures rna content on spectrophotometer.
3. screening the SBP class transcription factor induced by MeJA
0h, 1h, 3h, 6h, 9h, 12h are handled in hormone MeJA respectively and takes sweet wormwood blade for 24 hours, extract sweet wormwood according to step 2
Genome total serum IgE.The expression that SBP genoid is detected by Q-PCR generates thermal map file by MeV 4.9, referring to Fig. 1.
As shown in Figure 1, AaSPL2 is induced obviously by MeJA in sweet wormwood SPL, after MeJA is handled sweet wormwood plant 1 hour, expression quantity
It increases, between 3 hours to 24, expression quantity is also in raised trend, shows the induction of AaSPL2 response MeJA, and AaSPL2 may
Participate in the regulation of qinghaosu.
4, sweet wormwood different tissues expression pattern analysis
Sweet wormwood plant (identical as kind in step 1) different tissues position (COT, the cotyledon of 3 months sizes are taken respectively；ML,
Mature leaf；Meristem, separate living tissue；YL, tender leaf；Bud, bud), RNA is extracted according to step 2 method.It is examined by Q-PCR
The expression for surveying SBP genoid generates thermal map file by MeV 4.9, referring to fig. 2.Fig. 2 shows different groups in sweet wormwood
(COT, cotyledon are knitted in position；ML, mature leaf；Meristem, separate living tissue；YL, tender leaf；Bud, bud), AaSPL2 is presented
Different expressions.AaSPL2 expression quantity in colored and blade is higher, has class with artemisinin synthesis pathway key enzyme gene
As express spectra, screening obtain AaSPL2 be by MeJA induce important transcription factor.
Therefore, complex chart 1 and Fig. 2's as a result, show AaSPL2 may have regulation artemisinin synthesis potentiality.
The clone of 2 sweet wormwood AaSPL2 gene of embodiment
The sweet wormwood genome total serum IgE obtained is obtained into the first chain cDNA by reverse transcriptase XL (AMV) reverse transcription, according to institute
The coded sequence (as shown in SEQ ID NO:1) of sweet wormwood AaSPL2 gene is stated, the upstream and downstream that design amplifies complete encoder block is drawn
Object, AaSPL2-PF:atggagtggaattgggacaa, AaSPL2-PR:ttaatttgaacaaaagtagtcaaaa, and upper
Restriction endonuclease sites (this can be depending on the carrier of selection) is introduced respectively in trip and downstream primer, is carried to construct expression
Body.
Using the first chain cDNA as template, it is sequenced after PCR amplification.Determined dna sequence is by Shanghai Ying Junsheng
Object Technology Service Co., Ltd is completed using 3730 automatic sequencers.Sequencing result shows cloned sweet wormwood AaSPL2 gene
Sequence is consistent with sequence shown in SEQ ID NO:1.
The KOD reaction system of pcr amplification reaction is as shown in table 1.
Table 1
| Constituent | Content |
| 5×KOD buffer | 5μL |
| dNTP | 5μL |
| MgSO4 | 2μL |
| PF(10μM) | 1μL |
| PR(10μM) | 1μL |
| Template DNA | 1μL |
| KOD enzyme | 1μL |
| dH2O | up to 50μL |
PCR response procedures are as follows:
The present embodiment obtains the important transcription factor AaSPL2 of salicylic acid signal pathway using gene clone method from sweet wormwood
Gene, to provide an important key gene by turning AaSPL2 gene raising content of artemisinin in sweet wormwood.
The building of the plant binary expression vector of 3 gene containing AaSPL2 of embodiment
1. the building of intermediate vector pCAMBIA2300::p35S-gus-nos
It selects pBI121 (Clontech) and pCAMBIA2300 (Australian CAMBIA research institute) is primary element, structure
Build binary plant expression vector pCAMBIA2300::p35S-gus-nos.Specifically, HindIII and EcoRI double digestion pBI121
With pCAMBIA2300 plasmid；Recycle the gus expression cassette and pCAMBIA2300 large fragment of pBI121；Connect recovery product, conversion
Screening takes out plasmid enzyme restriction verifying, obtains binary plant expression vector pCAMBIA2300::p35S-gus-nos.Wherein, digestion is anti-
Answer system as shown in table 2.
Table 2
| Constituent | Content |
| 10 × buffer of digestion | 5μL |
| Carrier | 20μL |
| Restriction endonuclease 1 | 1μL |
| Interior enzyme 2 | 1μL |
| dH2O | up to 50μL |
2. the building of plant expression vector pCAMBIA2300::p35S-AaSPL2-nos
Using the pCAMBIA2300::p35S-gus-nos of building as expression vector, obtained with clone in embodiment 1
The gus gene of AaSPL2 gene replacement thereon, obtains plant expression vector pCAMBIA2300::p35S-AaSPL2-nos, building
Schematic diagram is as shown in Figure 3.Specifically, BamHI/SacI double digestion pCAMBIA2300::p35S-gus-nos, recycling
PCAMBIA2300::p35S-nos large fragment is arrived AaSPL2 gene swapping by seamless Cloning Kit (Nanjing Novi praises)
PCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2- is formed on pCAMBIA2300::p35S-nos
Nos, conversion, picking monoclonal extract plasmid and do PCR detection and digestion verification, obtain plant expression vector pCAMBIA2300::
p35S-AaSPL2-nos.Wherein, seamless cloning reaction system is as shown in table 3.
Table 3
| Constituent | Content |
| Gene PCR product | 1μL |
| Linearized vector | 3μL |
| Seamless clone Buffer | 4μL |
| Seamless clone enzyme | 2μL |
| dH2O | up to 20μL |
Salicylic acid signal pathway important gene AaSPL2 is operatively connectable to expression regulation sequence, shape by the present embodiment
At the plant expression vector of the gene containing AaSPL2, which can be used for improving sweet wormwood in sweet wormwood by metabolic engineering strategies
The content of element.
The Agrobacterium tumefaciens mediated AaSPL2 gene genetic of embodiment 4 converts sweet wormwood and obtains transgene abrotanum plant
1. the acquisition of the double base plant expression vector Agrobacterium tumefaciems engineering bacteria of gene containing AaSPL2
By the plant binary expression vector pCAMBIA2300::p35S-AaSPL2-nos of the gene containing AaSPL2 in embodiment 2
Being transferred to Agrobacterium tumefaciems, (such as EHA105 has the biomaterial of public offering for market, can purchase from Australian CAMBIA company
, strain number is Gambar 1), and carry out PCR verifying.Wherein, plasmid be transferred to Agrobacterium method it is as follows: 1 μ L plasmid adds
Enter into 50 μ L Agrobacterium competence, ice bath 30 minutes, freeze 5 minutes in liquid nitrogen, 37 DEG C heat shock 3 minutes, add LB culture
Base activates 3 hours in 37 DEG C of shaking tables, coated plate, identification.
The result shows that the binary expression vector of gene plant containing AaSPL2 is successfully building up in Agrobacterium tumefaciens strain.
2. Agrobacterium tumefaciens mediated AaSPL2 genetic transformation sweet wormwood
2.1. the preculture of explant
75% ethyl alcohol of seeds of southernwood impregnates 1min, then impregnates 20min with 20%NaClO, and aseptic water washing 3-4 times is used
Sterile blotting paper blots surface moisture, is inoculated in MS (Murashige and Skoog, 1962) solid medium of no hormone
In, 25 DEG C, 16h/8h (light/dark) illumination cultivation can be obtained sweet wormwood aseptic seedling.After seedling length to 5cm or so, clip
Tests for sterility explant is for converting.
2.2. the co-cultivation of Agrobacterium and explant
The blade explant is gone in co-culture medium (100 μm of ol/L of 1/2MS+AS), dropwise addition, which contains, to be activated
The binary expression vector of gene plant containing AaSPL2 Agrobacterium tumefaciems engineering bacteria 1/2MS suspension, make explant and bacterium
Liquid comes into full contact with, 28 DEG C of dark culture 3d.It is outstanding in the 1/2MS fluid nutrient medium of the Agrobacterium tumefaciems without target gene with dropwise addition
The blade explant of liquid is control.
2.3. the screening of resistance regeneration plant
The sweet wormwood explant for co-culturing 3d is transferred to germination screening and culturing medium (MS+6-BA 0.5mg/L+NAA
0.05mg/L+Kan 50mg/L+Cb 500mg/L) in 25 DEG C, 16h/8h illumination cultivation, squamous subculture is primary every two weeks, warp
It can be obtained Kan resistance Multiple Buds after crossing 2-3 subculture.Well-grown resistance Multiple Buds are cut and are transferred to root media
It is cultivated on (1/2MS+Cb 125mg/L) to taking root, to obtain Kan resistance regeneration sweet wormwood plant.
3. the PCR of transgene abrotanum plant is detected
(sweet wormwood is extracted by DNA extraction kit (Tiangeng) by template of the Kan resistance of acquisition regeneration sweet wormwood plant DNA
The DNA of plant), forward direction is separately designed according to expression cassette p35s-AaSPL2-nos sequence p35s and AaSPL2 where target gene
Design of primers and reverse primer detect target gene, detection primer PF:gagagactggtgatttcagcg, PR:
ttaatttgaacaaaagtagtcaaaa.Wherein, it using DNA as template, is expanded by TAKARA enzyme (Dalian treasured bioengineering),
Rtaq-MIX enzyme reaction system is as shown in table 4 and program is as follows:
Table 4
| Constituent | Content |
| 2×rtaq-MIX buffer | 5μL |
| PF(10μM) | 1μL |
| PR(10μM) | 1μL |
| Template DNA | 1μL |
| Rtaq enzyme | 1μL |
| dH2O | up to 20μL |
PCR response procedures are as follows:
The result shows that the specific DNA fragment of 1000bp, i.e. agarose can be amplified using designed PCR special primer
Purpose band is observed after gel electrophoresis in the UV lamp, the Kan resistance regeneration sweet wormwood plant of acquisition is to turn AaSPL2 gene
Sweet wormwood plant.And when using non-transformed sweet wormwood genomic DNA as template, do not amplify any segment.
The plant expression vector is converted Agrobacterium tumefaciems by the present embodiment, obtain for convert sweet wormwood containing AaSPL2
The Agrobacterium tumefaciens strain of gene plant expression vector converts sweet wormwood using constructed Agrobacterium tumefaciens strain, obtains through PCR
The transgene abrotanum plant of detection.The acquisition of transgene abrotanum plant is that the sweet wormwood strain of the higher artemislnin content of screening acquisition mentions
Direct material is supplied.
Embodiment 5 utilizes artemislnin content in HPLC-ELSD measurement transgene abrotanum
The preparation of 1.HPLC-ELSD condition and system suitability and standard solution
HPLC: using 2695 system of water alliance, and chromatographic column is C-18 reverse phase silica gel column
(SymmetryShieldTM C18,5 μm, 250 × 4.6mm, Waters), mobile phase is methanol: water, methanol: the volume ratio of water
For 70:30,30 DEG C of column temperature, flow velocity 1.0mL/min, 10 μ L of sample volume, sensitivity (AUFS=1.0), theoretical cam curve presses sweet wormwood
Plain peak, which calculates, is not less than 2000.
ELSD: 2420 system of water alliance is used, 40 DEG C of evaporative light scattering detector drift tube temperature, is amplified
Coefficient (gain) is 7, nebulizer gas pressure 5bar.
Precision weighs qinghaosu standard items (Sigma company) 2.0mg and is completely dissolved with 1mL methanol, obtains 2mg/mL sweet wormwood
Plain standard solution, be stored in -20 DEG C it is spare.
Mobile phase is methanol (methanol): water in the present invention, when ratio is 70%:30%, the retention time of qinghaosu
For 5.1min, peak type is good.Theoretical cam curve is calculated by qinghaosu is not less than 2000.
2. the production of standard curve
Above-mentioned standard product solution is distinguished to sample introduction 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l under corresponding chromatographic condition and records map
And chromatographic parameter, regression analysis is carried out to standard items content (X, μ g) with peak area (Y) respectively.It is green in the present invention by research
Good log-log linear relationship is presented in artemisin in 4-20 μ g range.The log-log equation of linear regression of Qinghaosu
Are as follows: Y=1.28e+000X+4.71e+000, R=0.979546.
3. the preparation of sample and the measurement of artemislnin content
The extraction process of qinghaosu is based on the method reported in Van Nieuwerburgh et al. (2006): taking a small amount of
Fresh sweet wormwood blade (1-2g fresh weight), is immersed into 10ml chloroform in 50ml test tube and sways 1 minute, leachate is fallen
Enter to make in new test tube chloroform volatilization completely, 3ml dehydrated alcohol is taken sufficiently to dissolve extract, is detected for HPLC.Meanwhile chloroform
Blade collection after extraction, which is put into 60 degree of baking ovens, is dried, and weighing (calculates the dry weight of sweet wormwood blade)；
Artemislnin content is measured using HPLC-ELSD, sample feeding volume is 20 μ l, substitutes into linear regression according to peak area
Equation calculation goes out the artemislnin content (mg) in sample, then divided by the sweet wormwood leaf dry weight (g) of sample, to calculate sweet wormwood plant
The content of middle qinghaosu.
After measured, the content of transfer AaSPL2 gene Artemisinin in Artemisia annuna of the present invention can reach 20.6mg/g DW, be
1.8 times (as shown in Figure 4) of non-transformed common sweet wormwood (10.9mg/g DW), AaSPL2 gene significantly improve sweet wormwood in sweet wormwood
Cellulose content.
The present embodiment determines artemislnin content in transgene abrotanum using HPLC-ELSD method, using conversion AaSPL2 base
The metabolic engineering strategies of cause obtain the sweet wormwood plant of qinghaosu high yield, provide a kind of ideal side for large-scale production qinghaosu
Method.
Sequence table
<110>Second Military Medical University, PLA
<120>a kind of method for improving content of artemisinin in sweet wormwood using AaSPL2 gene
<130>specification, claims
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 954
<212> DNA
<213>sweet wormwood (Artemisia annua)
<400> 1
atggagtgga attgggacaa taatgaagtt cccaagagtt tagcggtttc aagccacgaa 60
aatggcgagt atttgggggg tgaagatgtg caaaggagtt tttctaatga tataattgag 120
gaaggctcgg ttatatctgg tgaagcacta tttggtttga agctcgggca agagacatat 180
aatcaagata atcttagaat gagctcattc ccattagtac ctacctcttt gagttctccg 240
gttgtgaaaa aatctagagc ggtgtaccaa agttcacatt cgccacgttg tcaagttgag 300
gggtgcaacc ttgaccttgt gtcggctaaa gattaccacc gtcggcataa aatttgtgct 360
gatcattcta agagcccaaa agttgttgtt ggggggatgg aacgaaggtt ttgtcagcaa 420
tgtagcaggt tgcatgattt atcggagttt gacgatagga agcgtagttg tcgcaggcgc 480
ctctcggcac acaatgctag acgtcgtagg ccacaatcag aggacaagtt cagccccaca 540
catcgaagac catatatggg tttctttgtg aacggggtct cgacctcgac cccatcacca 600
aattcgacac cacaaagttc atctaacttt aaacgtgaag atgttgacat gatctcgagt 660
ggtgccttat cggacatctc gtcgtctttc catggggtca cacctagact tgctaaccac 720
gagggtttag atgcaaactc taactcgatc tgtaagatgg aagttcgaca ttctttctct 780
cttcaagcca cgagctcttg gggattcaac agtcgcgatg agccctcttc ctttgatcaa 840
ttcattaatg gacacaacgc cggcttgacg caacatggga caccactcga gatgcaacat 900
acccgaaaca ctcaaattcc acctcatgat tttgactact tttgttcaaa ttaa 954
Claims (10)
1. a kind of method for improving content of artemisinin in sweet wormwood using AaSPL2 gene, includes the following steps:
S1: sweet wormwood key gene AaSPL2 is obtained using gene clone method；
AaSPL2 gene: being operatively connectable to expression regulation sequence by S2, and the plant for forming the gene containing AaSPL2, which is expressed, to be carried
Body；
S3: converting Agrobacterium tumefaciems for the plant expression vector of the gene containing AaSPL2, obtain for convert sweet wormwood containing AaSPL2
The Agrobacterium tumefaciens strain of gene plant expression vector；
S4: converting sweet wormwood using constructed Agrobacterium tumefaciens strain, obtains the transgene abrotanum through PCR test positive and plants
Strain；
S5: HPLC-ELSD measurement is carried out to artemislnin content in the transgene abrotanum of acquisition, it is significant that screening obtains artemislnin content
The transgene abrotanum plant of raising.
2. the method according to claim 1 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that
Step S1 includes the following steps: to extract sweet wormwood genome total serum IgE；Sweet wormwood genome total serum IgE is obtained into the first chain by reverse transcription
cDNA；It using the first chain cDNA as template, is sequenced after PCR amplification, obtains and correct sweet wormwood AaSPL2 gene is sequenced；
The DNA sequence dna according to shown in SEQ ID NO.1 when PCR amplification, design amplifies the upstream and downstream primer of complete encoder block, described
The nucleotide sequence of upstream and downstream primer is respectively as follows: AaSPL2-PF:atggagtggaattgggacaa；AaSPL2-PR:
ttaatttgaacaaaagtagtcaaaa。
3. the method according to claim 1 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that
In step S2, it is described building the gene containing AaSPL2 plant expression vector include the following steps: select pBI121 and
PCAMBIA2300 is primary element, constructs binary plant expression vector pCAMBIA2300::p35S-gus-nos；BamHI is used again
With SacI double digestion carrier pCAMBIA2300::p35S-gus-nos, pCAMBIA2300::p35S-nos large fragment is recycled, it will
PCAMBIA2300::p35S-pCAMBIA2300: is formed in AaSPL2 gene swapping to pCAMBIA2300::p35S-nos:
P35S-gus-AaSPL2-nos, conversion, picking monoclonal extract plasmid and do PCR detection and digestion verification.
4. the method according to claim 3 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that
The building binary plant expression vector pCAMBIA2300::p35S-gus-nos include the following steps: with HindIII and
EcoRI double digestion pBI121 and pCAMBIA2300 plasmid；Recycle the gus expression cassette and pCAMBIA2300 large fragment of pBI121；
Recovery product is connected, transformation and selection takes out plasmid enzyme restriction verifying.
5. the method according to claim 1 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that
It is described to convert the preculture for including the following steps: explant in step S4；The co-cultivation of Agrobacterium and explant；Resistance regeneration
The screening of plant.
6. the method according to claim 5 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that
The preculture of the explant includes the following steps: that seeds of southernwood impregnates 1min with 75% ethyl alcohol, then is impregnated with 20%NaClO
20min, aseptic water washing 3-4 times blot surface moisture with sterile blotting paper, are inoculated in the MS solid medium of no hormone,
Illumination cultivation 16h, dark culture 8h, can be obtained sweet wormwood aseptic seedling at 25 DEG C, after seedling length to 5~8cm, clip tests for sterility
Explant is for converting.
7. the method according to claim 5 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that
The co-cultivation of the Agrobacterium and explant includes the following steps: the tests for sterility explant going to co-culture medium
In, it is added dropwise containing the activated binary expression vector of gene plant containing the AaSPL2 pCAMBIA2300::p35S-
The 1/2MS suspension of the Agrobacterium tumefaciems engineering bacteria of pCAMBIA2300::p35S-gus-AaSPL2-nos, makes explant and bacterium solution
It comes into full contact with, 28 DEG C of dark culture 3d.
8. the method according to claim 5 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that
The screening of the resistance regeneration plant includes the following steps: that the sweet wormwood explant by the co-cultivation 3d is transferred to germination screening
On culture medium at 25 DEG C illumination cultivation 16h, dark culture 8h, squamous subculture is primary every two weeks, can obtain after 2-3 subculture
Kan resistance Multiple Buds are obtained, well-grown resistance Multiple Buds are cut to be transferred on root media and are cultivated to taking root, Kan is obtained
Resistance regenerates sweet wormwood plant.
9. the method according to claim 1 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that
In step S4, the method for the PCR detection are as follows: separately design AaSPL2 gene in expression cassette p35s-AaSPL2-nos sequence
Specific primer carries out PCR amplification, observes that the positive strain of purpose band is after agarose gel electrophoresis in the UV lamp
Transgene abrotanum plant；The specific primer includes upstream primer, downstream primer, and particular sequence is respectively as follows: PF:
Gagagactggtgatttcagcg, PR:ttaatttgaacaaaagtagtcaaaa.
10. sweet wormwood AaSPL2 gene is improving the application in content of artemisinin in sweet wormwood.
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