A method of content of artemisinin in sweet wormwood is improved using AaSPL2 gene|一种利用AaSPL2基因提高青蒿中青蒿素含量的方法

Entry posted by Roger in Artemisinin Patent｜青蒿素专利 July 8

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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.

一种利用AaSPL2基因提高青蒿中青蒿素含量的方法，包括如下步骤：从青蒿中克隆水杨酸信号途径AaSPL2基因，构建含AaSPL2基因的植物表达载体，用根癌农杆菌介导，将AaSPL2基因转入青蒿并再生出植株，PCR检测外源目的基因AaSPL2的整合情况，再测定转基因青蒿中青蒿素的含量，筛选获得青蒿素含量提高的转基因青蒿植株。本发明获得的转基因青蒿中青蒿素的含量显著提高，最高达到非转化对照植株的1.8倍，为利用转基因青蒿大规模生产青蒿素打下了基础。

Description

A method of content of artemisinin in sweet wormwood is improved using AaSPL2 gene

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)

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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.

https://patents.google.com/patent/CN110438148A/en

一种利用AaSPL2基因提高青蒿中青蒿素含量的方法

技术领域

本发明属于生物技术领域。具体涉及的是一种提高青蒿素含量的方法，特别涉及是一种利用AaSPL2基因提高青蒿中青蒿素含量的方法。

背景技术

青蒿(Artemisia annua L.)是菊科蒿属的一年生草本植物。青蒿素(artemisinin)是从其地上部分分离的一种含有过氧桥结构的倍半萜内酯化合物，是目前世界上公认的最有效的治疗疟疾的药物，特别是对于脑型疟疾和抗氯喹疟疾具有速效和低毒的特点。目前，世界卫生组织推荐的最有效的治疗疟疾的方法就是青蒿素联合疗法(ACTs)。另外，随着对青蒿素药理研究的逐步深入，科学家发现青蒿素及其衍生物还具有抗炎、抗血吸虫、抗肿瘤以及免疫调节的功能。可见青蒿素是一种极具潜力的天然药物。

目前青蒿素的主要来源是从青蒿植株的地上部分提取，然而青蒿中青蒿素的含量非常低(0.01％-1％)，使得这种药物的大规模商业化生产受到了限制。由于青蒿素结构复杂，人工合成难度大，产量低，成本高，不具有可行性。有人尝试用组织培养和细胞工程的方法来生产青蒿素，然而青蒿素在愈伤组织中含量低于干重的0.1％，在芽中最高也只有干重的0.16％，而大多数研究在根中没有检测到青蒿素。因此利用组织培养及细胞工程来生产青蒿素的可行性也不高。

经对现有技术文献检索发现，Qian Shen等在《New Phytologist》(《新植物学家》，2016年210期1269-1281页)发表了题为“The jasmonate-responsiveAaMYC2transcription factor positively regulates artemisinin biosynthesis inArtemisia annua”(“受甲基茉莉酸响应的AaMYC2转录因子正调控青蒿中青蒿素的生物合成”)的论文，报道通过在青蒿中过表达受激素甲基茉莉酸响应的AaMYC2，使青蒿素含量提高了1.6倍。因此，通过过表达受甲基茉莉酸响应的基因为提高青蒿中青蒿素的含量提供了一条可行的方法。

发明内容

本发明的目的在于提供一种利用AaSPL2基因提高青蒿中青蒿素含量的方法，将青蒿中克隆出的AaSPL2基因转入青蒿中得到转基因AaSPL2青蒿，该转基因AaSPL2青蒿中青蒿素含量显著提高，从而实现利用青蒿大规模生产青蒿素。

本发明是通过以下技术方案实现的：本发明从青蒿中克隆AaSPL2基因，构建含AaSPL2基因的植物表达载体，用根癌农杆菌介导，将AaSPL2基因导入青蒿并再生出植株；PCR检测外源目的基因AaSPL2的整合情况，高效液相色谱-蒸发光散射检测器(HPLC-ELSD)测定青蒿中青蒿素含量，筛选获得青蒿素含量提高的转基因青蒿植株。

本发明提供的利用AaSPL2基因提高青蒿中青蒿素含量的方法，包括如下步骤：

S1：采用基因克隆方法获得青蒿关键酶基因AaSPL2；

S2：把AaSPL2基因可操作性地连接于表达调控序列，构建含AaSPL2基因的植物表达载体；

S3：将含AaSPL2基因的植物表达载体转化根癌农杆菌，获得用于转化青蒿的含AaSPL2基因植物表达载体的根癌农杆菌菌株；

S4：利用所构建的根癌农杆菌菌株转化青蒿，获得经PCR检测为阳性的转基因青蒿植株；

S5：对获得的转基因青蒿中青蒿素含量进行HPLC-ELSD测定，筛选获得青蒿素含量显著提高的转基因青蒿植株。

优选的，步骤S1：提取青蒿基因组总RNA；将青蒿基因组总RNA通过反转录获得第一链cDNA；根据如SEQ ID NO.1所示的青蒿AaSPL2基因的编码序列设计扩增出完整编码框的上下游引物，并在上游和下游引物上分别引入限制性内切酶位点，以便构建表达载体；以所述第一链cDNA为模板，经PCR扩增后进行测序，获得测序正确的青蒿AaSPL2基因，其中PCR扩增所用的上、下游引物的核苷酸序列分别为：AaSPL2-PF：atggagtggaattgggacaa，AaSPL2-PR：ttaatttgaacaaaagtagtcaaaa。

优选的，步骤S2中，所述构建含AaSPL2基因的植物表达载体包括如下步骤：选用pBI121和pCAMBIA2300为基本元件，构建双元植物表达载体pCAMBIA2300::p35S-gus-nos；再用BamHI和SacI双酶切载体pCAMBIA2300::p35S-gus-nos，回收pCAMBIA2300::p35S-nos大片段，通过无缝克隆试剂盒将AaSPL2基因交换到pCAMBIA2300::p35S-nos上形成pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos，转化，挑取单克隆，提取质粒做PCR检测和酶切验证。

更优选的，所述构建双元植物表达载体pCAMBIA2300::p35S-gus-nos包括如下步骤：用HindIII和EcoRI双酶切pBI121和pCAMBIA2300质粒；回收pBI121的gus表达盒和pCAMBIA2300大片段；连接回收产物，转化筛选，抽质粒酶切验证。

优选的，步骤S4中，所述转化包括如下步骤：外植体的预培养；农杆菌与外植体的共培养；抗性再生植株的筛选。

更优选的，所述外植体的预培养包括如下步骤：青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS固体培养基中，25℃下光照培养16h、暗培养8h，即可获得青蒿无菌苗，待苗长至5～8cm后，剪取无菌苗叶片外植体用于转化。

更优选的，所述农杆菌与外植体的共培养包括如下步骤：将所述无菌苗叶片外植体转到共培养培养基中，滴加含活化好的所述含AaSPL2基因植物双元表达载体pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3d。

更优选的，所述抗性再生植株的筛选包括如下步骤：将所述的共培养3d的青蒿外植体转入到发芽筛选培养基上于25℃下光照培养16h、暗培养8h，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽，将生长良好的抗性丛生芽剪下转入生根培养基上培养至生根，获得Kan抗性再生青蒿植株。

优选的，步骤S4中，所述PCR检测的方法为：分别设计合成表达盒p35s-AaSPL2-nos序列上AaSPL2基因的特异性引物进行PCR扩增，琼脂糖凝胶电泳后在紫外灯下观察到目的条带的阳性株系即为转基因青蒿植株；所述特异性引物包括上游引物、下游引物，具体序列分别为：PF：gagagactggtgatttcagcg，PR：ttaatttgaacaaaagtagtcaaaa。

步骤S5中，所述的HPLC-ELSD测定青蒿中青蒿素含量，方法为：色谱柱C-18反相硅胶柱，流动相为甲醇：水，甲醇:水的体积比为70：30，柱温30℃，流速1.0mL/min，进样量10μL，蒸发光散射检测器漂移管温度40℃，放大系数(gain)为7，载气压力5bar。

本发明步骤S3中所用根癌农杆菌为市场有公开出售的生物材料，可以从多家公司如澳大利亚CAMBIA公司购得。

本发明还提供青蒿AaSPL2基因在提高青蒿中青蒿素含量中的应用。

本发明证明：AaSPL2基因是水杨酸信号途径重要的转录因子及青蒿素代谢工程的重要靶点。本发明采用基因工程手段，用AaSPL2基因转化青蒿，打破了青蒿素生物合成的限速瓶颈，获得青蒿素高产的青蒿植株，建立了稳定提高青蒿中青蒿素含量的方法，为规模化生产青蒿素提供了一条新途径。

本发明的有益效果：

本发明提供的利用AaSPL2基因提高青蒿中青蒿素含量的方法，采用基因工程方法，将关键酶基因AaSPL2导入青蒿植株中，获得了青蒿素含量显著提高的转基因青蒿株系，转AaSPL2基因青蒿中青蒿素的含量最高可达到20.6mg/g DW，是非转化普通青蒿(10.9mg/gDW)的1.8倍，显著提高了青蒿中青蒿素含量，为青蒿素的规模化生产提供高产、稳定的新药源，具有重要意义。

附图说明

图1为本发明实施例1中AaSPL2基因受MeJA诱导图。

图2为本发明实施例1中AaSPL2基因在不同组织的表达谱。

图3为本发明实施例3植物表达载体：pCAMBIA2300::p35S-AaSPL2-nos的构建示意图。

图4为本发明实施例5中转基因青蒿中青蒿素的含量检测结果图。

具体实施方式

下面对本发明的实施例作详细说明：本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。

下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。

实施例1

激素MeJA在诱导次生代谢产物中具有重要作用。MeJA能诱导多种次生代谢产物的合成，如丹参中的丹酚酸、青蒿素等。SBP类转录因子是一类重要的转录因子，在调控植物生长发育、次生代谢中起关键作用。目前青蒿中SPL调控青蒿素的研究尚未有报道。

1.MeJA处理青蒿

通过100mM的激素MeJA处理30天大小的青蒿植株(采用产于重庆酉阳青蒿素含量较高的青蒿品种)，筛选受MeJA诱导的SBP类转录因子。分别在激素MeJA处理0h、1h、3h、6h、9h、12h和24h取青蒿叶片。置于液氮中，用于RNA的提取。

2.青蒿基因组总RNA的提取

取少量青蒿幼嫩叶片，用液氮速冻后，迅速用研钵研碎，加入盛有1mL TRIzol(TRIzol Reagents,GIBCO BRL,USA)的1.5mL Eppendorf管中，充分振荡后，于室温下放置5min，加200μL氯仿，用力振荡15sec，室温放置2-3min后，于4℃、12,000g离心15min；将上清液(约600μL)吸入干净的1.5mL Eppendorf管中，加入等体积的异丙醇，颠倒混匀，室温下放置10min后，于4℃、12,000g离心10min；弃上清，加1mL 75％乙醇清洗，振荡后，于4℃、7,500g离心5min；室温干燥15-20min后溶于适量(30-50μL)RNAase-free水中；用甲醛变性胶电泳鉴定总RNA质量，然后在分光光度计上测定RNA含量。

3.筛选受MeJA诱导的SBP类转录因子

分别在激素MeJA处理0h、1h、3h、6h、9h、12h和24h取青蒿叶片，按照步骤2提取青蒿基因组总RNA。通过Q-PCR检测SBP类基因的表达水平，通过MeV 4.9生成热图文件，参见图1。由图1可知，在青蒿SPL中AaSPL2受MeJA诱导明显，在MeJA处理青蒿植株1小时后，其表达量升高，在3小时到24之间，表达量也呈升高的趋势，表明AaSPL2响应MeJA的诱导，AaSPL2可能参与青蒿素的调控。

4、青蒿不同组织表达谱分析

分别取3个月大小的青蒿植株(与步骤1中品种相同)不同组织部位(COT，子叶；ML，成熟叶片；Meristem，分生组织；YL，嫩叶；Bud，花蕾)，按照步骤2方法提取RNA。通过Q-PCR检测SBP类基因的表达水平，通过MeV 4.9生成热图文件，参见图2。图2表明，在青蒿的不同组织部位中(COT，子叶；ML，成熟叶片；Meristem，分生组织；YL，嫩叶；Bud，花蕾)，AaSPL2呈现不同的表达水平。AaSPL2在花和叶片中表达量较高，与青蒿素合成途径关键酶基因具有类似的表达谱，筛选得到AaSPL2是受MeJA诱导的重要转录因子。

因此，综合图1和图2的结果，表明AaSPL2可能具有调控青蒿素合成的潜力。

实施例2青蒿AaSPL2基因的克隆

将所获的青蒿基因组总RNA通过反转录酶XL(AMV)反转录获得第一链cDNA，根据所述青蒿AaSPL2基因的编码序列(如SEQ ID NO:1所示)，设计扩增出完整编码框的上下游引物，AaSPL2-PF:atggagtggaattgggacaa，AaSPL2-PR:ttaatttgaacaaaagtagtcaaaa，并在上游和下游引物上分别引入限制性内切酶位点(这可视选用的载体而定)，以便构建表达载体。

以所述的第一链cDNA为模板，经PCR扩增后进行测序。DNA序列测定由上海英骏生物技术服务有限公司采用3730自动测序仪完成。测序结果表明，所克隆的青蒿AaSPL2基因序列与SEQ ID NO:1所示的序列一致。

PCR扩增反应的KOD反应体系如表1所示。

表1

组成成分	含量
		5×KOD buffer	5μL

dNTP	5μL
		MgSO<sub>4</sub>	2μL
PF(10μM)	1μL
		PR(10μM)	1μL
模板DNA	1μL
		KOD酶	1μL
dH<sub>2</sub>O	up to 50μL

PCR反应程序如下：

本实施例采用基因克隆方法从青蒿中获得水杨酸信号途径重要转录因子AaSPL2基因，为通过转AaSPL2基因提高青蒿中青蒿素含量提供了一个重要关键酶基因。

实施例3含AaSPL2基因的植物双元表达载体的构建

1.中间载体pCAMBIA2300::p35S-gus-nos的构建

选用pBI121(Clontech)和pCAMBIA2300(澳大利亚CAMBIA研究所)为基本元件，构建双元植物表达载体pCAMBIA2300::p35S-gus-nos。具体地，HindIII和EcoRI双酶切pBI121和pCAMBIA2300质粒；回收pBI121的gus表达盒和pCAMBIA2300大片段；连接回收产物，转化筛选，抽质粒酶切验证，得到双元植物表达载体pCAMBIA2300::p35S-gus-nos。其中，酶切反应体系如表2所示。

表2

组成成分	含量
		酶切10×buffer	5μL
载体	20μL

内切酶1	1μL
		内酶2	1μL
dH2O	up to 50μL

2.植物表达载体pCAMBIA2300::p35S-AaSPL2-nos的构建

以构建的pCAMBIA2300::p35S-gus-nos为表达载体，用实施例1中克隆得到的AaSPL2基因替换其上的gus基因，得到植物表达载体pCAMBIA2300::p35S-AaSPL2-nos，构建示意图如图3所示。具体地，BamHI/SacI双酶切pCAMBIA2300::p35S-gus-nos，回收pCAMBIA2300::p35S-nos大片段，通过无缝克隆试剂盒(南京诺维赞)将AaSPL2基因交换到pCAMBIA2300::p35S-nos上形成pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos，转化，挑取单克隆，提取质粒做PCR检测和酶切验证，得到植物表达载体pCAMBIA2300::p35S-AaSPL2-nos。其中，无缝克隆反应体系如表3所示。

表3

组成成分	含量
		基因PCR产物	1μL
线性化载体	3μL
		无缝克隆Buffer	4μL
无缝克隆酶	2μL
		dH<sub>2</sub>O	up to 20μL

本实施例将水杨酸信号途径重要基因AaSPL2可操作性地连接于表达调控序列，形成含AaSPL2基因的植物表达载体，该表达载体可用于通过代谢工程策略来提高青蒿中青蒿素的含量。

实施例4根癌农杆菌介导AaSPL2基因遗传转化青蒿获得转基因青蒿植株

1.含AaSPL2基因双元植物表达载体根癌农杆菌工程菌的获得

将实施例2中含AaSPL2基因的植物双元表达载体pCAMBIA2300::p35S-AaSPL2-nos转入根癌农杆菌(如EHA105，为市场有公开出售的生物材料，可以从澳大利亚CAMBIA公司购得，菌株编号为Gambar 1)，并进行PCR验证。其中，质粒转入农杆菌的方法如下：1μL质粒加入到50μL农杆菌感受态中，冰浴30分钟，液氮中冷冻5分钟，37℃热激3分钟，再加入LB培养基在37℃摇床活化3小时，涂板，鉴定。

结果表明，含AaSPL2基因植物双元表达载体已成功构建到根癌农杆菌菌株中。

2.根癌农杆菌介导AaSPL2基因转化青蒿

2.1.外植体的预培养

青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS(Murashige and Skoog,1962)固体培养基中，25℃、16h/8h(light/dark)光照培养，即可获得青蒿无菌苗。待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化。

2.2.农杆菌与外植体的共培养

将所述的叶片外植体转到共培养培养基(1/2MS+AS 100μmol/L)中，滴加含活化好的所述含AaSPL2基因植物双元表达载体的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3d。以滴加在不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。

2.3.抗性再生植株的筛选

将共培养3d的青蒿外植体转入到发芽筛选培养基(MS+6-BA 0.5mg/L+NAA0.05mg/L+Kan 50mg/L+Cb 500mg/L)上于25℃、16h/8h光照培养，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽。将生长良好的抗性丛生芽剪下转入生根培养基(1/2MS+Cb 125mg/L)上培养至生根，从而获得Kan抗性再生青蒿植株。

3.转基因青蒿植株的PCR检测

以获得的Kan抗性再生青蒿植株DNA为模板(通过DNA提取试剂盒(天根)提取青蒿植株的DNA)，根据目的基因所在表达盒p35s-AaSPL2-nos序列p35s和AaSPL2分别设计正向引物设计和反向引物对目的基因进行检测，检测引物为PF：gagagactggtgatttcagcg，PR：ttaatttgaacaaaagtagtcaaaa。其中，以DNA为模板，通过TAKARA酶(大连宝生物工程)扩增，rtaq-MIX酶反应体系如表4所示及程序如下：

表4

组成成分	含量
		2×rtaq-MIX buffer	5μL
PF(10μM)	1μL
		PR(10μM)	1μL
模板DNA	1μL
		rtaq酶	1μL
dH<sub>2</sub>O	up to 20μL

PCR反应程序如下：

结果表明，利用所设计的PCR特异引物，能扩增出1000bp的特异DNA片段，即琼脂糖凝胶电泳后在紫外灯下观察到目的条带的，获得的Kan抗性再生青蒿植株为转AaSPL2基因青蒿植株。而以非转化青蒿基因组DNA为模板时，没有扩增出任何片段。

本实施例将所述的植物表达载体转化根癌农杆菌，获得用于转化青蒿的含AaSPL2基因植物表达载体的根癌农杆菌菌株，利用所构建的根癌农杆菌菌株转化青蒿，获得经PCR检测的转基因青蒿植株。转基因青蒿植株的获得为筛选获得较高青蒿素含量的青蒿株系提供了直接素材。

实施例5利用HPLC-ELSD测定转基因青蒿中青蒿素含量

1.HPLC-ELSD条件及系统适用性以及标准溶液的配制

HPLC：采用water alliance 2695系统，色谱柱为C-18反相硅胶柱(SymmetryShieldTM C18，5μm，250×4.6mm，Waters)，流动相为甲醇：水，甲醇：水的体积比为70：30，柱温30℃，流速1.0mL/min，进样量10μL，灵敏度(AUFS＝1.0)，理论塔板数按青蒿素峰计算不低于2000。

ELSD：采用water alliance 2420系统，蒸发光散射检测器漂移管温度40℃，放大系数(gain)为7，载气压力5bar。

精密称取青蒿素标准品(Sigma公司)2.0mg用1mL甲醇完全溶解，得到2mg/mL青蒿素标准品溶液，保存于-20℃备用。

本发明中流动相为甲醇(methanol)：水，比例为70％：30％时，青蒿素的保留时间为5.1min，峰型良好。理论塔板数按青蒿素计算不低于2000。

2.标准曲线的制作

将上述标准品溶液在相应色谱条件下分别进样2μl，4μl，6μl，8μl，10μl记录图谱及色谱参数，分别以峰面积(Y)对标准品含量(X,μg)进行回归分析。通过研究，本发明中青蒿素在4-20μg范围内呈现良好的log-log线性关系。青蒿素对照品的log-log线性回归方程为：Y＝1.28e+000X+4.71e+000，R＝0.979546。

3.样品的制备和青蒿素含量的测定

青蒿素的提取过程基于Van Nieuwerburgh et al.(2006)中报道的方法：取少量新鲜的青蒿叶片(1-2g鲜重)，于50ml试管中将其浸没在10ml氯仿中摇荡1分钟，将浸出液倒入新的试管中使氯仿挥发完全，取3ml无水乙醇充分溶解提取物，用于HPLC检测。同时，氯仿提取后的叶片收集放入60度烘箱进行烘干，称重(计算青蒿叶片的干重)；

采用HPLC-ELSD测定青蒿素含量，样品进样体积为20μl，根据峰面积代入线形回归方程计算出样品中的青蒿素含量(mg)，再除以样品的青蒿叶干重(g)，从而计算出青蒿植株中青蒿素的含量。

经测定，本发明中转AaSPL2基因青蒿中青蒿素的含量最高可达到20.6mg/g DW，是非转化普通青蒿(10.9mg/g DW)的1.8倍(如图4所示)，AaSPL2基因显著提高了青蒿中青蒿素含量。

本实施例采用HPLC-ELSD法测定了转基因青蒿中青蒿素含量，采用转化AaSPL2基因的代谢工程策略获得了青蒿素高产的青蒿植株，为规模化生产青蒿素提供了一种理想方法。

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<110> 中国人民解放军第二军医大学

<120> 一种利用AaSPL2基因提高青蒿中青蒿素含量的方法

<130> 说明书，权利要求书

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 954

<212> DNA

<213> 青蒿(Artemisia annua)