CN104152463A - Coding sequence of AaMYBL1 protein of artemisia apiacea and application thereof - Google Patents
Coding sequence of AaMYBL1 protein of artemisia apiacea and application thereof Download PDFInfo
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Abstract
The invention relates to a coding sequence of an AaMYBL1 protein of artemisia apiacea and an application thereof. The amino acid sequence coded by the coding sequence AaMYBL1 of an MYB-like type transcription factor of artemisia apiacea is shown as SEQ ID NO:4. The AaMYBL1 which codes a R3MYB type transcription factor takes part in regulation of density of glandular hairs of artemisia apiacea. An interference vector of the AaMYBL1 transcription factor of artemisia apiacea is transformed into artemisia apiacea by means of the transgenic technology, so that the density of the glandular hairs on the surface of artemisia apiacea can be effectively regulated, thus, the content of artemisinin is improved. The density of the glandular hairs on the surface of a blade of non-transgenic common artemisia apiacea is 24/square millimeter, and the density of the glandular hairs of a blade of transgenic artemisia apiacea inhibiting AaMYBL1 genetic expression is increased to 34/square millimeter; the quantity of total glandular hairs of each blade is increased from 61947 to 93683; correspondingly, the content of artemisinin is improved from 8mg/g DW of non-transgenic artemisia apiacea to 12mg/g DW. The coding sequence provided by the invention is of significance for providing a high-yield stable novel medicine source for scaled production of artemisinin.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of sweet wormwood AaMYBL1 albumen coded sequence and application thereof.
Background technology
Plant metabolism is divided into nascent metabolism and secondary metabolism, nascent meta-bolites (as carbohydrate, lipid and nucleic acid) is present in all plants, that to maintain cell activities necessary, and Secondary Metabolism of Plant product refers in plant that a large class is not the necessary small molecules organic compound of growth and development of plants, its generation and distribution have kind, histoorgan and the specificity of growing.Most plants blade surface is not smooth, is permitted eurypalynous hair shape structure but have.Be divided into two large classes according to its function and morphology: a class is the cilium without secreting function; Another kind of is the glandular hairs with secreting function.The main representative of last class is the cotton fibre of cotton, and it is the unicellular cilium of cotton seeds epidermis specialization.And secretor type glandular hairs a kind of multi-cellular structure that to be plant epidermis cell specifications go out is found the research of glandular hairs in recent years, many valuable secondary metabolites are all that specificity is synthetic and store in this structure.Such as with peppermint essential oil, the large class natural essence spices that Flos Rosae Rugosae quintessence oil is representative; Chinese herbal medicine effective ingredients taking Artemisinin as representative; Natural phant additive taking hops as representative.
Its secondary metabolites content producing in natural plant is extremely low, and uses the method for chemosynthesis, and technical process complexity, cost are high, and a lot of secondary metabolite adds edible cosmetic product industry to, uses natural vegetable material safer.Therefore, researchist starts the method for the raising Secondary Metabolism of Plant product content of exploring other, among all multi-methods, improves glandular hairs density and is considered to a kind of directly effective means.In glandular hairs, the synthetic secondary metabolite of specificity has a common feature, participates in only specifically expressing in glandular hairs of the synthetic key gene of these secondary metabolites.Therefore, utilizing engineered method regulation and control glandular hairs density is the plant genetic operational means that has much using value.
In recent years, in the simple gland hair developmental regulation of nonsecreting type, researchist has obtained a lot of achievements.Such as, in model plant Arabidopis thaliana, the developmental regulation mechanism of unicellular cilium is substantially clear.In the cotton that has Important Economic to be worth, the developmental regulation mechanism of unicellular cilium (cotton fibre) has also obtained a lot of achievements in research.And in secondary metabolism field, also fewer about the report of secretor type glandular hairs.Particularly, in the research of Chinese traditional herbs sweet wormwood, research mainly concentrates on the transcriptional control aspect to synthetic key gene, does not does not also regulate and control the report of glandular hairs density.
If by the variety analysis to sweet wormwood cultivar, the technology such as the expression analysis of transcription factor, from sweet wormwood, clone the gene that can regulate and control sweet wormwood glandular hairs density, so just can use genetic engineering means to improve sweet wormwood blade epidermal gland gross density, thereby improve the content of important secondary metabolite Artemisinin.By the variety analysis to two regional sweet wormwood cultivars, from sweet wormwood, clone a R3MYB class transcription factor, adopt genetic engineering means, this transcription factor interference carrier is transformed to sweet wormwood, can significantly improve sweet wormwood glandular hairs density, thereby improve the content of the secondary metabolites Artemisinin in sweet wormwood.A kind of new strategy and target gene are provided for improving artemislnin content.Provide theoretical basis for further studying the growth of sweet wormwood glandular hairs.
Summary of the invention
The object of the invention is to overcome deficiency of the prior art, a kind of sweet wormwood AaMYBL1 albumen coded sequence is provided, this genes encoding R3MYB class transcription factor (AaMYBL1), the density of its participation regulation and control sweet wormwood glandular hairs; Utilize transgenic technology that sweet wormwood AaMYBL1 transcription factor interference carrier is transformed to the glandular hairs density that sweet wormwood can Effective Regulation sweet wormwood epidermis, thereby improve the content of Artemisinin.Blade table cutaneous gland gross density at the common sweet wormwood of non-transgenic is 24 every square millimeter, suppresses the blade glandular hairs density of the transgene abrotanum of AaMYBL1 genetic expression and brings up to 34 every square millimeter.And the every total glandular hairs quantity of blade is brought up to 93683 (Fig. 1) from 61947.Corresponding artemislnin content is brought up to 12mg/g DW (Fig. 2) from the 8mg/g DW of non-transgenic sweet wormwood with it, this invention for providing high yield for the large-scale production of Artemisinin, to stablize source new drugs significant.
The present invention realizes by following technical scheme,
First aspect, the present invention relates to a kind of sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1, and the aminoacid sequence of described encoding sequence coding is as shown in SEQ ID NO:4.
Preferably, the nucleotide sequence of described encoding sequence is as shown in SEQ ID NO:3.
Second aspect, the present invention relates to a peptide species, and the aminoacid sequence of described polypeptide is as shown in SEQ ID NO:4.
The third aspect, the present invention relates to a kind of aforesaid sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1 in the application improving in artemislnin content, and described encoding sequence improves artemislnin content by improving sweet wormwood glandular hairs density.
Preferably, described application comprises the steps:
Step 1, is connected in sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1 on expression of plants regulating and controlling sequence, builds the plant expression vector containing sweet wormwood MYB-like class transcription factor encoding sequence;
Step 2, proceeds to Agrobacterium by the expression vector in step 1, and Agrobacterium is proceeded to sweet wormwood;
Step 3, by antibiotic-screening, obtains the transformant that contains sweet wormwood sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1, regeneration of transgenic plant, and the artemislnin content of described transfer-gen plant is improved.
Preferably, in step 2, described in proceed to and be specially: adopt freeze-thaw method to proceed to.
Fourth aspect, the present invention relates to a kind of method that improves content of artemisinin in sweet wormwood, comprises the steps:
(1) two sweet wormwood cultivar MYB class transcription factors are analyzed, from sweet wormwood cDNA library, be cloned into sweet wormwood R3MYB class transcription factor AaMYBL1;
(2) AaMYBL1 gene is connected in to expression regulation sequence operably, forms the plant RNA interference carrier containing AaMYBL1 gene;
(3) the plant interference expression vector containing AaMYBL1 gene is transformed to agrobacterium tumefaciens EH105, obtain the agrobacterium tumefaciens bacterial strain with this interference expression vector;
(4) utilize constructed agrobacterium tumefaciens bacterial strain to transform sweet wormwood, obtain resistance seedling through kantlex screening, then be transgene abrotanum seedling through the plant of PCR test positive;
(5) transgene abrotanum obtaining is carried out to glandular hairs density and pass through, obtain the transgene abrotanum that glandular hairs density significantly improves, and then obtain the sweet wormwood plant that artemislnin content improves.
Preferably, in step (4), the described transgene abrotanum plant detecting through PCR refers to, the detection primer of the synthetic AaMYBL1 gene of design respectively, carry out DNA cloning, the positive strain of observing object band under ultraviolet ray is transgene abrotanum plant, and described glandular hairs Density Detection refers under fluorescent microscope, sweet wormwood secretor type glandular hairs show bright fluorescence under specific wavelength, add up its quantity and density after taking pictures again.
Wherein, artemislnin content in the transgene abrotanum obtaining is carried out to HPLC-ELSD mensuration.
The present invention, by two sweet wormwood variety analysis, clones AaMYBL1 gene from sweet wormwood, builds the plant interference expression vector containing AaMYBL1 gene, with agrobacterium tumefaciens EH105 mediation, adopts leaf dish method that AaMYBL1 gene interference expression vector is transformed to sweet wormwood; PCR detects the integration of external source goal gene AaMYBL1, by fluorescence microscope and statistics glandular hairs density, has obtained the transgene abrotanum that glandular hairs density significantly improves; High performance liquid chromatography-light scattering detector (HPLC-ELSD) is measured content of artemisinin in sweet wormwood, and the artemislnin content in the transgene abrotanum that significantly improves of sweet wormwood epidermal gland gross density that shows to obtain also significantly improves.
In the present invention, can select various carrier known in the art, as commercially available carrier, comprise plasmid, clay etc.In the time producing sweet wormwood AaMYBL1 protein polypeptide of the present invention, sweet wormwood AaMYBL1 albumen coded sequence operationally can be connected in to expression regulation sequence, thereby form sweet wormwood AaMYBL1 protein expression vector.
As used herein, " being operationally connected in " refers to so a kind of situation, and some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA is operationally connected in polypeptid DNA so; If transcribing of promotor control sequence, it is to be operationally connected in encoding sequence so; If when ribosome bind site is placed in the position that can make its translation, it is to be operationally connected in encoding sequence so.Generally, " being operationally connected in " means adjacent, means in reading frame adjacent for secretion leader sequence.
In the present invention, related Agrobacterium is agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain EH105, this bacterial strain is open purchase (derive from Australian CAMBIA company, strain number is Gambar1) from the market.
Brief description of the drawings
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
The expression that Fig. 1 suppresses AaMYBL1 in sweet wormwood has improved glandular hairs density.
The expression that Fig. 2 suppresses AaMYBL1 in sweet wormwood has improved Artemisinin and dihydroartemisinic acid content.
Embodiment
Below embodiments of the invention are elaborated: the present embodiment is implemented under taking technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular clone: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
the clone of embodiment 1, sweet wormwood AaMYBL1 gene
1. the extraction of the total RNA of sweet wormwood genome
Get sweet wormwood leaf tissue, be placed in liquid nitrogen and grind, add in 1.5mL Eppendorf (EP) centrifuge tube that fills lysate, fully after vibration, according to the specification sheets extracted total RNA of TIANGEN test kit.Identify total RNA quality with denaturing formaldehyde gel electrophoresis, then on spectrophotometer, measure rna content.
2. the clone of sweet wormwood AaMYBL1 gene
Taking total RNA of being extracted as template, synthetic cDNA under the effect of PowerScript ThermoScript II; According to the sequences Design gene-specific primer of AaMYBL1 gene (SEQ ID NO:1 and SEQ ID NO:2), the AaMYBL1 gene that increases from total cDNA by PCR, and order-checking.
By above-mentioned steps, obtain the complete encoding sequence of this transcription factor in sweet wormwood (SEQ ID NO:3) and derived its albumen coded sequence (SEQ ID NO:4), wherein, initiator codon is ATG, terminator codon is TGA.
embodiment 2, containing the structure of the plant binary interference expression vector of AaMYBL1 gene
1. the structure of intermediate carrier pENTY-AaMYBL1
At non-conservative zone design upstream primer and the downstream primer of AaMYBL1 gene, in order to build interference carrier.Before upstream primer ATG base, add tetra-bases of CACC to build Gateway entry vector.According to the pENTR of Invitrogen company
tM/ D-
the operation steps of Cloning Kit, first obtains the fragment of AaMYBL1 with flush end enzymatic amplification, be connected to pENTR/D-TOPO carrier after reclaiming purifying by Gateway clone technology.
2. the structure of expression of plants interference carrier pHELLSGATE-AaMYBL1i
According to the LR of Invitrogen company
the operation of II Enzyme test kit, recombinate two of rna interference vector pHELLSGATE of the interference fragment of AaMYBL1 in pENTR-AaMYBL1 carrier can be formed in the recombination site of hairpin structure, obtain the rna interference vector pHELLSGATE-AaMYBL1i of AaMYBL1.
Sweet wormwood AaMYBL1 gene is connected in operably expression regulation sequence by the present embodiment, forms the expression of plants interference carrier containing AaMYBL1 hairpin structure, and this carrier can be used for regulating and controlling by metabolic engineering strategy the content of Artemisinin in Artemisia annuna.
embodiment 3, Agrobacterium tumefaciens mediated AaMYBL1 interference carrier genetic transformation sweet wormwood obtain transgene abrotanum plant
1. contain the acquisition of the agrobacterium tumefaciens engineering bacteria of AaMYBL1 interference expression vector
Adopt freeze-thaw method to proceed to agrobacterium tumefaciens (as EHA105 the plant binary interference expression vector containing AaMYBL1 in embodiment 2, for there is the biomaterial of public sale in market, can buy from Australian CAMBIA company, strain number is Gambar1), the performing PCR of going forward side by side checking.Result shows, is successfully building up in agrobacterium tumefaciens bacterial strain containing the plant binary interference expression vector of AaMYBL1.
2. Agrobacterium tumefaciens mediated AaMYBL1 gene transformation sweet wormwood
2.1. the preculture of explant
75% alcohol immersion 1min for seeds of southernwood, soak 20min with 20%NaClO again, aseptic water washing 3-4 time, blot surface-moisture with aseptic thieving paper, be inoculated in MS (the Murashige and Skoog without hormone, 1962), in solid medium, 25 DEG C, 16h/8h (light/dark) illumination cultivation, can obtain sweet wormwood aseptic seedling.After seedling grows to 5cm left and right, clip aseptic seedling leaf explant is for transforming.
2.2. the common cultivation of Agrobacterium and explant
By described leaf explant, forward in common culture medium (1/2MS+AS100 μ mol/L), drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing AaMYBL1 plant binary interference expression vector having activated, explant is fully contacted, 28 DEG C of dark 3d that cultivate with bacterium liquid.To drip at the leaf explant of the 1/2MS of the agrobacterium tumefaciens without goal gene liquid nutrient medium suspension as contrast.
2.3. the screening of resistance regeneration plant
The sweet wormwood explant of described common cultivation 3d is transferred to germination screening culture medium (MS+6-BA0.5mg/L+NAA0.05mg/L+Kan50mg/L+Cb500mg/L) upper in 25 DEG C, 16h/8h illumination cultivation, every two weeks succeeding transfer culture once, can obtain Kan resistance Multiple Buds after 2-3 subculture.Well-grown resistance Multiple Buds is cut to proceed on root media (1/2MS+Cb125mg/L) and be cultured to and take root, the sweet wormwood plant thereby acquisition Kan resistance is regenerated.
3. the PCR of transgene abrotanum plant detects
Design respectively forward primer design and reverse primer detects goal gene according to the 35S promoter region of expression cassette upstream, goal gene place and AaMYBL1.Result shows, utilizes designed PCR special primer, can amplify specific DNA fragment.And during as template, do not amplify any fragment taking non-transformed sweet wormwood genomic dna.
Described plant expression vector is transformed agrobacterium tumefaciens by the present embodiment, obtain the agrobacterium tumefaciens bacterial strain containing AaMYBL1 plant binary interference expression vector for transforming sweet wormwood, utilize constructed agrobacterium tumefaciens bacterial strain to transform sweet wormwood, obtain the transgene abrotanum plant detecting through PCR.The acquisition of transgene abrotanum plant obtains higher artemislnin content sweet wormwood strain for screening provides direct material.
embodiment 4, transgene abrotanum epidermal gland gross density and total glandular hairs are counted quantitative statistics
Use the BX51 model microscope of Olympus Corp, under the exciting light that is 450nm-480nm at wavelength, observe non-transgenic sweet wormwood and the blade that turns AaMYBL1i interference carrier sweet wormwood.Get the sweet wormwood blade of equal size, in 5 different position random sampling, statistics glandular hairs density.Measure the sweet wormwood blade total area, calculate the total glandular hairs quantity of each blade.
The glandular hairs density that turns in the present invention AaMYBL1i interference carrier sweet wormwood significantly improves.In the time that the glandular hairs density of non-transgenic sweet wormwood is 24 every square millimeter, suppresses the blade glandular hairs density of the transgene abrotanum of AaMYBL1 genetic expression and bring up to 34 every square millimeter.And the every total glandular hairs quantity of blade is brought up to 93683 (Fig. 1) from 61947.Fig. 1: the expression that suppresses AaMYBL1 in sweet wormwood has improved glandular hairs density.A, AaMYBL1 expression amount in half-quantitative detection AaMYBL1-RNAi sweet wormwood.B, AaMYBL1 expression amount in quantitative PCR detection AaMYBL1-RNAi sweet wormwood.C, fluorescence microscope wild-type sweet wormwood blade glandular hairs.D, fluorescence microscope AaMYBL1-RNAi sweet wormwood blade glandular hairs.E, the total glandular hairs quantity statistics of each blade.*, P<0.01 (T inspection).F, glandular hairs Statistics of Density.*, P<0.01 (T inspection).
embodiment 5, utilize HPLC-ELSD to measure artemislnin content in transgene abrotanum
The preparation of 1.HPLC-ELSD condition and system suitability and standardized solution
HPLC: adopt water alliance2695 system, chromatographic column is C-18 reverse phase silica gel post (SymmetryShieldTM C18,5 μ m, 250 × 4.6mm, Waters), moving phase is methyl alcohol: water, methyl alcohol: the volume ratio of water is 70:30,30 DEG C of column temperatures, flow velocity 1.0mL/min, sample size 10 μ L, sensitivity (AUFS=1.0), theoretical plate number is calculated and is not less than 2000 by Artemisinin peak.
ELSD: adopt water alliance2420 system, 40 DEG C of light scattering detector drift tube temperatures, scale-up factor (gain) is 7, nebulizer gas pressure 5bar;
Precision takes Artemisinin standard substance (Sigma company) 2.0mg 1mL methyl alcohol and dissolves completely, obtains 2mg/mL Artemisinin standard solution, be stored in-20 DEG C for subsequent use.
In the present invention, moving phase is methyl alcohol (methanol): water, and when ratio is 70%:30%, the retention time of Artemisinin is 5.1min, peak type is good.Theoretical plate number is calculated and is not less than 2000 by Artemisinin.
2. the making of typical curve
By described reference substance solution sample introduction 2 μ l respectively under corresponding chromatographic condition, 4 μ l, 6 μ l, 8 μ l, 10 μ l record collection of illustrative plates and chromatographic parameter, and with peak area (Y), to standard substance content, (X, μ g) carries out regression analysis respectively.By research, in the present invention, Artemisinin presents good log-log linear relationship within the scope of 4-20 μ g.The log-log equation of linear regression of Qinghaosu is: Y=1.28e+000X+4.71e+000, R=0.979546.
3. the preparation of sample and the mensuration of artemislnin content
Upper sweet wormwood plant, in and bottom get altogether the sweet wormwood blade that 2g is fresh, in 45 DEG C of baking ovens, dry to constant weight.Then strike inferior lobe and bud from the branch of drying, clay into power.Take about 0.1g dry powder in 2mL Eppendorf pipe, add 2mL ethanol, with 40W ultrasonication 30min, the centrifugal 10min of 5000rpm, gets 0.22 μ m membrane filtration for supernatant, can be used for the content of HPLC-ELSD mensuration Artemisinin.
Adopt HPLC-ELSD to measure artemislnin content, sample feeding volume is 20 μ l, go out the artemislnin content (mg) in sample according to peak area substitution linear regression Equation for Calculating, again divided by the artemisia leaf dry weight (g) of sample, thereby calculate the content of Artemisinin in sweet wormwood plant.
The transfer-gen plant that turns in the present invention AaMYBL1i interference carrier can significantly improve content of artemisinin in sweet wormwood.In the time that non-transformed common sweet wormwood content is 8mg/g DW, the content that the contemporaneously turns AaMYBL1i interference carrier Artemisinin in Artemisia annuna on average reaches 12mg/g DW, and its content is 1.5 times (Fig. 2) of non-transformed sweet wormwood content.Fig. 2: the expression that suppresses AaMYBL1 in sweet wormwood has improved Artemisinin and dihydroartemisinic acid content.A, turns in AaMYBL1RNAi sweet wormwood, uses HPLC to detect Artemisinin.B, turns in AaMYBL1RNAi sweet wormwood, uses HPLC to detect dihydroartemisinic acid content.Result is three mean values that repeat, and error line represents standard deviation.T-test inspection (*, P<0.01) for statistical study.
The present embodiment adopts HPLC-ELSD method to measure artemislnin content in transgene abrotanum, adopt conversion AaMYBL1i interference carrier metabolic engineering strategy to find that the expression of AaMYBL1 gene and the content of Artemisinin have obvious incidence relation, for the content that utilizes this gene to carry out expression study and then raising Artemisinin in Artemisia annuna provides strong experimental evidence.
Sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1 of the present invention can realize and improve artemislnin content in plant, and described encoding sequence is connected on expression of plants regulation and control carrier, builds the plant expression vector containing described encoding sequence; Expression vector is proceeded to Agrobacterium, Agrobacterium is proceeded to sweet wormwood; By antibiotic-screening, obtain the transformant that contains described encoding sequence, regeneration of transgenic plant; In the transgene abrotanum that the present invention obtains, the content of Artemisinin is subject to remarkable regulation and control, in the time that non-transformed common sweet wormwood content is 8mg/g DW, contemporaneously turns the content average out to 12mg/g DW of AaMYBL1i interference carrier Artemisinin in Artemisia annuna, and its content is 1.5 times of non-transformed sweet wormwood content.The invention provides the transcription factor encoding sequence of a regulation and control content of artemisinin in sweet wormwood, for utilizing this encoding sequence scale operation Artemisinin to lay a solid foundation.
Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (8)
1. a sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1, is characterized in that, the aminoacid sequence of described encoding sequence coding is as shown in SEQ ID NO:4.
2. sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1 as claimed in claim 1, is characterized in that, the nucleotide sequence of described encoding sequence is as shown in SEQ ID NO:3.
3. a peptide species, is characterized in that, the aminoacid sequence of described polypeptide is as shown in SEQ ID NO:4.
4. sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1 as claimed in claim 1 is in the application improving in artemislnin content, and described encoding sequence improves artemislnin content by improving sweet wormwood glandular hairs density.
5. application as claimed in claim 4, is characterized in that, described application comprises the steps:
Step 1, is connected in sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1 on expression of plants regulating and controlling sequence, builds the plant expression vector containing sweet wormwood MYB-like class transcription factor encoding sequence;
Step 2, proceeds to Agrobacterium by the expression vector in step 1, and Agrobacterium is proceeded to sweet wormwood;
Step 3, by antibiotic-screening, obtains the transformant that contains sweet wormwood sweet wormwood MYB-like class transcription factor encoding sequence AaMYBL1, regeneration of transgenic plant, and the artemislnin content of described transfer-gen plant is improved.
6. application according to claim 5, is characterized in that, in step 2, described in proceed to and be specially: adopt freeze-thaw method to proceed to.
7. a method that improves content of artemisinin in sweet wormwood, is characterized in that, comprises the steps:
(1) two sweet wormwood cultivar MYB class transcription factors are analyzed, from sweet wormwood cDNA library, be cloned into sweet wormwood R3MYB class transcription factor AaMYBL1;
(2) AaMYBL1 gene is connected in to expression regulation sequence operably, forms the plant RNA interference carrier containing AaMYBL1 gene;
(3) the plant interference expression vector containing AaMYBL1 gene is transformed to agrobacterium tumefaciens EH105, obtain the agrobacterium tumefaciens bacterial strain with this interference expression vector;
(4) utilize constructed agrobacterium tumefaciens bacterial strain to transform sweet wormwood, obtain resistance seedling through kantlex screening, then be transgene abrotanum seedling through the plant of PCR test positive;
(5) transgene abrotanum obtaining is carried out to glandular hairs density and pass through, obtain the transgene abrotanum that glandular hairs density significantly improves, and then obtain the sweet wormwood plant that artemislnin content improves.
8. the method for raising content of artemisinin in sweet wormwood as claimed in claim 7, it is characterized in that, in step (4), the described transgene abrotanum plant detecting through PCR refers to, the detection primer of the synthetic AaMYBL1 gene of design respectively, carry out DNA cloning, the positive strain of observing object band under ultraviolet ray is transgene abrotanum plant, described glandular hairs Density Detection refers under fluorescent microscope, sweet wormwood secretor type glandular hairs show bright fluorescence under specific wavelength, add up its quantity and density after taking pictures again.
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| CN106349352B (en) * | 2016-10-27 | 2019-09-24 | 上海交通大学 | Sweet wormwood transport protein AaPDR3 and its application |
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