CN102499037B - Method for rapid propagation of genetically modified sweet wormwood by hydroponics - Google Patents
Method for rapid propagation of genetically modified sweet wormwood by hydroponics Download PDFInfo
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Classifications
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- Y02P60/216—
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A method for rapid propagation of genetically modified sweet wormwood by hydroponics belongs to the technical field of hydroponic planting and includes: placing explants of genetically modified sweet wormwood in hydroponic induction culture solution for light-tight cultivation to obtain adventitious roots which are transplanted to transplant matrix; and obtaining a great amount of genetically modified sweet wormwood plants through domestication. By the method, a great amount of genetically modified sweet wormwood seedlings can be propagated in a short time, genetic stability of the sweet wormwood is retained, and significance to large-scale popularization of genetically modified sweet wormwood, guarantee of sufficient high-quality sweet wormwood seedlings and increase of sweet wormwood yield is realized.
Description
Technical field
What the present invention relates to is the method in a kind of water planting planting technology field, specifically a kind of method that adopts the water culture technology rapidly breeding transgene abrotanum.
Background technology
Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.Qinghaosu (artemisinin) is a kind of sesquiterpene lactone compound that contains the peroxide bridge structure that separates from its acrial part, be the medicine of present the most effective treatment malaria of generally acknowledging in the world, particularly have quick-acting and characteristics low toxicity for encephalic malaria and anti-chloroquine malaria.At present, the method for the most effective treatment malaria of world health organisation recommendations is exactly qinghaosu conjoint therapy (ACTs).In addition, along with to the qinghaosu pharmacological research progressively deeply, scientist finds that qinghaosu and derivative thereof also have anti-inflammatory, anti-schistosome, antitumor and immunoregulatory function.As seen qinghaosu is a kind of natural drug that has potentiality.
The main source of qinghaosu is the acrial part extraction from the sweet wormwood plant at present, however the content of Artemisinin in Artemisia annuna very low (0.01%-1%), so that the large-scale commercial applications production of this medicine is restricted.Because the qinghaosu complex structure, manually synthetic difficulty is large, yields poorly, and cost is high, does not have feasibility.In addition, qinghaosu content in callus is lower than 0.1% of dry weight, and the highest in bud also only have 0.16% of dry weight, and most researchers does not detect qinghaosu in root.Therefore utilize the feasibility of tissue cultivation and cell engineering production qinghaosu also not high.
Along with qinghaosu biosynthesis pathway research gradually deeply and the development of Plant secondary metabolic engineering, the content that utilizes transgenic technology to improve Artemisinin in Artemisia annuna is a kind of method that has much prospect.2009, the classmates such as this laboratory Zhang have delivered at " Biotechnol.Appl.Biochem. " (" biotechnology and applied biochemistry ") and have been entitled as " Development of transgenic Artemisia annua (Chinese wormwood) plants with an enhanced content of artemisinin; an effective anti-malarial drug, by hairpin-RNA mediated gene silencing " (" making up the RNAi carrier also utilizes transgenic technology that the content of anti-malaria medicaments qinghaosu in transgene abrotanum is improved.") paper, reported by transgenic technology and can obviously improve this experimental result of active ingredient artemislnin content in the sweet wormwood.At present, this laboratory has obtained a plurality of different lines and the higher transgene abrotanum of artemislnin content.Yet transgene abrotanum is generally heterozygote, does not possess genetic stability, and gene separates among the offspring, is unfavorable for agricultural production.Cultivating pure line cultivar from transgene abrotanum needs long time, and expends a large amount of manpower and materials, and this is so that the spread of transgene abrotanum has been subject to certain restriction.
Have at present the method by micro adventitious bud technology rapidly breeding transgene abrotanum plant in the available data.Yet, micro adventitious bud technology rapidly breeding transgene abrotanum plant need to be through the detoxification of transgene abrotanum explant, inducing of transgene abrotanum micro adventitious bud, the elongation of transgene abrotanum micro adventitious bud, taking root and a plurality of steps such as transplanting of transgene abrotanum regeneration plant of transgene abrotanum indefinite bud, process are comparatively complicated, and wherein multistep needs sterile working, careless slightly with regard to causing that easily the sweet wormwood explant pollutes, cause the numerous failure of expansion.And in the During Detoxification of transgene abrotanum explant, need to use the poisonous reagents such as mercuric chloride, easily operating personnel's health is caused damage, surrounding environment is polluted.
By the water culture technology rapidly breeding transgene abrotanum, for keeping the genetic stability of transgene abrotanum, significant to the output of the spread of transgene abrotanum, the southernwood seedlings that ensures sufficient high-quality, raising qinghaosu.And can overcome all drawbacks in the existing transgene abrotanum water planting multiplication technique, save time, low consumption is a kind of biotechnology that has application prospect.The relevant report of the employing water culture technology rapidly breeding transgene abrotanum that present not yet discovery and theme of the present invention are mentioned.
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of method that adopts the water culture technology rapidly breeding transgene abrotanum is provided, the method of prescription and water planting fast-propagation by the water planting culture fluid, set up the method with the water culture technology rapidly breeding transgene abrotanum, can breed at short notice a large amount of transgene abrotanum seedlings, and the genetic stability that keeps transgene abrotanum, significant for the output of the spread of transgene abrotanum, the southernwood seedlings that ensures sufficient high-quality, raising qinghaosu.
The present invention is achieved by the following technical solutions, and the present invention obtains being transplanted to transplanting medium behind the adventive root by the transgene abrotanum explant being placed water planting induce the culture fluid lucifuge to cultivate, and obtains the transgene abrotanum plant by domestication.
Described transgene abrotanum explant refers to: the plant binary expression vector that contains the sweet wormwood endogenous gene changes Agrobacterium tumefaciems over to (such as EHA105, the biomaterial that public offering is arranged for market, can buy from Australian CAMBIA company, strain number is Gambar 1), Agrobacterium tumefaciens mediated lower, pass through again the preculture of explant, the common cultivation of Agrobacterium and explant, resulting transgenic positive plant after the PCR of the screening of resistance regeneration plant and transgene abrotanum plant detects.
Described water planting induces the component of culture fluid to be: 1g/L full water soluble fertilizer (spend intact, 20-20-20+TE, general fertilizer) and 10mg/L heteroauxin (IAA), surplus is water.
Described lucifuge is cultivated and referred to: the transgene abrotanum explant branch that the children is tender is transferred to water planting and is induced in the culture fluid, and lucifuge is cultivated and taken root in 14 days under 25 ± 3 ℃ of environment, i.e. adventive root.
Described transplanting refers to: choose the plant of robust growth, be transplanted in the cave dish or flowerpot that transplanting medium is housed.
Described transplanting medium is that volume ratio is 1: 1 perlite: the mixture of peat.
The present invention adopts the method for water planting, but Fast-propagation goes out a large amount of transgene abrotanum seedlings, and the genetic stability that keeps transgene abrotanum, for the spread of transgene abrotanum, the southernwood seedlings of the sufficient high-quality of guarantee, and the output of raising qinghaosu is significant.Simultaneously, this method can obtain the transgene abrotanum tip of a root a large amount of, easy to drawing materials, for the chromosomal preparation of transgene abrotanum and adopt fluorescence in situ hybridization technique that the transgene abrotanum plant is carried out chromosome mapping to lay a solid foundation.
Embodiment
The below elaborates to embodiments of the invention, and present embodiment is implemented under take 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.
Embodiment 1
The structure that contains the plant binary expression vector of sweet wormwood endogenous gene
1. the structure of intermediate carrier pCAMBIA2300::p35S-gus-nos
Selecting pBI121 and pCAMBIA2300 is primary element, makes up double base plant expression vector pCAMBIA2300::p35S-gus-nos.Particularly, HindIII and EcoRI double digestion pBI121 and pCAMBIA2300 plasmid; Reclaim gus expression cassette and the pCAMBIA2300 large fragment of pBI121; Connect and reclaim product, transformation and selection is taken out the plasmid enzyme restriction checking.
2. the structure of plant expression vector pCAMBIA2300::p35S-sweet wormwood endogenous gene-no
Take described pCAMBIA2300::p35S-gus-nos as expression vector, replace gus gene on it with the sweet wormwood endogenous gene.Particularly, BamHI/SacI double digestion pGEMT-easy+ sweet wormwood endogenous gene and pCAMBIA2300::p35S-gus-nos reclaim sweet wormwood endogenous gene and pCAMBIA2300::p35S-gus-nos large fragment, connect to transform, the picking monoclonal, the extraction plasmid is done the PCR detection and enzyme is cut checking.
Present embodiment is connected in expression regulation sequence operably with the sweet wormwood endogenous gene, forms the plant expression vector that contains the sweet wormwood endogenous gene, and this expression vector can be used for improving by the metabolic engineering strategy content of Artemisinin in Artemisia annuna.
Embodiment 2
Agrobacterium tumefaciens mediated sweet wormwood endogenous gene genetic transformation sweet wormwood obtains the transgene abrotanum plant
1. contain the acquisition of sweet wormwood endogenous gene double base plant expression vector Agrobacterium tumefaciems engineering bacteria
Change the plant binary expression vector that contains the sweet wormwood endogenous gene among the embodiment 1 over to Agrobacterium tumefaciems (such as EHA105, the biomaterial that public offering is arranged for market, can buy from Australian CAMBIA company, strain number is Gambar 1), the performing PCR of going forward side by side checking.The result shows, contains sweet wormwood endogenous gene plant binary expression vector and successfully is building up in the Agrobacterium tumefaciems bacterial strain.
2. Agrobacterium tumefaciens mediated sweet wormwood endogenous gene transforms sweet wormwood
2.1. the preculture of explant
Seeds of southernwood is with 75% alcohol immersion 1min, soak 20min with 20%NaClO again, aseptic water washing 3-4 time, blot surface moisture with aseptic blotting paper, be inoculated in MS (the Murashige and Skoog without hormone, 1962) in the solid culture medium, 25 ℃, 16h/8h (light/dark) illumination cultivation can obtain the sweet wormwood aseptic seedling.After seedling grew to about 5cm, clip aseptic seedling leaf explant was used for transforming.
2.2. the common cultivation of Agrobacterium and explant
With described leaf explant, forward in the common culture medium (1/2MS+AS 100 μ mol/L), dropping contains the good described 1/2MS suspension that contains the Agrobacterium tumefaciems engineering bacteria of sweet wormwood endogenous gene plant binary expression vector of activation, and explant is fully contacted with bacterium liquid, 28 ℃ of dark 3d that cultivate.Take drip without the leaf explant of the 1/2MS liquid nutrient medium suspension of the Agrobacterium tumefaciems of genes of interest as contrast.
2.3. the screening of resistance regeneration plant
It is upper in 25 ℃, 16h/8h illumination cultivation that the described sweet wormwood explant of cultivating altogether 3d is transferred to germination screening and culturing base (MS+6-BA 0.5mg/L+NAA0.05mg/L+Kan 50mg/L+Cb 500mg/L), per two all subcultures are cultivated once, can obtain Kan resistance Multiple Buds through behind 2-3 subculture.Well-grown resistance Multiple Buds cut to change over to be cultured on the root media (1/2MS+Cb 125mg/L) take root, thereby obtain Kan resistance regeneration sweet wormwood plant.
3. the PCR of transgene abrotanum plant detects
Design respectively the forward primer design and reverse primer detects genes of interest according to genes of interest place expression cassette p35s-sweet wormwood endogenous gene-no sequence p35s and sweet wormwood endogenous gene.The result shows, utilizes designed PCR special primer, can amplify special dna fragmentation.And during as template, do not amplify any fragment take non-transformed sweet wormwood genomic DNA.
Present embodiment transforms Agrobacterium tumefaciems with described plant expression vector, obtain to be used for transforming the Agrobacterium tumefaciems bacterial strain that contains sweet wormwood endogenous gene plant expression vector of sweet wormwood, utilize constructed Agrobacterium tumefaciems bacterial strain to transform sweet wormwood, obtain the transgene abrotanum plant that detects through PCR.The acquisition of transgene abrotanum plant provides direct material for the sweet wormwood strain that screening obtains higher artemislnin content.
Embodiment 3
Utilize HPLC-ELSD to measure artemislnin content in the transgene abrotanum
1.HPLC-ELSD the preparation of condition and system suitability and standard liquid
HPLC: adopt water alliance 2695 systems, chromatographic column is C-18 reverse phase silica gel post (SymmetryShieldTM C18,5 μ m, 250 * 4.6mm, Waters), mobile phase is methyl alcohol: water, and methyl alcohol: the volume ratio of water is 70: 30,30 ℃ of column temperatures, flow velocity 1.0mL/min, sample size 10 μ L, sensitivity (AUFS=1.0), theoretical cam curve is calculated by the qinghaosu peak and is not less than 2000.
ELSD: adopt water alliance 2420 systems, 40 ℃ of EISD drift tube temperatures, amplification coefficient (gain) is 7, nebulizer gas pressure 5bar;
Precision takes by weighing qinghaosu standard items (Sigma company) 2.0mg and dissolves fully with 1mL methyl alcohol, obtains 2mg/mL qinghaosu standard solution, be stored in-20 ℃ for subsequent use.
Mobile phase is methyl alcohol (methanol) among the present invention: water, and when ratio was 70%: 30%, the retention time of qinghaosu was 5.1min, the peak type is good.Theoretical cam curve is calculated by qinghaosu and is not less than 2000.
2. the making of calibration curve
With described reference substance solution difference sample introduction 2 μ l under corresponding chromatographic condition, 4 μ l, 6 μ l, 8 μ l, 10 μ l record collection of illustrative plates and chromatographic parameter carry out regression analysis with peak area (Y) to standard items content (X, μ g) respectively.By research, qinghaosu presents good log-log linear relation among the present invention in 4-20 μ g scope.The log-log equation of linear regression of Qinghaosu is: Y=1.28e+000X+4.71e+000, R=0.979546.
3. the mensuration of the preparation of sample and artemislnin content
Upper the sweet wormwood plant, in and the bottom get altogether the fresh sweet wormwood blade of 2g, in 45 ℃ of baking ovens, dry to constant weight.Then the branch from oven dry strikes inferior lobe and bud, clays into power.Take by weighing about 0.1g dry powder in 2mL Eppendorf pipe, add 2mL ethanol, process 30min with the 40W ultrasonic, the centrifugal 10min of 5000rpm gets supernatant with 0.22 μ m membrane filtration, namely can be used for the content that HPLC-ELSD measures qinghaosu.
Adopt HPLC-ELSD to measure artemislnin content, the sample feeding volume is 20 μ l, calculate artemislnin content (mg) in the sample according to peak area substitution linear regression equation, again divided by the artemisia leaf dry weight (g) of sample, thereby calculate the content of qinghaosu in the sweet wormwood plant.
Embodiment 4
1) drawing materials of transgene abrotanum explant and inducing of water planting
Clip contains the tender stem segments of growing point as explant from the transgene abrotanum plant.The transgene abrotanum explant is transferred to water planting induce culture fluid, it is 1g/L full water soluble fertilizer (spend intact, 20-20-20+TE, general fertilizer)+10mg/L heteroauxin (IAA) that described water planting is induced culture fluid.Cultivation temperature is 25 ± 3 ℃, and lucifuge is cultivated and grow adventive root after 14 days around explant.Length is 1-2cm.The water planting inductivity reaches more than 80%.
2) transplanting of transgene abrotanum regeneration plant
Choose the plant of robust growth, the culture fluid of flush away root is transplanted in the cave dish that transplanting medium is housed, and the component pearl rock in the described transplanting medium and peat formula rate are 1: 1.By taming for 1 week, can obtain the normal transgene abrotanum plant of raised growth, transplanting survival rate is 100%.
By research, a kind of method that adopts the water culture technology rapidly breeding transgene abrotanum is provided in the present invention, for Fast-propagation goes out a large amount of transgene abrotanum seedlings, and the genetic stability that keeps transgene abrotanum, significant for the output of the spread of transgene abrotanum, the southernwood seedlings that ensures sufficient high-quality, raising qinghaosu.Simultaneously, this method can obtain the transgene abrotanum tip of a root a large amount of, easy to drawing materials, for the chromosomal preparation of transgene abrotanum and adopt fluorescence in situ hybridization technique that the transgene abrotanum plant is carried out chromosome mapping to lay a solid foundation.
Claims (5)
1. method that adopts the water culture technology rapidly breeding transgene abrotanum, it is characterized in that, obtain being transplanted to transplanting medium behind the adventive root by the transgene abrotanum explant being placed water planting induce the culture fluid lucifuge to cultivate, obtain a large amount of transgene abrotanum plant by domestication;
Described transgene abrotanum explant refers to: the plant binary expression vector that contains the sweet wormwood endogenous gene changes Agrobacterium tumefaciems over to, Agrobacterium tumefaciens mediated lower, pass through again the preculture of explant, the common cultivation of Agrobacterium and explant, the tender stem segments that contains growing point of clip on the resulting transgenic positive plant after the PCR of the screening of resistance regeneration plant and transgene abrotanum plant detects;
Described water planting induces the component of culture fluid to be: 1 g/L full water soluble fertilizer and 10 mg/L heteroauxins, surplus is water.
2. method according to claim 1 is characterized in that, described Agrobacterium tumefaciems is the EHA105 type.
3. method according to claim 1 is characterized in that, described lucifuge is cultivated and referred to: the transgene abrotanum explant branch that the children is tender is transferred to water planting and induced in the culture fluid, and lucifuge is cultivated and taken root in 14 days under 25 ± 3 ℃ of environment, i.e. adventive root.
4. method according to claim 1 is characterized in that, described transplanting refers to: choose the plant of robust growth, be transplanted in the cave dish or flowerpot that transplanting medium is housed.
5. method according to claim 1 is characterized in that, described transplanting medium is that volume ratio is the perlite of 1:1: the mixture of peat.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103404215A CN102499037B (en) | 2011-11-01 | 2011-11-01 | Method for rapid propagation of genetically modified sweet wormwood by hydroponics |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103404215A CN102499037B (en) | 2011-11-01 | 2011-11-01 | Method for rapid propagation of genetically modified sweet wormwood by hydroponics |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102499037A CN102499037A (en) | 2012-06-20 |
| CN102499037B true CN102499037B (en) | 2013-05-01 |
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| CN110100736B (en) * | 2019-06-20 | 2020-09-22 | 南京农业大学 | Water culture propagation method for thesium Chinese tissue culture seedlings |
| CN114766338B (en) * | 2022-04-19 | 2023-04-07 | 永州市农业科学研究所 | Method for improving sweet wormwood inbred line cultivation and hybrid parent combining ability detection efficiency by utilizing water culture cuttage |
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| CN101180952B (en) * | 2007-12-13 | 2011-03-30 | 中国科学院植物研究所 | Tissue culture rapid breeding method of Chinese medicine abrotanum |
| CN101213915B (en) * | 2008-01-15 | 2012-05-23 | 重庆大学 | Abrotanum vegetative propagation method capable of remaining high content of arteannuin |
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