CN101792774A - Agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method - Google Patents
Agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method Download PDFInfo
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- CN101792774A CN101792774A CN201010143761A CN201010143761A CN101792774A CN 101792774 A CN101792774 A CN 101792774A CN 201010143761 A CN201010143761 A CN 201010143761A CN 201010143761 A CN201010143761 A CN 201010143761A CN 101792774 A CN101792774 A CN 101792774A
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Abstract
The invention discloses an agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method, which belongs to the technical field of biotechnology. The method lays a foundation for deeply researching the medicinal value of dioscorea zingiberensis by transgene technology. The agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method comprises the following steps: (1) obtaining an embryonic callus acceptor material through a high-frequency regeneration system; (2) picking individual agrobacterium tumefaciens colonies, and adding the colonies into a YEP culture medium to perform culture; (3) adding the induced callus into the agrobacterium tumefaciens liquid, infecting the callus, taking an explant out and transferring the explant to a co-culture medium to perform culture and transition culture, and then transferring the cultured explant to a screening culture medium to perform screening; and (4) picking resistant callus to perform differentiation and rooting.
Description
Technical field
The present invention relates to technical field of biotechnology, particularly a kind of dioscorea zingiberensis transgene breeding technique.
Background technology
Rhizome of Peltate Yam (Dioscorea Zingiberensis C.H Wright) is a kind of important medicinal plant of monocotyledons Dioscoreaceae Wild yam, endemic species for China, diosgenin in its root stock (being called for short saponin) is an important source material of making steroid hormone class medicine, and content is the highest in Wild yam.The structure of diosgenin can obtain thousands of kinds of steroid hormone class medicines and aqueous saponin preparation after transforming, be called " medicinal gold " by the world of medicine.Therefore, Rhizome of Peltate Yam has very high economic worth as important medicine source plant resource.In recent years, along with the increase of Rhizome of Peltate Yam demand with excavate the guidance of the science that lacks in the process, make wild Rhizome of Peltate Yam resource day by day exhausted, the quality serious degradation.In addition, Rhizome of Peltate Yam is mainly bred by root stock, and needs kind of amount big, and the cost height has seriously hindered Rhizome of Peltate Yam plantation industrialized development.Therefore, cultivating the high-load Rhizome of Peltate Yam new lines of stable root stock high yield or sapogenin, is the main path of alleviating the current demand and supply contraction and solving cultivation back quality deterioration.
Rhizome of Peltate Yam mainly carries out vegetative propagation, and is bigger by traditional cross breeding method improvement proterties difficulty, and somaclonal variation, selection by mutation has blindness again.The method of present domestic raising diosgenin output mainly is to optimize cultivation technique, tissue culture and improve aspect such as planting environment.Wang Zhian, Wang Rizhao screen high yield saponin plant system with the method for tissue culture, and the field becomes seedling 6830 strains, and sapogenin average content 2.48% is compared with wild Rhizome of Peltate Yam, and saponin content has decline to a certain degree.Flourish all one's life, thank to green rosy cloud and utilize the Rhizome of Peltate Yam somaclonal variation to carry out the screening study of high yield clone respectively, but have the problem of plant regeneration difficulty, be difficult to obtain the new variety of plant of can big area promoting.Zhou Yuan etc. are by the Rhizome of Peltate Yam tetraploid plant of the method acquisition of in-vitro inducing, though reveal the feature that tangible cell volume is big, quantity is many in the test-tube plantlet phase table, but transplant to the land for growing field crops, its organ does not show huge feature, with the liploid plant no significant difference, and effective medicinal components does not detect.These technique means in the market do not realize the improvement of Rhizome of Peltate Yam on hereditary kind, and the saponin high yield plant that obtains by these screening screenings exists problems such as content instability.
Transgenic technology is a kind of technique means of effective orderly improvement proterties, has been widely used in the character improvement of various plants.
The objective of the invention is to utilize transgenic technology, carry out the improvement of Rhizome of Peltate Yam genetically engineered, alleviate the problem that exists in the current Rhizome of Peltate Yam industry, contribute for promoting Rhizome of Peltate Yam industry and featured agriculture Sustainable development.
Summary of the invention
The purpose of this invention is to provide a kind of agriculture bacillus mediated dioscorea zingiberensis transgene method, for laying a good foundation by the pharmaceutical use of transgenic technology further investigation Rhizome of Peltate Yam.
A kind of agriculture bacillus mediated technical scheme that dioscorea zingiberensis transgene method adopted of the present invention is finished in the following way: agriculture bacillus mediated dioscorea zingiberensis transgene method may further comprise the steps:
(1) obtains the embryo callus subculture acceptor material by high frequency regenerating system;
(2) the single bacterium colony of picking Agrobacterium joins in the YEP liquid nutrient medium and cultivates;
(3) callus that induces is joined in the Agrobacterium bacterium liquid, change on the common culture medium and to cultivate, after transition cultivates, change the enterprising row filter of screening culture medium over to through infecting, take out explant;
(4) selecting resistant calli breaks up and takes root.
The concrete operations step of a kind of agriculture bacillus mediated dioscorea zingiberensis transgene method of the present invention is:
(1) the Rhizome of Peltate Yam stem tuber is planted in the soil, when treating that also over-ground part length was to the 60cm left and right sides after the Rhizome of Peltate Yam stem tuber was sprouted, get its stem section, be cut into the single-unit stem that contains single bud, be inoculated in axillalry bud and induce solid medium MS+BA 1.0～2.0mg/L+NAA 0～0.5mg/L, pH 5.8,25 ± 1 ℃ of temperature, the dark photoperiod of 16h illumination/8h, illumination cultivation aseptic seedling in the culturing room of 1500～2000Lx light intensity (same under culturing room's condition);
(2) after aseptic seedling grows, get Rhizome of Peltate Yam aseptic seedling blade or stem section, be inoculated in solid callus inducing medium MS+BA 2.0～4.0mg/L+NAA 0～0.5mg/L, pH 5.8, and dark culturing can obtain callus after 1 month;
(3) bacterial concentration OD is adjusted in preparation engineering bacterium, cultivation and activation
600Value is 0.3～0.7, gets active high callus and puts into bacterium liquid, infects 15～45 minutes, blots bacterium liquid, places common cultivation solid medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L+AS 200 μ mol/L, and pH 5.3, cultivate 2～3 days;
(4) place the last transition of solid transitional culture medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L+cef, 400～500mg/L to cultivate a week earlier after taking off bacterium, the back is added 40～50mg/L Hyg and is carried out screening and culturing in substratum;
(5) getting resistant calli is placed on division culture medium MS+BA 2.0～3.0mg/L+NAA 0.2～0.5mg/L+cef 400～500mg/L+Hyg10～50mg/L, break up the root media 1/2MS+NAA 0.1～0.2mg/L+Hyg40～50mg/L root induction that places macroelement to reduce by half the unrooted regrowth that grows to 3cm under the illumination condition;
When (6) treating that regrowth grows fibrous root more than 10, open and seal film, one week of hardening, with distilled water flush away root substratum, be transplanted in the flowerpot that fills nutrition soil, with the MS macroelement mother liquor sprinkling plant of 50 times of dilutions, plastic covering film, remove plastic film after one week, obtain the resistance regeneration plant;
(7) the gus gene test material is taken from the resistant calli that obtains through after 1～4 step, will be through the blade of the resistance regeneration plant that obtains after 1～6 step, extract genomic dna with the SDS method, do positive control with plasmid DNA, DNA and the ddH2O of unconverted plant do negative control, carry out pcr amplification, detect the resistance regeneration plant.
In above-mentioned agriculture bacillus mediated dioscorea zingiberensis transgene method, used Rhizome of Peltate Yam stem tuber picks up from Xianju, Zhejiang Chinese yam planting base, plants in the Zhejiang Normal University nursery.
Description of drawings
Fig. 1 is a callus GUS coloration result photo.Wherein, the left side is unconverted callus in the photo, the callus of right side for transforming in the photo.
Fig. 2 is the structure iron that contains the T-DNA section among the segmental vector plasmid D1-6 of purpose.
Fig. 3 is to Rhizome of Peltate Yam resistant calli HPT primer PCR amplification scintigram.
Fig. 4 is to Rhizome of Peltate Yam resistant calli RRM1 primer PCR amplification scintigram.
Fig. 5 is the photo of Rhizome of Peltate Yam resistant calli screening and culturing after 1 month.
Fig. 6 is a dioscorea zingiberensis transgene regeneration plant aseptic seedling photo.
Fig. 7 is a dioscorea zingiberensis transgene regeneration plant photo.
Embodiment
Below by embodiment the present invention is made further and to specify, but the invention is not restricted to these embodiment.
One, induces the Rhizome of Peltate Yam callus
1, the Rhizome of Peltate Yam of the surface sterilization stem section of growing directly from seeds of learning from else's experience is cut into the single-unit stem that contains single bud, and be inoculated in axillalry bud and induce solid medium MS+BA1.0～2.0mg/L+NAA 0.5mg/L, 5.8,25 ± 1 ℃ of pH, illumination cultivation goes out aseptic seedling;
2, get each 120 of aseptic seedling blade and stem sections, blade is cut into about 0.5cm
2Size, stem section are cut into about 1cm size and are inoculated in solid callus inducing medium MS+BA 3.0mg/L, and 25 ± 1 ℃, dark culturing, month later 139 of the callus that obtain.
Two, Agrobacterium is infected conversion
1, engineering bacteria, cultivation and the activation of preparation band gus gene, the single bacterium colony of picking Agrobacterium joins that concussion is cultured to OD in the YEP liquid nutrient medium
600Value 0.4～0.6;
2, embryo callus is joined in the Agrobacterium bacterium liquid, infected 20～30 minutes, take out explant, change common cultivation solid-based MS+BA3.0mg/L+AS 200 μ mol/L over to, pH 5.3;
3, cultivate altogether after 2～3 days and take off bacterium, change solid transitional culture medium MS+BA 3.0mg/L+cef 400～500mg/L over to;
4, transition changes on solid screening culture medium MS+BA 3.0mg/L+cef 400～500mg/L+Hyg 40～50mg/L after one to two week of cultivation and filters out 6 of resistant callis.
Three, the check of resistant calli
1, the resistant calli that filters out is put in the GUS staining fluid that has configured, 37 ℃ are soaked the color of observing resistant calli after 24 hours;
2, experimental result shows, gus gene successfully is transferred in the callus, sees Fig. 1.
Embodiment 2
One, induces the Rhizome of Peltate Yam callus
1, the Rhizome of Peltate Yam of the surface sterilization stem section of growing directly from seeds of learning from else's experience is cut into the single-unit stem that contains single bud, and be inoculated in axillalry bud and induce solid medium MS+BA2.0mg/L+NAA 0.5mg/L, 5.8,25 ± 1 ℃ of pH, illumination cultivation goes out aseptic seedling;
2, get aseptic seedling stem sections, be inoculated in solid callus inducing medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L, 25 ± 1 ℃, dark culturing is inoculated 360, month later 294 of the callus that obtain;
Two, Agrobacterium is infected conversion
1, the engineering bacteria of preparation agrobacterium tumefaciens bacterial strain EHA105, expression vector is D2-8, cultivates and activation, the single bacterium colony of picking Agrobacterium joins that concussion is cultured to OD value 0.3～0.7 in the YEP liquid nutrient medium;
2, embryo callus is joined in the Agrobacterium bacterium liquid, infected 20～30 minutes, take out explant, change common cultivation solid-based MS+BA2.0mg/L～3.0mg/L+NAA 0～0.5mg/L+AS 200 μ mol/L over to, pH 5.3;
3, cultivate altogether after 2～3 days and take off bacterium, change solid transitional culture medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L+cef500mg/L over to;
4, transition changes on solid screening culture medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L+cef 500mg/L+Hyg40～50mg/L after one to two week of cultivation and filters out resistant calli.
Three, the check of resistant calli
Get resistant calli, extract genomic dna with the SDS method, PCR detects primer according to the design of hygromycin gene rFCA-RRM2 gene fragment.D2-8 makes positive control with plasmid, and non-transformed plant and ddH2O carry out PCR and detect as negative control.The result shows, the resistant calli of choosing has amplified the purpose fragment of 845bp and 330bp respectively, identical with the clip size of plasmid DNA amplification, and no purpose fragment in the amplified production with the negative contrast of unconverted plant leaf DNA illustrates that tentatively foreign gene has been incorporated in the genome of resistant calli.
One, induces the Rhizome of Peltate Yam callus
1, the Rhizome of Peltate Yam of the surface sterilization stem section of growing directly from seeds of learning from else's experience is cut into the single-unit stem that contains single bud, and be inoculated in axillalry bud and induce solid medium MS+BA2.0mg/L+NAA 0.5mg/L, 5.8,25 ± 1 ℃ of pH, illumination cultivation goes out aseptic seedling;
2, get aseptic seedling stem sections, be inoculated in solid callus inducing medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L, 25 ± 1 ℃, dark culturing can obtain callus after one month.
Two, Agrobacterium is infected conversion
1, the engineering bacteria of preparation agrobacterium tumefaciens bacterial strain EHA105, expression vector is D1-6, cultivates and activation, the single bacterium colony of picking Agrobacterium joins that concussion is cultured to OD value 0.3～0.7 in the YEP liquid nutrient medium;
2, embryo callus is joined in the Agrobacterium bacterium liquid, infected 15～45 minutes, take out explant, change common cultivation solid-based MS+BA2.0mg/L+NAA 0.5mg/L+AS 200 μ mol/L over to, pH 5.3;
3, cultivate altogether after 2～3 days and take off bacterium, change solid transitional culture medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L+cef 400～500mg/L over to;
4, transition changes on solid screening culture medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L+cef 400～500mg/L+Hyg 40～50mg/L after one to two week of cultivation and filters out resistant calli.
Three, the acquisition of resistant transgenic plant
1, resistant calli is transferred in the division culture medium that contains the 40mg/L Totomycin;
2, after 2 months, only produce green bud point and do not have seedling differentiation;
3, will select to press remove 1 month after resistant calli begin differentiation;
4, more sturdy when seedling, stretch out many blades after, changed over to and select to have pressed among root media 1/2MS+NAA 0.1～0.2mg/L+Hyg 40mg/L;
5, treat that the resistance regrowth grows the root of many sturdy, adularescent fine hair, plant growth condition is good, transplants after the hardening, and 90% plant grows newborn branches and leaves.
Four, the check of resistant plant
Get the blade of resistant plant, extract genomic dna with the SDS method, PCR detects primer according to the design of hygromycin gene rFCA-RRM1 gene fragment.D1-6 makes positive control with plasmid, and non-transformed plant and ddH2O carry out PCR and detect as negative control.The result shows, the resistant calli of choosing has amplified the purpose fragment of 845bp and 154bp respectively, identical with the clip size of plasmid DNA amplification, and no purpose fragment in the amplified production with the negative contrast of unconverted plant leaf DNA illustrates that tentatively foreign gene has been incorporated in the genome of resistant calli.
Utilize method of the present invention successfully to obtain 10 strains of transgenosis Rhizome of Peltate Yam positive plant.
Claims (2)
1. agriculture bacillus mediated dioscorea zingiberensis transgene method is characterized in that agriculture bacillus mediated dioscorea zingiberensis transgene method may further comprise the steps:
(1) obtains the embryo callus subculture acceptor material by high frequency regenerating system;
(2) the single bacterium colony of picking Agrobacterium joins in the YEP liquid nutrient medium and cultivates;
(3) callus that induces is joined in the Agrobacterium bacterium liquid, change on the common culture medium and to cultivate, after transition cultivates, change the enterprising row filter of screening culture medium over to through infecting, take out explant;
(4) selecting resistant calli breaks up and takes root.
2. a kind of agriculture bacillus mediated dioscorea zingiberensis transgene method according to claim 1 is characterized in that the concrete operations step of agriculture bacillus mediated dioscorea zingiberensis transgene method is:
(1) the Rhizome of Peltate Yam stem tuber is planted in the soil, when treating that also over-ground part length was to the 60cm left and right sides after the Rhizome of Peltate Yam stem tuber was sprouted, get its stem section, be cut into the single-unit stem that contains single bud, be inoculated in axillalry bud and induce solid medium MS+BA 1.0～2.0mg/L+NAA 0～0.5mg/L, pH 5.8,25 ± 1 ℃ of temperature, the dark photoperiod of 16h illumination/8h, illumination cultivation aseptic seedling in the culturing room of 1500～2000Lx light intensity (same under culturing room's condition);
(2) after aseptic seedling grows, get Rhizome of Peltate Yam aseptic seedling blade or stem section, be inoculated in solid callus inducing medium MS+BA 2.0～4.0mg/L+NAA 0～0.5mg/L, pH 5.8, and dark culturing can obtain callus after 1 month;
(3) bacterial concentration OD is adjusted in preparation engineering bacterium, cultivation and activation
600Value is 0.3～0.7, gets active high callus and puts into bacterium liquid, infects 15～45 minutes, blots bacterium liquid, places common cultivation solid medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L+AS 200 μ mol/L, and pH 5.3, cultivate 2～3 days;
(4) place the last transition of solid transitional culture medium MS+BA 2.0～3.0mg/L+NAA 0～0.5mg/L+cef, 400～500mg/L to cultivate a week earlier after taking off bacterium, the back is added 40～50mg/L Hyg and is carried out screening and culturing in substratum;
(5) getting resistant calli is placed on division culture medium MS+BA 2.0～3.0mg/L+NAA 0.2～0.5mg/L+cef 400～500mg/L+Hyg10～50mg/L, break up the root media 1/2MS+NAA 0.1～0.2mg/L+Hyg40～50mg/L root induction that places macroelement to reduce by half the unrooted regrowth that grows to 3cm under the illumination condition;
When (6) treating that regrowth grows fibrous root more than 10, open and seal film, one week of hardening, with distilled water flush away root substratum, be transplanted in the flowerpot that fills nutrition soil, with the MS macroelement mother liquor sprinkling plant of 50 times of dilutions, plastic covering film, remove plastic film after one week, obtain the resistance regeneration plant;
(7) the gus gene test material is taken from the resistant calli that obtains through after 1～4 step, will be through the blade of the resistance regeneration plant that obtains after 1～6 step, extract genomic dna with the SDS method, do positive control with plasmid DNA, DNA and the ddH20 of unconverted plant do negative control, carry out pcr amplification, detect the resistance regeneration plant.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728481A (en) * | 2018-04-28 | 2018-11-02 | 河南师范大学 | A kind of method of RHIIZOMA DIOSCOREAE from Henan of China genetic transformation |
| CN110904148A (en) * | 2019-11-19 | 2020-03-24 | 湖北大学 | Plant expression vector for synthesizing taxadiene, strain DZGGPPSTS and dioscorea zingiberensis and preparation method thereof |
| CN112931205A (en) * | 2021-03-04 | 2021-06-11 | 广东丰绿源生物医药科技有限公司 | Dioscorea composita tissue culture breeding method |
| CN114711144A (en) * | 2022-05-05 | 2022-07-08 | 天津博奥聚能生物科技有限公司 | Rapid propagation method and culture medium for dioscorea composita tissue culture seedlings |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000014260A1 (en) * | 1998-09-03 | 2000-03-16 | University Of Florida | Methods for controlling viral diseases in plants |
| CN1551916A (en) * | 2001-09-05 | 2004-12-01 | ������ɽ���� | Seed specific 7Salpha promoter for expressing genes in plants |
| CN101358234A (en) * | 2008-08-02 | 2009-02-04 | 江苏省中国科学院植物研究所 | ISSR-SCARs marker for identifying main confusion matrix original dioscorea collettii hook.F. with chinese herb rhizoma dioscoreae hypoglaucae |
-
2010
- 2010-04-12 CN CN201010143761A patent/CN101792774A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000014260A1 (en) * | 1998-09-03 | 2000-03-16 | University Of Florida | Methods for controlling viral diseases in plants |
| CN1551916A (en) * | 2001-09-05 | 2004-12-01 | ������ɽ���� | Seed specific 7Salpha promoter for expressing genes in plants |
| CN101358234A (en) * | 2008-08-02 | 2009-02-04 | 江苏省中国科学院植物研究所 | ISSR-SCARs marker for identifying main confusion matrix original dioscorea collettii hook.F. with chinese herb rhizoma dioscoreae hypoglaucae |
Non-Patent Citations (2)
| Title |
|---|
| 任椒矫: "盾叶薯蓣（Dioscorea zingiberensis C.H.Wright）再生体系的建立和农杆菌介导的rFCA-RRM1基因片段转化研究", 《浙江师范大学硕士学位论文》, 31 December 2009 (2009-12-31) * |
| 任椒矫等: "盾叶薯蓣高频再生体系的建立", 《浙江师范大学学报（自然科学版）》, vol. 32, no. 2, 31 December 2009 (2009-12-31), pages 216 - 221 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728481A (en) * | 2018-04-28 | 2018-11-02 | 河南师范大学 | A kind of method of RHIIZOMA DIOSCOREAE from Henan of China genetic transformation |
| CN110904148A (en) * | 2019-11-19 | 2020-03-24 | 湖北大学 | Plant expression vector for synthesizing taxadiene, strain DZGGPPSTS and dioscorea zingiberensis and preparation method thereof |
| CN112931205A (en) * | 2021-03-04 | 2021-06-11 | 广东丰绿源生物医药科技有限公司 | Dioscorea composita tissue culture breeding method |
| CN114711144A (en) * | 2022-05-05 | 2022-07-08 | 天津博奥聚能生物科技有限公司 | Rapid propagation method and culture medium for dioscorea composita tissue culture seedlings |
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Application publication date: 20100804 |