CN104593409A - Chinese chestnut transgenic method based on agrobacterium rhizogenes mediation - Google Patents
Chinese chestnut transgenic method based on agrobacterium rhizogenes mediation Download PDFInfo
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Abstract
The invention provides a Chinese chestnut transgenic method based on agrobacterium rhizogenes mediation, which comprises the following steps: infecting hypocotyledonary axis of yanshanhong chestnut aseptic seedlings, a chestnut variety mainly cultivated in Beijing, by utilizing agrobacterium rhizogenes MSU440 containing DsRed reporter gene, to obtain a co-transformed plant, thus further obtaining transformed hairy roots. By adopting the method of transforming the hypocotyledonary axis of yanshanhong chestnut, not only can the transformation period (one month) be greatly shortened, but also the experimental results can be directly observed; by performing root development study through yanshanhong chestnut in-vitro hairy root systems, cultivation conditions can be artificially controlled to eliminate the influence brought by environment factors; in addition, the method can overcome the observation and sampling difficulties of the yanshanhong chestnut seedling roots due to soil environment, so that the situation that the chestnut root study lags behind relatively is hopefully changed. The invention provides a fast and reliable method for verifying the chestnut gene function, and also provides a new transformation method for the genetic improvement of chestnut genes.
Description
Technical field
The invention belongs to biological technical field, relate to plant transgenic technology, relate generally to a kind of Chinese chestnut transgenic method based on Agrobacterium rhizogenes mediation.
Background technology
Chinese chestnut is one of important dry fruit fruit tree in the world.At present, at home, focus mostly in physiological-biochemical level to the research of Chinese chestnut, as fruit attributional analysis etc., and less about the report of gene excavating and functional analysis; Secondly more to the research of Chinese chestnut over-ground part, as bud differentiation etc., and less to the research of root system.For the research of Chinese chestnut critical biological proterties, gene excavating and functional verification play a part indispensable, and in Chinese Chestnut, transformation system is not also set up, and the gene function checking based on transformation system have not been reported.Lack the transformation system of a set of stability and high efficiency at present, become the bottleneck of restriction Chinese chestnut molecular biology research.
Agrobacterium tumefaciens (Agrobacterium tumefaciens) and Agrobacterium rhizogenes (Agrobacterium rhizogenes) are all Gram-negative soil bacterias, apply its Ti-plasmids and Ri plasmid respectively to infect plants wound and enter cell, and it contains one section of T-DNA is inserted into (Zhou, 2010 in Plant Genome; Cheng, 2011), the T-DNA of Agrobacterium rhizogenes there is rol A-D gene group, T-DNA has tms gene, the injury that can bring out infected plant produces a large amount of hairly root, and the gene on Ri plasmid T-DNA does not affect the regeneration of plant, transformation efficiency is high, succeeds in Radix Glycyrrhizae (GlycyrrhizauralensisFisch.), tobacco (NicotianatabacumL.) etc. at present.The hairly root of Agrobacterium rhizogenes induction belongs to Hormone autotrophy, have that active constituent content is high, Physiology and biochemistry and genetic stability, be easy to carry out the features such as operation control, and the hairly root of inducing can be identified from morphological level, root is grown thickly more, multi-branched, many hairs, and apogetropism, these all can differentiate with unconverted.
At present, Chinese chestnut transgenosis have not been reported, and therefore, for the genetic improvement of Chinese chestnut and gene excavating and functional analysis, it is very necessary for setting up a set of transgenic technology that is agriculture bacillus mediated, efficient stable.The object of the invention is to utilize Agrobacterium rhizogenes to set up ' Yanshan Mountain Chinese chestnut ' hairly root induction system, thus a kind of transgenic method based on Agrobacterium rhizogenes mediation is provided.
Summary of the invention
The transgenic method that the present invention can provide a kind of Agrobacterium rhizogenes to mediate, obtains ' Yanshan Mountain Chinese chestnut ' hairly root.
Technical scheme proposed by the invention comprises the following steps:
(1) Chinese chestnut aseptic seedling is prepared;
(2) Agrobacterium rhizogenes activation, Agrobacterium rhizogenes is rule on LB solid medium, to obtain single bacterium colony, picking list bacterium colony on LB liquid nutrient medium, through light culture;
(3) infect explant, Chinese chestnut aseptic seedling hypocotyl is excised, smears the Agrobacterium rhizogenes activated in wound, then place it on Dual culture base, through illumination cultivation;
(4) Dual culture;
(5) hairy root culture;
(6) fluoroscopic examination.
Further, wherein said Agrobacterium rhizogenes is MSU440, tool 35S::DsRed reporter gene on carrier.
Further, wherein the middle LB solid medium of step (2) or LB liquid nutrient medium contain 50 ~ 100mg/L spectinomycin (spectinomycin).
Further, wherein in step (2), Agrobacterium rhizogenes is rule on LB solid medium, be inverted through 28 ± 2 DEG C of dark places after cultivation 48-72h and obtain single bacterium colony.
Further, wherein the light culture system described in step (2) through 28 ± 2 DEG C, shaking speed 200 ± 20rpm light culture 48h.
Further, wherein step (3) also to comprise with the syringe needle speckling with Agrobacterium in the wound of Chinese chestnut aseptic seedling radicle and wound circumference, then places it in and is covered with on the Dual culture substratum of 1/2 aseptic filter paper, Parafilm film sealing 2/3.
Further, wherein the Dual culture system of step (4) is through 21 ± 2 DEG C of illumination 16h, light culture 8h, so repeatedly cultivates 5d.
Further, wherein step (1) comprises and strips ' Yanshan Mountain Hong Li ' fruit and seed, clear water rinses rear 70-75% ethanol disinfection 2 ± 1min well, then 3-4% hypochlorite disinfectant 15-20min, aseptic distillation water rinse more than 3 times, be inoculated in xylophyta basic medium (WPM substratum), 25 ± 2 DEG C of light culture 2-3d obtain aseptic seedling.
Further, wherein the succeeding transfer culture of step (5) comprise by contaminate after explant change on hairy root culture base from Dual culture substratum, Parafilm film seals entirely, 24 DEG C ~ 25 DEG C illumination 16h, light culture 8h, cultivates 25d so repeatedly, changes weekly a subculture.
Further, wherein step (6) comprise adopt Stereo fluorescence microscope Fluirescence observation is carried out to cotransformation plant.
The transgenic technology that the present invention is mediated by Agrobacterium rhizogenes, (1 month) hairly root can be obtained at short notice, save search time, and transformation efficiency is higher, about 60% (positive plant sum accounts for the per-cent always infecting plant sum) can be reached.
Specific implementation method:
Now in conjunction with specific embodiments, further each step of the present invention is described in detail:
1, containing the expression vector MSU440 Agrobacterium rhizogenes of 35S::DsRed reporter: presented by TonBisseling seminar of Wageningen University.
2, ' the acquisition of Yanshan Mountain Hong Li ' aseptic seedling explant: ' Yanshan Mountain Hong Li ' picks up from Chinese chestnut testing station of Huairou District, Beijing City forestry bureau, strip ' Yanshan Mountain Hong Li ' fruit and seed, clear water rinses rear 70%-75% ethanol disinfection 2min (shaking table shakes) well, then 3-4% hypochlorite disinfectant 15-20min (shaking table shakes), aseptic distillation water rinse more than 3 times, be inoculated in WPM substratum, 25 ± 1 DEG C of light culture 2-3d obtain aseptic seedling;
3, the activation of Agrobacterium rhizogenes: Agrobacterium rhizogenes is being rule containing on the LB solid medium of 50mg/L spectinomycin, 28 DEG C of dark places obtain single bacterium colony after being inverted and cultivating 48-72h, picking list bacterium colony contains on 50mg/L spectinomycin (spectinomycin) LB liquid nutrient medium in fresh, 28 DEG C of 220rpm light culture 48h.Drawing bacterium liquid coats on the LB solid medium containing 50mg/L spectinomycin (spectinomycin), for infecting explant after 48-72h again;
4, explant is infected: first by ' Yanshan Mountain Hong Li ' aseptic seedling hypocotyl is excised, the Agrobacterium rhizogenes MSU440 activated is smeared in wound, its wound and wound circumference is stabbed again with the syringe needle speckling with Agrobacterium, then place it in and be covered with on the Dual culture substratum of 1/2 aseptic filter paper, Parafilm film sealing 2/3.
5, Dual culture: 21 ± 1 DEG C of illumination 16 hours, light culture 8h, cultivates 5d so repeatedly.
6, hairy root culture: change on hairy root culture base, the same step of method (4), Parafilm film seals entirely, 24 DEG C ~ 25 DEG C illumination 16 hours, light culture 8h, so repeatedly, cultivates 25d, changes weekly a subculture.
7, fluoroscopic examination: utilize Stereo fluorescence microscope to carry out Fluirescence observation to cotransformation plant, result shows: turning 35S::DsRed plant root visible whole piece root all has stronger red fluorescence.
In the present invention main medium and reagent as follows, wherein LB substratum dissolve after not adjust pH:
Xylophyta basic medium (WPM substratum): WPM+3% sucrose+0.7% agar
Dual culture substratum: g/L (see table 1)
Table 1
| Composition/L | Gram or mL/L | Gram or mL/L |
| Macroelement 10x | 100mL | 200mL |
| 15mM Fe-Citrate trianion | 1mL | 2mL |
| Trace element | 1mL | 2mL |
| CaSO 4·2H 2O | 0.25g | 0.5g |
Above reagent is joined in sterilizing bottle, is settled to 1L, adjust pH to 6.6 ± 0.2, then add 1% agar, 121 DEG C of sterilizing 15 ~ 20min, after cool to room temperature, add 125 μ L NH
4nO
3(1M).
Wherein: macroelement 10X (g/1L): KH
2pO
4: 1.2, K
2hPO
4: 3.6, MgSO
47H
2o:2.5, NaSO
4: 1.0
Trace element (mg/100mL): MnSO
4: 100, ZnSO
47H
2o:25, CuSO
45H
2o:25, H
3bO
4: 25, NaMoO
42H
20:0.5
Hairy root culture base (see table 2)
Table 2
| Composition | 1L |
| 20x conc.SH-A | 50mL |
| 20x conc.UM-C | 50mL |
| 1% sucrose | 10g |
| 1M MES pH5.8 | 3mL |
Above reagent is joined in sterilizing bottle, is settled to 1L, adjust pH to 5.8 ± 0.2 with 1M KOH, then add 1% agar, 121 DEG C of sterilizing 30min, after cool to room temperature, add 500 μ g/mL cefotaxime sodiums (Cefotaxine).
Wherein, 20X conc.SH-A, see table 3
Table 3
| Composition | g/liter | Conc.in medium(mg/l) |
| KNO 3 | 50 | 2500 |
| MgSO 4·7H 2O | 8 | 400 |
| NH 4H 2PO 4 | 6 | 300 |
| CaCl 2·2H 2O | 4 | 200 |
| MnSO 4·H 20 | 0.15 | 10 |
| H 3BO 3 | 0.1 | 5 |
| ZnSO 4·7H 2O | 0.02 | 1 |
| KI | 0.02 | 1 |
| CuSO 4·5H 2O | 0.004 | 0.2 |
| NaMoO 4·2H 2O | 0.002 | 0.1 |
| CoCl 2·6H 2O | 0.002 | 0.1 |
| FeSO 4·7H 2O* | 0.3 | 15 |
| Na 2EDTA* | 0.4 | 20 |
* be first dissolved in 100ml water at 50 DEG C joining before in total solution.
20x conc.UM-C, see table 4
Table 4
| Micro-element | g/liter | Conc.in medium(mg/l) |
| Inositol | 2 | 100 |
| Nicotinic acid | 0.1 | 5 |
| Vb6 | 0.2 | 10 |
| Vb1 | 0.2 | 10 |
| Glycine | 0.04 | 2 |
SH-A and UM-C is kept at-20 DEG C; 1M MES pH value of solution 5.8 (1M KOH) need be got out in advance, be kept at-20 DEG C.
The present invention proposes a kind of Chinese chestnut transgenic method based on Agrobacterium rhizogenes mediation, the Agrobacterium rhizogenes MSU440 containing 35S::DsRed reporter gene is utilized to infect Beijing main cultivation Cultivars of Chinese Chestnut " Yanshan Mountain Hong Li " aseptic seedling hypocotyl, obtain cotransformation plant, and then obtain transformed hairy root." Yanshan Mountain Hong Li " the Regenerated from Hypocotyl Explants method adopting the present invention to propose not only can shorten the transformation period (1 month) greatly, also can see experimental result intuitively; The research carrying out root system development aspect with " Yanshan Mountain Hong Li " in vitro hairly root system can manual control culture condition, eliminates the impact that environmental factors is brought; In addition, the method can overcome, and ' Yanshan Mountain Hong Li ' seedling root system is given because of edatope and is observed, samples the difficulty brought, thus is expected to change the present situation that Chinese chestnut root system research falls behind relatively.The present invention is that the checking of Chinese chestnut gene function provides a kind of fast and reliable method, also for the genetic improvement of Chinese chestnut gene provides a kind of new method for transformation.
Claims (10)
1. a Chinese chestnut transgenic method for Agrobacterium rhizogenes mediation, is characterized in that, comprise the following steps:
Preparation Chinese chestnut aseptic seedling;
Agrobacterium rhizogenes activate, Agrobacterium rhizogenes is rule on LB solid medium, to obtain single bacterium colony, picking list bacterium colony on LB liquid nutrient medium, through light culture;
Infect explant, Chinese chestnut aseptic seedling hypocotyl is excised, smears the Agrobacterium rhizogenes activated in wound, then place it on Dual culture base, through illumination cultivation;
Dual culture;
Hairy root culture;
Fluoroscopic examination.
2. the Chinese chestnut transgenic method of Agrobacterium rhizogenes mediation according to claim 1, wherein said Agrobacterium rhizogenes is MSU440, tool 35S: on carrier:
dsRedreporter gene.
3. the Chinese chestnut transgenic method of Agrobacterium rhizogenes mediation according to claim 1, wherein in step (2), LB solid medium or LB liquid nutrient medium contain 50 ~ 100mg/L spectinomycin (spectinomycin).
4. according to the Chinese chestnut transgenic method of the Agrobacterium rhizogenes mediation described in claim 1 or 3, wherein in step (2), Agrobacterium rhizogenes is rule on LB solid medium, be inverted through 28 ± 2 DEG C of dark places after cultivation 48-72h and obtain single bacterium colony.
5., according to the Chinese chestnut transgenic method of the Agrobacterium rhizogenes mediation described in claim 1 or 3, wherein the light culture system described in step (2) is through 28 ± 2 DEG C, shaking speed 200 ± 20rpm light culture 48h.
6. the Chinese chestnut transgenic method of Agrobacterium rhizogenes mediation according to claim 1, wherein step (3) also comprises and repeatedly piercing through at the wound of Chinese chestnut aseptic seedling radicle and wound circumference with the syringe needle speckling with Agrobacterium, then place it in and be covered with on the Dual culture substratum of 1/2 aseptic filter paper, Parafilm film sealing 2/3.
7. the Chinese chestnut transgenic method of Agrobacterium rhizogenes mediation according to claim 1, wherein the Dual culture system of step (4) is through 21 ± 2 DEG C of illumination 16h, light culture 8h, so repeatedly cultivates 5d.
8. the Chinese chestnut transgenic method of Agrobacterium rhizogenes mediation according to claim 1, wherein step (1) comprises and strips ' Yanshan Mountain Hong Li ' fruit and seed, clear water rinses rear 70-75% ethanol disinfection 2 ± 1min well, then 3-4% hypochlorite disinfectant 15-20min, aseptic distillation water rinse more than 3 times, be inoculated in xylophyta basic medium (WPM substratum), 25 ± 2 DEG C of light culture 2-3d obtain aseptic seedling.
9. the Chinese chestnut transgenic method of Agrobacterium rhizogenes mediation according to claim 1, wherein the hairy root culture of step (5) comprise by contaminate after explant change on hairy root culture base from Dual culture substratum, Parafilm film seals entirely, 24 DEG C ~ 25 DEG C illumination 16h, light culture 8h, so repeatedly cultivate 25d, change weekly a subculture.
10. the Chinese chestnut transgenic method of Agrobacterium rhizogenes according to claim 1 mediation, wherein step (6) comprises and adopts Stereo fluorescence microscope to carry out Fluirescence observation to cotransformation plant.
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Cited By (6)
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| CN104855288A (en) * | 2015-05-11 | 2015-08-26 | 中国科学院西北高原生物研究所 | Establishment method of Tibetan drug przewalskia tangutica maxim autonomous rooting system |
| CN105861543A (en) * | 2016-06-29 | 2016-08-17 | 云南纳博生物科技有限公司 | Method for building agrobacterium tumefaciens mediated peperomiate trophylla efficient transfection system |
| CN105941149A (en) * | 2016-05-20 | 2016-09-21 | 北京农学院 | Castanea mollissima somatic embryo induction method |
| CN107034231A (en) * | 2017-06-12 | 2017-08-11 | 北京农学院 | A kind of gene transformation method of Chinese Chestnut |
| CN109652444A (en) * | 2019-02-26 | 2019-04-19 | 中国科学院武汉植物园 | The peach root system stable conversion method and its application that agrobacterium rhizogenes mediates |
| CN109679993A (en) * | 2019-02-22 | 2019-04-26 | 北京林业大学 | A kind of construction method for the genetically modified plants that agrobacterium rhizogenes mediates |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104855288A (en) * | 2015-05-11 | 2015-08-26 | 中国科学院西北高原生物研究所 | Establishment method of Tibetan drug przewalskia tangutica maxim autonomous rooting system |
| CN105941149A (en) * | 2016-05-20 | 2016-09-21 | 北京农学院 | Castanea mollissima somatic embryo induction method |
| CN105941149B (en) * | 2016-05-20 | 2018-06-29 | 北京农学院 | A kind of abductive approach of Chinese chestnut somatic embryo |
| CN105861543A (en) * | 2016-06-29 | 2016-08-17 | 云南纳博生物科技有限公司 | Method for building agrobacterium tumefaciens mediated peperomiate trophylla efficient transfection system |
| CN105861543B (en) * | 2016-06-29 | 2019-08-23 | 云南纳博生物科技有限公司 | A method of establishing mediated by agriculture bacillus fourleaf peperomia herb rotaring redyeing system |
| CN107034231A (en) * | 2017-06-12 | 2017-08-11 | 北京农学院 | A kind of gene transformation method of Chinese Chestnut |
| CN107034231B (en) * | 2017-06-12 | 2021-01-19 | 北京农学院 | Gene transformation method of Chinese chestnut |
| CN109679993A (en) * | 2019-02-22 | 2019-04-26 | 北京林业大学 | A kind of construction method for the genetically modified plants that agrobacterium rhizogenes mediates |
| CN109679993B (en) * | 2019-02-22 | 2020-11-27 | 北京林业大学 | Construction method of agrobacterium rhizogenes-mediated transgenic plant |
| CN109652444A (en) * | 2019-02-26 | 2019-04-19 | 中国科学院武汉植物园 | The peach root system stable conversion method and its application that agrobacterium rhizogenes mediates |
| CN109652444B (en) * | 2019-02-26 | 2022-04-22 | 中国科学院武汉植物园 | Agrobacterium rhizogenes-mediated stable transformation method for peach root system and application thereof |
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Application publication date: 20150506 |