CN104593410A - Agrobacterium rhizogenes mediated strawberry gene transferring method - Google Patents
Agrobacterium rhizogenes mediated strawberry gene transferring method Download PDFInfo
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- CN104593410A CN104593410A CN201510003996.6A CN201510003996A CN104593410A CN 104593410 A CN104593410 A CN 104593410A CN 201510003996 A CN201510003996 A CN 201510003996A CN 104593410 A CN104593410 A CN 104593410A
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Abstract
The invention provides an Agrobacterium rhizogenes mediated strawberry gene transferring method. Agrobacterium rhizogenes MSU440 containing 35S::DsRed reporter gene is utilized to infect forest strawberry aseptic seedling hypocotyl to obtain a co-transformation plant, so as to obtain a transformed hairy root. According to the proposed forest strawberry hypocotyl transformation method, the transformation period which is one and half months can be shortened greatly, and the experimental result is also intuitive; an in-vitro strawberry hairy root system can provide a rapid and reliable method for strawberry gene function identification.
Description
Technical field
The invention belongs to biological technical field, relate to plant transgenic technology, relate generally to a kind of strawberry transgenic method based on Agrobacterium rhizogenes mediation.
Background technology
Strawberry is the Rosaceae (Rosaccae) Fragaria (Fragaria) perennial root perennial herb fruit tree.The domestic research to strawberry at present focuses mostly in Physiology and biochemistry, and less for the report of gene excavating and functional analysis; Secondly, more as fruit color etc. to the fruit research of strawberry, and less to the root system research of strawberry.In addition, forest strawberry (Fragaria vesca) sequenced genomes completes, and carries out gene annotation, and this is significant to excavating the functional gene controlling important character.Lack the transformation system of a set of stability and high efficiency at present, become the bottleneck of restriction strawberry molecular biology research.
Agrobacterium tumefaciens (Agrobacterium tumefaciens) and Agrobacterium rhizogenes (Agrobacteriumrhizogenes) 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 (Glycyrrhizauralensis Fisch.), 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, strawberry transgenosis have not been reported, and therefore, for the genetic improvement of strawberry 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 forest strawberry 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 forest strawberry hairly root.
Technical scheme proposed by the invention comprises the following steps:
(1) strawberry 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, strawberry 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 comprises the strawberry aseptic seedling radicle after infecting to be placed on 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 forest strawberry seed, deepfreeze, 4 DEG C of Refrigerator stores 7 days; Seed disinfection: 75% Ethanol Treatment 5min, the NaClO reprocessing 5min of 1%, sterile distilled water rinses 2-3 time; Be inoculated on MS base, light culture 10 days.
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 first quarter moon) hairly root can be obtained at short notice, save search time, and transformation efficiency is higher, about 70% (positive plant sum accounts for the per-cent always infecting explant plant sum) can be reached.
Embodiment
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 Ton Bisseling seminar of Wageningen University.
2, forest strawberry seed, deepfreeze, 4 DEG C of Refrigerator stores 7 days; Seed disinfection: 75% Ethanol Treatment 5min, the clorox NaClO reprocessing 5min of 1%, sterile distilled water rinses 2-3 time; Be inoculated on MS base, light culture 10 days.
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 excised by forest strawberry aseptic seedling hypocotyl, smear the Agrobacterium rhizogenes MSU440 activated in wound, 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 (MS substratum): MS+1% sucrose+0.9% agar
Dual culture substratum: g/L (see table 1)
Table 1
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 NH4NO3 (1M).
Wherein: macroelement 10X (g/1L): KH2PO4:1.2, K2HPO4:3.6, MgSO47H2O:2.5, NaSO4:1.0
Trace element (mg/100mL): MnSO4:100, ZnSO47H2O:25, CuSO45H2O:25, H3BO4:25, NaMoO42H20:0.5
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
* be first dissolved in 100ml water at 50 DEG C joining before in total solution.
20x conc.UM-C, see table 4
Table 4
SH-A and UM-C is kept at-20 DEG C; 1M MES pH value of solution 5.8 (1MKOH) need be got out in advance, be kept at-20 DEG C.
The present invention proposes a kind of strawberry transgenic method based on Agrobacterium rhizogenes mediation, utilize the Agrobacterium rhizogenes MSU440 containing 35S::DsRed reporter gene to infect forest strawberry aseptic seedling hypocotyl, obtain cotransformation plant, and then obtain transformed hairy root.The strawberry Regenerated from Hypocotyl Explants method adopting the present invention to propose not only can shorten the transformation period (1 first quarter moon) greatly, and experimental result is also more directly perceived.Strawberry in vitro hairly root system is that the checking of strawberry gene function provides a kind of fast and reliable method.
The above; be only patent of the present invention preferably embodiment; but the protection domain of patent of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope of patent diselosesll of the present invention; the change that can expect easily or replacement, within the protection domain that all should be encompassed in patent of the present invention.
Claims (10)
1. a strawberry transgenic method for Agrobacterium rhizogenes mediation, is characterized in that, comprise the following steps:
(1) strawberry 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.
2. the strawberry transgenic method of Agrobacterium rhizogenes mediation according to claim 1, wherein said Agrobacterium rhizogenes is MSU440, tool 35S: on carrier:
dsRedreporter gene.
3. the strawberry transgenic method of Agrobacterium rhizogenes mediation according to claim 1, wherein said LB solid medium or LB liquid nutrient medium are containing 50 ~ 100mg/L spectinomycin (spectinomycin).
4. according to the strawberry transgenic method of the Agrobacterium rhizogenes mediation described in claim 1 or 3, wherein 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 strawberry transgenic method of the Agrobacterium rhizogenes mediation described in claim 1 or 3, wherein said light culture system is through 28 ± 2 DEG C, shaking speed 200 ± 20rpm light culture 48h.
6. the strawberry transgenic method of Agrobacterium rhizogenes according to claim 1 mediation, wherein the strawberry aseptic seedling radicle after infecting is placed on and is covered with on the Dual culture substratum of 1/2 aseptic filter paper by step (3), Parafilm film sealing 2/3.
7. the strawberry 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 strawberry transgenic method of Agrobacterium rhizogenes according to claim 1 mediation, wherein step (1) comprises forest strawberry seed, deepfreeze, 4 DEG C of Refrigerator stores 7 days; Seed disinfection: 75% Ethanol Treatment 5min, the NaClO reprocessing 5min of 1%, sterile distilled water rinses 2-3 time; Be inoculated on MS base, light culture 10 days.
9. the strawberry 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 strawberry 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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| CN111235176A (en) * | 2020-01-17 | 2020-06-05 | 北京农学院 | Genetic transformation system |
| CN114561422A (en) * | 2022-02-28 | 2022-05-31 | 安徽农业大学 | Induction method of transgenic hairy roots growing on strawberry stolons and application of transgenic hairy roots |
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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 |
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| CN114561422A (en) * | 2022-02-28 | 2022-05-31 | 安徽农业大学 | Induction method of transgenic hairy roots growing on strawberry stolons and application of transgenic hairy roots |
| CN114561422B (en) * | 2022-02-28 | 2023-12-12 | 安徽农业大学 | Induction method of transgenic hairy roots grown by strawberry stolon and application thereof |
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Application publication date: 20150506 |