CN109652438A - A kind of Wheat genetic transformation method based on wheat seed embryo injection Agrobacterium - Google Patents
A kind of Wheat genetic transformation method based on wheat seed embryo injection Agrobacterium Download PDFInfo
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- CN109652438A CN109652438A CN201811590260.3A CN201811590260A CN109652438A CN 109652438 A CN109652438 A CN 109652438A CN 201811590260 A CN201811590260 A CN 201811590260A CN 109652438 A CN109652438 A CN 109652438A
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- wheat
- agrobacterium
- genetic transformation
- transformation method
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
Abstract
The invention discloses a kind of Wheat genetic transformation methods based on wheat seed embryo injection Agrobacterium, are related to genetic transfoumation field, and the Wheat genetic transformation method is the following steps are included: 1) receptor prepares；2) prepared by bacteria suspension；3) embryo injects Agrobacterium-mediated Transformation.Cause to solve traditional Efficiency of Wheat Transformation technology due to needing excised cotyledon with regenerated process at high cost, the problem that period is long and regeneration rate is low, target gene is imported wheat living body by being directly injected into the method for carrying target gene Agrobacterium in embryo by the present invention, combining target gene PCR detection technique has obtained heritable transgenic progeny, the valuable instrument and equipments such as particle gun are not needed, it is low in cost, common laboratory can operate, and there is good regeneration rate, inorganization culture and regenerative process, period is shorter, and without wheat genotypes Dependence Problem, it draws materials simultaneously conveniently, it can operate throughout the year.
Description
Technical field
The present invention relates to genetic transfoumation field, specifically a kind of wheat genetic based on wheat seed embryo injection Agrobacterium
Method for transformation.
Background technique
Wheat is one of cereal crops important in the world, is the important cultivation cereal of grass family Triticum.In order to improve
The yield of wheat, it usually needs using Efficiency of Wheat Transformation technology by external source target gene as required in vitro using artificial weight
Group imports wheat, and then supplements and improve traditional crossbreeding, substantially reduces breeding cycle.Wherein, Efficiency of Wheat Transformation
Technology imports target gene in wheat body, and stablize the technology of heredity and expression objective trait, is that wheat transgenic is educated
Kind, gene editing breeding, the basis of gene cloning and functional study.
Currently, Efficiency of Wheat Transformation technology mainly has via Particle Bombardment Transformation and Agrobacterium-mediated genetic transformation method two major classes,
These two types of method for transformation require excised cotyledon and regenerated process, cause the at high cost of this process, period long and again
Raw rate is low.Therefore, a kind of Wheat genetic transformation method based on wheat seed embryo injection Agrobacterium of simple and effective low cost is designed,
The problem of as current urgent need to resolve.
Summary of the invention
The purpose of the present invention is to provide a kind of Wheat genetic transformation methods based on wheat seed embryo injection Agrobacterium, with solution
Certainly the problems mentioned above in the background art, by directly with mature seed embryos be conversion object, by directly being infused in embryo
Enter to carry target gene Agrobacterium and target gene is imported into wheat living body, combining target gene PCR detection technique, having obtained can
The transgenic progeny of heredity.
To achieve the above object, the invention provides the following technical scheme:
A kind of Wheat genetic transformation method based on wheat seed embryo injection Agrobacterium, which is characterized in that it includes following step
It is rapid:
1) receptor prepares: the healthy seed for choosing receptor is rinsed 3-5 times with distilled water, is then with mass concentration
1.5% sodium hypochlorite carries out soaking disinfection 15min, then uses distilled water immersion 10min, then extremely with distilled water flushing seed
No thimerosal smells are placed in the culture dish of paving two layers of filter paper, are added water to not having seed just and hair of sprouting at 25 DEG C
Bud obtains chitting piece A, spare；
2) prepared by bacteria suspension: Agrobacterium tumefaciems progress Multiplying culture being obtained bacterium solution, is subsequently poured into the EP pipe of 50ml simultaneously
Centrifugation 10min is carried out with the rate of 4000rpm at 4 DEG C, then removes supernatant and 10ml is added or so fresh antibiotic-free
YEB culture solution adjusts OD value to 0.9-1.2, acetosyringone is then added to final concentration of 200 μM, then in incubated at room temperature 12
Hour is activated, and Agrobacterium bacterium solution B is obtained, spare；
3) embryo injects Agrobacterium-mediated Transformation: (the germination when coleoptile length in picking step 1) reaches 0.5-1.0cm
Time is 42-48 hour) chitting piece A, put to air-drying at superclean bench air port 1 hour, then with the medical skin test needle of 1ml
It draws Agrobacterium bacterium solution B obtained in the step 2) of 0.5ml and injection bacterium solution is carried out to chitting piece A, then bacterium solution will be injected
Chitting piece A carry out air-dry 30 minutes, then transfer load in the nutritive cube for filling vermiculite and turf, after sprinkling profoundly water at room temperature after
It is continuous to cultivate.
As a further solution of the present invention: in step 1), the receptor is that (foreign countries turn Common Wheat Varieties Bobwhite
Gene often uses receptor kind) and Yanzhan4110 (domestic large area varieties of plant).
As further scheme of the invention: in step 2), the Agrobacterium tumefaciems is by beta-glucuronidase
(GUS) encoding gene is connected to the Agrobacterium tumefaciems containing pWMB110 plasmid obtained on plasmid vector pWMB110.
As further scheme of the invention: in step 2), the Multiplying culture is to draw containing pWMB110 plasmid
Agrobacterium tumefaciems is added into the YEB fluid nutrient medium of 50ml, then carries out shaking bacterium 12 hours in 28 DEG C of rates with 200rpm,
?.
As further scheme of the invention: the YEB fluid nutrient medium includes that the concentration of 50 μ l is the card of 50mg/ml
The concentration of that mycin and 50 μ l are the rifampin of 50mg/ml.
As the present invention further scheme: in step 3), the injection bacterium solution be by the plumule of chitting piece A with
Embryo is upward, fixes chitting piece A with tweezers on the other hand, and the other hand injects bacterium solution into embryo along the plumule of chitting piece A, directly
To have bacterium solution from plumule end ooze out.
As further scheme of the invention: in step 3), the weight ratio of the vermiculite and turf is 2:1.
Wheat genetic transformation method the answering in wheat transgenic breeding based on wheat seed embryo injection Agrobacterium
With.
Compared with prior art, the beneficial effects of the present invention are:
1, the present invention is by carrying target base by being directly injected into embryo directly with mature seed embryos to convert object
Because target gene is imported wheat living body by the method for Agrobacterium, combining target gene PCR detection technique has obtained heritable turn
Progeny, transformant frequency reach 26.6%, solve traditional Efficiency of Wheat Transformation technology because needing excised cotyledon
Lead to the problem that at high cost, the period is long and regeneration rate is low with regenerated process；
2, the present invention provides a kind of novel genetic transfoumations for avoiding excised cotyledon process, turn with common wheat
Genetic method is compared, and is had three advantages: 1) not being needed the valuable instrument and equipments such as particle gun, low in cost, common laboratory is equal
It can operate, and there is good regeneration rate；2) inorganization culture and regenerative process, the period is shorter, and relies on without wheat genotypes
Problem；3) materials are convenient, can operate throughout the year, simple and effective low cost has a vast market foreground.
Detailed description of the invention
Fig. 1 is the signal that plasmid vector pWMB110 in the Wheat genetic transformation method of Agrobacterium is injected based on wheat seed embryo
Figure.
Fig. 2 is to inject T in the Wheat genetic transformation method of Agrobacterium based on wheat seed embryo0For transgenic wheat blade GUS
Dyeing detection figure.
Fig. 3 is to inject T in the Wheat genetic transformation method of Agrobacterium based on wheat seed embryo1For transgenic wheat Bar gene
It detects and schemes with gus gene PCR.
Specific embodiment
Present invention will be explained in further detail in the following with reference to the drawings and specific embodiments.Following embodiment will be helpful to
Those skilled in the art further understands the present invention, but the invention is not limited in any way.It should be pointed out that ability
For the those of ordinary skill in domain, without departing from the inventive concept of the premise, various modifications and improvements can be made.These
Belong to protection scope of the present invention.
Embodiment 1
A kind of Wheat genetic transformation method based on wheat seed embryo injection Agrobacterium, which is characterized in that it includes following step
It is rapid:
1) receptor prepares: healthy seed distilled water flushing 3-5 times for choosing receptor, is then 1.5% with mass concentration
Sodium hypochlorite carry out soaking disinfection 15min, then distilled water immersion 10min is used, then with distilled water flushing seed to without disappearing
Venom smell is placed in the culture dish of 2 layers of filter paper of paving, is added water to not having seed just and germination of sprouting at 25 DEG C, must be sent out
Bud seed A, it is spare；The receptor is Common Wheat Varieties Bobwhite (external transgenosis often uses receptor kind) and Yanzhan4110
(domestic large area varieties of plant)；
2) prepared by bacteria suspension: Agrobacterium tumefaciems progress Multiplying culture being obtained bacterium solution, is subsequently poured into the EP pipe of 50ml simultaneously
Centrifugation 10min is carried out with the rate of 4000rpm at 4 DEG C, then removes supernatant and 10ml is added or so fresh antibiotic-free
YEB culture solution adjusts OD value to 0.9-1.2, acetosyringone is then added to final concentration of 200 μM, then in incubated at room temperature 12
Hour is activated, and Agrobacterium bacterium solution B is obtained, spare；
Wherein, the Agrobacterium tumefaciems is that beta-glucuronidase (GUS) encoding gene is connected to plasmid vector
The Agrobacterium tumefaciems containing pWMB110 plasmid obtained on pWMB110, sees Fig. 1；The Multiplying culture is to draw to contain
The Agrobacterium tumefaciems of pWMB110 plasmid is added into the YEB fluid nutrient medium of 50ml, then 28 DEG C of rates with 200rpm into
Row shakes bacterium 12 hours；The YEB fluid nutrient medium includes that the concentration of 50 μ l is the kanamycins and 50 μ l of 50mg/ml
Concentration is the rifampin of 50mg/ml；
3) embryo injects Agrobacterium-mediated Transformation: (the germination when coleoptile length in picking step 1) reaches 0.5-1.0cm
Time is 42-48 hour) chitting piece A, put to air-drying at superclean bench air port 1 hour, then with the medical skin test needle of 1ml
It draws Agrobacterium bacterium solution B obtained in the step 2) of 0.5ml and injection bacterium solution is carried out to chitting piece A, then bacterium solution will be injected
Chitting piece A carry out air-dry 30 minutes, then transfer load in the nutritive cube for filling vermiculite and turf, after sprinkling profoundly water at room temperature after
It is continuous to cultivate；
Wherein, the injection bacterium solution is that the plumule of chitting piece A and embryo is upward, fixes seed with tweezers on the other hand,
The other hand injects bacterium solution into embryo along seed embryo bud scale, until there is bacterium solution to ooze out from plumule end；The vermiculite and turf
Weight ratio be 2:1.
In the present embodiment, the Wheat genetic transformation method based on wheat seed embryo injection Agrobacterium is in wheat transgenic
Application in breeding, the wheat seed embryo by providing a kind of simple and effective low cost inject Agrobacterium tumefaciems genetic transforming method,
Target gene is imported wheat living body, combining target gene by the Agrobacterium by being directly injected into carrying target gene in embryo
PCR detection technique has obtained heritable transgenic progeny.
Embodiment 2
To the conversion present age (T cultivated in embodiment 10) seedling progress PCR detection, bacterium will be specifically injected in step 3)
The chitting piece A of liquid takes each 2cm leaf section of 1-3 piece leaf to be mixed, using CATB miniprep dna extraction side when length to 31 heart of leaf
Method extracts each transformed plant DNA, (is located on pWMB110 plasmid with gus gene (beta-glucuronidase) and Bar gene respectively
The gene of the polynucleotides encoding herbicide resistant in the region T-DNA) special primer carry out PCR amplification, detect positive plant, then transplant to big
It plants to obtain T in field0For positive plant；
Wherein, the special primer of the gus gene of PCR detection includes upstream primer and downstream primer, and PCR amplification piece
Segment length is 995bp, the upstream primer are as follows: 5'-CAAGGAAATCCGCAACCATATC-3', the downstream primer are as follows: 5'-
TCAAACGTCCGAATCTTCTCCC-3'；
The special primer of the Bar gene of the PCR detection includes upstream primer and downstream primer, and pcr amplified fragment is long
Degree is 429bp, the upstream primer are as follows: 5'-ACCATCGTCAACCACTACATCG-3', the downstream primer are as follows: 5'-
GCTGCCAGAAACCACGTCATG-3'。
Embodiment 3
To T obtained in embodiment 20For the main fringe set generation selfing of positive plant, T is harvested1For seed, every fringe takes 10 T1Generation
Seed indoor germination takes the 1st leaf to extract DNA, is carried out using the PCR detection method of gus gene and Bar gene in embodiment 2
It identifies transformed plant, obtains T1For transformed plant.
Embodiment 4
To T obtained in embodiment 31The detection of gus protein histochemical method is carried out for transformed plant, specifically takes T1For PCR
Positive plant root and blade carry out gus gene detection of expression with gus protein histochemical staining method, wherein dyeing liquor includes 50
X-Gluc (the chloro- 3- indoles-beta-glucosidase acid esters of the bromo- 4- of the 5-) mother liquor of μ l and 450 μ l base fluids (PBS of 50mM, pH7.0),
Colouring method is that ready test material is immersed in dyeing liquor, and 1 hour is kept the temperature in 25-37 DEG C to staying overnight, then by blade etc.
Green material is transferred to decolourize 2-3 times into 70% ethyl alcohol, until negative control material is white, then in stereoscopic microscopic observation, white
Blue portion in background is Gus gene expression site.
Embodiment 5
Inject according to the Wheat genetic transformation method based on wheat seed embryo injection Agrobacterium described in embodiment 1
Then 857 Mature Embryos Amongs carry out PCR detection, specific T according to 2 the method for embodiment0It is detected for transgenic plant PCR
The results are shown in Table 1, therefrom detects GUS positive plant 203, positive rate averagely reaches 23.69%, and Yanzhan4110 positive rate
Slightly above Bobwhite, but without significant difference.
1 T of table0For transgenic plant PCR testing result table
Further, GUS dyeing is carried out to the embryo after injection, in T0For visible blue area in rotaring gene plant blade
Domain, it was demonstrated that gus gene is in T0For can be with transient expression in transgenic plant, (blue region, that is, gus protein be instantaneous as shown in Figure 2
Express position；Receptor is wheat breed Bobwhite), the blue region is the dark parts at middle part in figure.
Further, to 203 T0For the T of GUS positive plant1In generation, has carried out the PCR detection of GUS and Bar gene, therefrom
Detect positive T1For transgenic wheat 54, T is accounted for0For the 26.6% of positive transformants plant, that is, there is 26.6% T0Positive turn of generation
Change strain and obtained heritable transgenic progeny, (in Fig. 3, left figure is Bar gene PCR amplified band as shown in Figure 3
(429bp), right figure are gus gene PCR amplification band (995bp)；The T that upper figure is Bobwhite1For transgenic plant PCR amplification
As a result, the following figure is the T of Yanzhan41101For transgenic plant PCR amplification result；Arrow show PCR specific amplified band；1-10
For T1Numbered for different transgenic plants), it is calculated by injection embryo quantity, T1Reach 6.30% for genetic transformation rate.
Further, to T1Gus protein histochemical stain is carried out for gus gene positive plant, as a result root system and blade
Gus protein blue signal, PCR detection and gus protein histochemical stain is presented the result shows that embryo of the invention injects root
Cancer agrobacterium genetic transformation method can obtain the transgenic wheat for stablizing heredity.
Present invention has the advantages that it is conversion object with mature seed embryos that the present invention, which passes through directly, by straight in embryo
Connect injection carry target gene Agrobacterium method by target gene import wheat living body, combining target gene PCR detection technique,
Obtained heritable transgenic progeny, transformant frequency reaches 26.6%, solve traditional Efficiency of Wheat Transformation technology because
Excised cotyledon is needed to lead to the problem that at high cost, the period is long and regeneration rate is low with regenerated process；The present invention provides
A kind of novel genetic transfoumation for avoiding excised cotyledon process is big with three compared with common wheat transgenic method
Advantage: 1) not needing the valuable instrument and equipments such as particle gun, and low in cost, common laboratory can operate, and have it is good again
Raw rate；2) inorganization culture and regenerative process, the period is shorter, and without wheat genotypes Dependence Problem；3) materials are convenient, a Nian Si
Season can operate, and simple and effective low cost has a vast market foreground.
Better embodiment of the invention is explained in detail above, but the present invention is not limited to above-mentioned embodiment party
Formula within the knowledge of one of ordinary skill in the art can also be without departing from the purpose of the present invention
Various changes can be made.There is no necessity and possibility to exhaust all the enbodiments.And it thus amplifies out apparent
Variation or variation be still in the protection scope of this invention.
Claims (8)
1. it is a kind of based on wheat seed embryo injection Agrobacterium Wheat genetic transformation method, which is characterized in that it the following steps are included:
1) receptor prepares: the healthy seed for choosing receptor is rinsed with distilled water, the secondary chlorine for being then 1.5% with mass concentration
Sour sodium carries out soaking disinfection 15min, then uses distilled water immersion 10min, then with distilled water flushing seed to no thimerosal gas
Taste is placed in the culture dish of paving two layers of filter paper, is added water and is not crossed seed and germination of sprouting at 25 DEG C, obtains chitting piece A, standby
With；
2) prepared by bacteria suspension: Agrobacterium tumefaciems progress Multiplying culture being obtained bacterium solution, then with the rate of 4000rpm at 4 DEG C
Carry out centrifugation 10min, then remove supernatant and YEB culture solution is added, then be added acetosyringone to final concentration of 200 μM,
It was activated again at incubated at room temperature 12 hours, obtains Agrobacterium bacterium solution B, it is spare；
3) embryo injects Agrobacterium-mediated Transformation: the chitting piece A in picking step 1) is put small to air-drying 1 at superclean bench air port
When, it then draws Agrobacterium bacterium solution B obtained in the step 2) of 0.5ml and injection bacterium solution is carried out to chitting piece A, then will inject
The chitting piece A of bacterium solution air-dry 30 minutes, is then transferred load in the nutritive cube for filling vermiculite and turf, in room after sprinkling profoundly water
Continue to cultivate under temperature.
2. the Wheat genetic transformation method according to claim 1 based on wheat seed embryo injection Agrobacterium, which is characterized in that
In step 1), the receptor is wheat breed Bobwhite and Yanzhan4110.
3. the Wheat genetic transformation method according to claim 1 or 2 based on wheat seed embryo injection Agrobacterium, feature exist
In in step 2), the Agrobacterium tumefaciems is that beta-glucuronidase encoding gene is connected on plasmid vector pWMB110
The Agrobacterium tumefaciems containing pWMB110 plasmid arrived.
4. the Wheat genetic transformation method according to claim 3 based on wheat seed embryo injection Agrobacterium, which is characterized in that
In step 2), the Multiplying culture is to draw the Agrobacterium tumefaciems containing pWMB110 plasmid to be added to the YEB Liquid Culture of 50ml
In base, then carry out shaking bacterium 12 hours in 28 DEG C of rates with 200rpm.
5. the Wheat genetic transformation method according to claim 4 based on wheat seed embryo injection Agrobacterium, which is characterized in that
The YEB fluid nutrient medium includes the Li Fu that the concentration of the kanamycins that the concentration of 50 μ l is 50mg/ml and 50 μ l are 50mg/ml
It is flat.
6. the Wheat genetic transformation method according to claim 5 based on wheat seed embryo injection Agrobacterium, which is characterized in that
In step 3), the injection bacterium solution be by the plumule of chitting piece A and embryo it is upward, fix chitting piece A with tweezers on the other hand,
The other hand injects bacterium solution into embryo along the plumule of chitting piece A, until there is bacterium solution to ooze out from plumule end.
7. the Wheat genetic transformation method according to claim 6 based on wheat seed embryo injection Agrobacterium, which is characterized in that
In step 3), the weight ratio of the vermiculite and turf is 2:1.
8. a kind of Wheat genetic transformation method as claimed in claim 1 based on wheat seed embryo injection Agrobacterium is small
Application in wheat transgenic breeding.
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| TNSN08356A1 (en) * | 2008-09-17 | 2009-12-29 | Centre De Biotechnologie De Sfax | A METHOD FOR " in embryo" transformation with agrobacterium of wheat and production of transgenic fertile plans over-expressing the AISAP gene, isolated from the halophyte grass " A littoralis", for improvement of abiotic stress tolerance |
| CN102021198A (en) * | 2009-09-11 | 2011-04-20 | 辽宁师范大学 | Method for transforming exogenous gene by coleoptile of germinating seed |
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Application publication date: 20190419 |