CN105177039A - Method for transferring exogenous genes into betula platyphylla Suks. seedlings for transient expression - Google Patents
Method for transferring exogenous genes into betula platyphylla Suks. seedlings for transient expression Download PDFInfo
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- CN105177039A CN105177039A CN201510701208.0A CN201510701208A CN105177039A CN 105177039 A CN105177039 A CN 105177039A CN 201510701208 A CN201510701208 A CN 201510701208A CN 105177039 A CN105177039 A CN 105177039A
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Abstract
The invention provides a method for transferring exogenous genes into betula platyphylla Suks. seedlings for transient expression, relating to methods for transferring exogenous genes into betula platyphylla Suks. seedlings. The method comprises the following steps: I. carrying out oscillation washing with a Tween sterile water solution and then putting the betula platyphylla Suks. seedlings in a hyperosmotic solution to be soaked; II. infecting and soaking the betula platyphylla Suks. seedlings in transformation culture media; and III. carrying out oscillation washing with mixed liquor of mannitol and dithiothreitol, then sucking all the moisture and planting the betula platyphylla Suks. seedlings in soil. The method lays the foundation for researches for more effectively analyzing the functions of stress resistance genes and promoters of betula platyphylla Suks.
Description
Technical field
The present invention relates to a kind of method that foreign gene proceeds to white birch seedling.
Background technology
White birch (BetulaplatyphyllaSuks.) is a cosmopolitan species of north temperate zone, and rapidly, adaptability and resistance are comparatively strong in growth, are that Effect of Anti is against mechanism and the ideal biomaterial carrying out adversity gene clone.Because the breeding cycle of white birch is long, genetic improvement process is slow, so adopt traditional breeding technique can not meet the needs of white birch research.
Transient expression (transientexpression) just can detect the exogenous gene expression mode of its expression product at short notice after referring to and importing foreign gene.Agriculture bacillus mediated instant expression method is applied in a lot of transgenic plant, compared with traditional transgenic protocol, have the cycle short, safe and efficient, operate simple and easy, expense is relatively low, transformation efficiency advantages of higher.But be all only at present utilize agriculture bacillus mediated between leaf, root, stipes or the indefinite bud that formed of the callus of isolated culture; Again indefinite bud is cultivated one's ability and obtain complete plant.
Summary of the invention
The invention provides the instant expression method that a kind of foreign gene proceeds to Drought stress, the method for explant, utilizes agriculture bacillus mediated with the Drought stress of whole strain.
Realize the transient expression that foreign gene proceeds to Drought stress according to the following steps:
One, by white birch seedling concentration be 0.5ml/L Tween aseptic aqueous solution concussion cleaning 2min, then put into high sepage and soak 10min;
Two, the white birch seedling soaked through too high sepage being put into bioconversion medium, is infect under the condition of 120r/min to soak 4h at rotating speed;
Three, by the mixed solution concussion washing 2 times of the white birch seedling N.F,USP MANNITOL after infecting and dithiothreitol (DTT) (DTT), then aseptic filter paper suck dry moisture is used, plant again in soil, overlay film moisturizing obtained transient transformant after 3 days, namely realized the transient expression that foreign gene proceeds to Drought stress;
Wherein, step one middle and high infiltration liquid is the 1/4MS substratum containing 250g/L sucrose;
In step 2, bioconversion medium is 1/2MS+AS100 μM+N.F,USP MANNITOL 100mM+KT1.5mg/L+NAA0.5mg/L+ sucrose 50g/L+Tween200.1ml/L+ Agrobacterium, and bioconversion medium OD value is 0.6 ~ 1.0; ;
In step 3 N.F,USP MANNITOL and dithiothreitol (DTT) mixed solution, the concentration of N.F,USP MANNITOL is 170mM, the concentration of dithiothreitol (DTT) is 1mM.
Seedling is the seedling strain obtained by seminal propagation, and it is different from cuttage seeding or Graft that vegetative propagation obtains; Seedling has the features such as vigorous, the well developed root system of growth, life-span be longer.The object of the present invention is to provide and a kind ofly foreign gene is proceeded to the method for carrying out transient expression in Drought stress, with agriculture bacillus mediated, by building plant over-express vector, making foreign gene carry out expression analysis in white birch seedling.The present invention adopts seedling to carry out genetic transformation, has the advantage that the genetic transformation cycle is short, efficiency is high, and for effectively analyzing white birch adversity gene function, Assay of promoter activity, Subcellular Localization, manufacture target protein lays the foundation.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of plant over-express vector pBI121-proBpDREB1B::GUS in embodiment 1.
Fig. 2 is proBpDREB1B promoter fragment amplification gel electrophoresis figure in embodiment 1.
Fig. 3 is pBI121-proBpDREB1B::GUS Escherichia coli bacteria liquid pcr amplification gel electrophoresis figure in embodiment 1.
Fig. 4 is pBI121-proBpDREB1B::GUS plasmid HindIII, XbaI double digestion gel electrophoresis figure in embodiment 1.
Fig. 5 is pBI121-proBpDREB1B::GUS Agrobacterium bacterium liquid pcr amplification gel electrophoresis figure in embodiment 1.
Fig. 6 is that after control group in embodiment 1 (Drought stress that infects of Agrobacterium transformed with PBI121 empty carrier) GUS dyeing, white birch is grown directly from seeds the GUS coloration result figure of earth culture seedling.
Fig. 7 is that after control group in embodiment 1 (Drought stress that infects of Agrobacterium transformed with PBI121 empty carrier) GUS dyeing, white birch is grown directly from seeds the GUS coloration result figure of earth culture seedling leaf.
The observation figure that Fig. 8 is negative control group in embodiment 1 (infecting Drought stress with the Agrobacterium not containing PBI121 empty carrier) GUS dyeing, the rear white birch of decolouring grows directly from seeds earth culture seedling.
Fig. 9 is that in embodiment 1, instantaneous conversion white birch white birch after earth culture seedling (Drought stress that infects of Agrobacterium transformed with plant over-express vector pBI121-proBpDREB1B::GUS) GUS dyeing, decolouring of growing directly from seeds is grown directly from seeds the observation figure of earth culture seedling.
Figure 10 is proBpbZIP38 promoter fragment amplification gel electrophoresis figure in embodiment 2.
Figure 11 is pBI121-proBpbZIP38::GUS Escherichia coli bacteria liquid pcr amplification gel electrophoresis figure in embodiment 2.
Figure 12 is pBI121-proBpbZIP38::GUS plasmid HindIII, XbaI double digestion gel electrophoresis figure in embodiment 2.
Figure 13 is pBI121-proBpbZIP38::GUS Agrobacterium bacterium liquid pcr amplification gel electrophoresis figure in embodiment 2.
Figure 14 is that in embodiment 2, instantaneous conversion white birch white birch after earth culture seedling (Drought stress that infects of Agrobacterium transformed with plant over-express vector pBI121-proBpbZIP38::GUS) GUS dyeing of growing directly from seeds is grown directly from seeds the GUS coloration result figure of earth culture seedling.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment realizes the transient expression that foreign gene proceeds to Drought stress according to the following steps:
One, by white birch seedling concentration be 0.5ml/L Tween aseptic aqueous solution concussion cleaning 2min, then put into high sepage and soak 10min;
Two, the white birch seedling soaked through too high sepage being put into bioconversion medium, is infect under the condition of 120r/min to soak 4h at rotating speed;
Three, by the mixed solution concussion washing 2 times of the white birch seedling N.F,USP MANNITOL after infecting and DTT, then use aseptic filter paper suck dry moisture, then plant in soil, overlay film moisturizing obtained transient transformant after 3 days, namely realized the transient expression that foreign gene proceeds to Drought stress;
Wherein, step one middle and high infiltration liquid is the 1/4MS substratum containing 250g/L sucrose;
In step 2, bioconversion medium is 1/2MS+AS (Syringylethanone) 100 μMs+N.F,USP MANNITOL 100mM+KT (kinetin) 1.5mg/L+NAA (naphthylacetic acid) 0.5mg/L+ sucrose 50g/L+Tween200.1ml/L+ Agrobacterium, and bioconversion medium OD value is 0.6 ~ 1.0;
In step 3 N.F,USP MANNITOL and dithiothreitol (DTT) mixed solution, the concentration of N.F,USP MANNITOL is 170mM, the concentration of dithiothreitol (DTT) is 1mM.
White birch seedling is put into the surface tension of Tween aseptic aqueous solution concussion cleaning in order to eliminate explant (Drought stress) by present embodiment step one.
Present embodiment, by the acting in conjunction of Agrobacterium and high sepage, realizes infecting the Drought stress with integral skin.
The mixed solution concussion washing of the white birch seedling N.F,USP MANNITOL after infecting in present embodiment step 3 and DTT, is soaked to prevent the transient transformant obtained from again contacting with water.
Present embodiment bioconversion medium take 1/2MS as minimum medium, additional AS (Syringylethanone) 100 μMs, N.F,USP MANNITOL 100mM, KT (kinetin) 1.5mg/L, NAA (naphthylacetic acid) 0.5mg/L, sucrose 50g/L, Tween200.1ml/L and Agrobacterium bacterium liquid, adding Agrobacterium bacterium liquid to bioconversion medium OD value is 0.6 ~ 1.0.
Embodiment two: the difference of present embodiment and embodiment one is: the white birch seedling in step one be 4 week age white birch to grow directly from seeds earth culture seedling.Other step and parameter identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one or two is: at every turn shake in step 3 that washing time is 3min, rotating speed is 120r/min.Other step and parameter identical with embodiment one or two.
Embodiment four: the difference of one of present embodiment and embodiment one to three is: the Agrobacterium in step 2 Agrobacterium bacterium liquid contains the plasmid of band reporter gene.Other step and parameter identical with one of embodiment one to three.
Embodiment five: the difference of one of present embodiment and embodiment one to four is: plasmid is pBI121 plasmid.Other step and parameter identical with one of embodiment one to four.
Embodiment six: the difference of one of present embodiment and embodiment one to five is: the plasmid of Agrobacterium comprises BpDREB1B gene promoter or BpbZIP38 gene promoter.Other step and parameter identical with one of embodiment one to five.
Embodiment 1
One, white birch BpDREB1B gene promoter clone
With white birch DNA for template, according to BpDREB1B gene promoter gene order, design primer (pBI121-BPDREB1B-GUSF:5 '-CCCAAGCTTAAAAGGAGTCTCACTAG-3 '; PBI121-BPDREB1B-GUSR:5 '-GCTCTAGAAGTAAAGGATGCTCTTCT-3 '), clone obtains BpDREB1B promoter gene fragment, and as shown in SEQIDNO:1, length is 814bp.
Two, the 35S promoter of carrier pBI121 plasmid is replaced by BpDREB1B gene promoter orientation, with restriction enzyme HindIII and XbaI, double digestion is carried out to BpDREB1B promoter gene fragment and the pBI121 plasmid containing GUS (β-glucuronidase) gene, then connect and build plant over-express vector pBI121-proBpDREB1B::GUS (as shown in Figure 1).
With plant over-express vector pBI121-proBpDREB1B::GUS transformation of E. coli and Agrobacterium, again through PCR and double digestion checking (double digestion gel electrophoresis figure as shown in Figure 2-5), prove the plant expression vector successfully built containing proBpDREB1B::GUS.
Three, the Agrobacterium of merit is changed in culturing step two, then centrifugal, collection Agrobacterium.
Four, earth culture seedling concentration of being grown directly from seeds by white birch in 4 week age is the Tween aseptic aqueous solution concussion cleaning 2min of 0.5ml/L, then puts into high sepage and soaks 10min;
Five, the white birch seedling soaked through too high sepage being put into bioconversion medium, is infect under the condition of 120r/min to soak 4h at rotating speed;
Six, by the mixed solution concussion washing 2 times of the white birch seedling N.F,USP MANNITOL after infecting and DTT, each concussion washing time is 3min, rotating speed is 120r/min; Then use aseptic filter paper suck dry moisture, then to plant in soil and overlay film moisturizing 3 days, namely realize the transient expression that foreign gene proceeds to Drought stress;
Wherein, step 4 middle and high infiltration liquid is the 1/4MS substratum containing 250g/L sucrose;
In step 5, bioconversion medium is 1/2MS+AS (Syringylethanone) 100 μMs+N.F,USP MANNITOL 100mM+KT (kinetin) 1.5mg/L+NAA (naphthylacetic acid) 0.5mg/L+ sucrose 50g/L+Tween200.1ml/L+ Agrobacterium, and bioconversion medium OD value is 0.6 ~ 1.0;
In step 6 N.F,USP MANNITOL and dithiothreitol (DTT) mixed solution, the concentration of N.F,USP MANNITOL is 170mM, the concentration of dithiothreitol (DTT) is 1mM.
Experiment
(50ml staining fluid is 0.1mol/L by 20ml concentration to carry out GUS dyeing to the instantaneous conversion nursery stock of the present embodiment acquisition, pH value is 7.0 sodium phosphate buffers, 1ml concentration is the EDTA solution of 0.5mol/L, the TritonX-100 of 0.5ml, 42.5mg yellow prussiate of potash, the Tripotassium iron hexacyanide of 33mg, the paraxin of 5mg, 50mgX-Gluc 10ml dissolve with methanol solution, and the water of surplus is formulated), 37 DEG C are incubated overnight, then plant is placed in destainer (dehydrated alcohol: glacial acetic acid volume ratio is 3:1) decolouring 2h ~ 4h, again the plant after decolouring being stored in volumetric concentration is in the ethanol of 70%, observe staining conditions, control group adopts PBI121 empty carrier to carry out GUS dyeing, negative control group infects Drought stress with the Agrobacterium not containing PBI121 empty carrier, experimental result is as shown in Fig. 6 ~ 9.Be blueness after the Drought stress that the Agrobacterium that in Fig. 6 and Fig. 7, PBI121 empty carrier transforms is infected and blade dyeing, the agriculture bacillus mediated Drought stress of the inventive method is described, plasmid successfully imports Drought stress.Fig. 8 infects Drought stress with the Agrobacterium not containing PBI121 empty carrier, and after GUS dyeing, decolouring, white birch grows directly from seeds earth culture seedling for white; With the Drought stress that the Agrobacterium that plant over-express vector pBI121-proBpDREB1B::GUS transforms is infected in Fig. 9, blueness is presented in the grow directly from seeds local of earth culture seedling leaf and root of white birch after GUS dyeing, decolouring, illustrate under the driving of BpDREB1B gene promoter, Reporter gene GUS is specifically expressing in tender leaf and root, external source reporter gene successfully proceeds to Drought stress and realizes transient expression, experimental result also shows that BpDREB1B gene promoter not only has expression activity, and has certain tissue expression specificity.
Embodiment 2
One, white birch BpbZIP38 gene promoter clone
With white birch DNA for template, according to BpbZIP38 gene promoter gene order, design primer (pBI121-bZIP38-GUSF:5 '-CCCAAGCTTACGGGGTGGATCAAGACCTC-3 '; PBI121-bZIP38-GUSR:5 '-GCTCTAGAATTTTGAAAACTTGAAGCAGTG-3 '), clone obtains BpbZIP38 promoter gene fragment, and as shown in SEQIDNO:2, length is 1862bp.
Two, the 35S promoter of carrier pBI121 plasmid is replaced by BpbZIP38 gene promoter orientation, with restriction enzyme HindIII and XbaI, double digestion is carried out to BpbZIP38 promoter gene fragment and the pBI121 plasmid containing GUS (β-glucuronidase) gene, then connect and build plant over-express vector pBI121-proBpbZIP38::GUS (as shown in Figure 1).
With plant over-express vector pBI121-proBpbZIP38::GUS transformation of E. coli and Agrobacterium, again through PCR and double digestion checking (double digestion gel electrophoresis figure is as Suo Shi Figure 10 ~ 13), prove the plant expression vector successfully built containing proBpbZIP38::GUS.
Three, the Agrobacterium of merit is changed in culturing step two, then centrifugal, collection Agrobacterium.
Four, earth culture seedling concentration of being grown directly from seeds by white birch in 4 week age is the Tween aseptic aqueous solution concussion cleaning 2min of 0.5ml/L, then puts into high sepage and soaks 10min;
Five, the white birch seedling soaked through too high sepage being put into bioconversion medium, is infect under the condition of 120r/min to soak 4h at rotating speed;
Six, by the mixed solution concussion washing 2 times of the white birch seedling N.F,USP MANNITOL after infecting and DTT, each concussion washing time is 3min, rotating speed is 120r/min; Then use aseptic filter paper suck dry moisture, then to plant in soil and overlay film moisturizing 3 days, namely realize the transient expression that foreign gene proceeds to Drought stress;
Wherein, step 4 middle and high infiltration liquid is the 1/4MS substratum containing 250g/L sucrose;
In step 5, bioconversion medium is 1/2MS+AS (Syringylethanone) 100 μMs+N.F,USP MANNITOL 100mM+KT (kinetin) 1.5mg/L+NAA (naphthylacetic acid) 0.5mg/L+ sucrose 50g/L+Tween200.1ml/L+ Agrobacterium, and bioconversion medium OD value is 0.6 ~ 1.0;
In step 6 N.F,USP MANNITOL and dithiothreitol (DTT) mixed solution, the concentration of N.F,USP MANNITOL is 170mM, the concentration of dithiothreitol (DTT) is 1mM.
Experiment
(50ml staining fluid is 0.1mol/L by 20ml concentration to carry out GUS dyeing to the instantaneous conversion nursery stock of the present embodiment acquisition, pH value is 7.0 sodium phosphate buffers, 1ml concentration is the EDTA solution of 0.5mol/L, the TritonX-100 of 0.5ml, 42.5mg yellow prussiate of potash, the Tripotassium iron hexacyanide of 33mg, the paraxin of 5mg, 50mgX-Gluc 10ml dissolve with methanol solution, and the water of surplus is formulated), 37 DEG C are incubated overnight, then plant is placed in destainer (dehydrated alcohol: glacial acetic acid volume ratio is 3:1) decolouring 2h ~ 4h, again the plant after decolouring being stored in volumetric concentration is in the ethanol of 70%, observe staining conditions, result as shown in figure 14, under the driving of BpbZIP38 gene promoter, Reporter gene GUS equally can in tender leaf and root specifically expressing, external source reporter gene successfully proceeds to Drought stress and realizes transient expression, experimental result also shows that BpbZIP38 gene promoter has expression activity, and there is certain tissue expression specificity.
Claims (6)
1. foreign gene proceeds to the instant expression method of Drought stress, it is characterized in that realizing the transient expression that foreign gene proceeds to Drought stress according to the following steps:
One, by white birch seedling concentration be 0.5ml/L Tween aseptic aqueous solution concussion cleaning 2min, then put into high sepage and soak 10min;
Two, the white birch seedling soaked through too high sepage being put into bioconversion medium, is infect under the condition of 120r/min to soak 4h at rotating speed;
Three, by the mixed solution concussion washing 2 times of the white birch seedling N.F,USP MANNITOL after infecting and dithiothreitol (DTT), then aseptic filter paper suck dry moisture is used, plant in soil, overlay film moisturizing obtained transient transformant after 3 days again, namely realized the transient expression that foreign gene proceeds to Drought stress;
Wherein, step one middle and high infiltration liquid is the 1/4MS substratum containing 250g/L sucrose;
In step 2, bioconversion medium is 1/2MS+AS100 μM+N.F,USP MANNITOL 100mM+KT1.5mg/L+NAA0.5mg/L+ sucrose 50g/L+Tween200.1ml/L+ Agrobacterium, and bioconversion medium OD value is 0.6 ~ 1.0;
In step 3 N.F,USP MANNITOL and dithiothreitol (DTT) mixed solution, the concentration of N.F,USP MANNITOL is 170mM, the concentration of dithiothreitol (DTT) is 1mM.
2. foreign gene according to claim 1 proceeds to the instant expression method of Drought stress, the white birch seedling that it is characterized in that in step one be 4 week age white birch to grow directly from seeds earth culture seedling.
3. foreign gene according to claim 1 proceeds to the instant expression method of Drought stress, it is characterized in that in step 3, each concussion washing time is 3min, rotating speed is 120r/min.
4. foreign gene according to claim 1 proceeds to the instant expression method of Drought stress, it is characterized in that the Agrobacterium in step 2 Agrobacterium bacterium liquid contains the plasmid of band reporter gene.
5. foreign gene according to claim 4 proceeds to the instant expression method of Drought stress, it is characterized in that plasmid is pBI121 plasmid.
6. the foreign gene according to claim 1 or 4 proceeds to the instant expression method of Drought stress, it is characterized in that the plasmid of Agrobacterium comprises BpDREB1B gene promoter or BpbZIP38 gene promoter.
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| CN113430194A (en) * | 2020-11-30 | 2021-09-24 | 东北林业大学 | White birch gene editing method based on CRISPR/Cas9 |
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