CN105177039B - Transient expression method for exogenous gene transferred birch seedling - Google Patents
Transient expression method for exogenous gene transferred birch seedling Download PDFInfo
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Abstract
A transient expression method for exogenous gene transferred birch seedlings relates to a method for exogenous gene transferred birch seedlings. The invention provides a transient expression method for transferring exogenous genes into white birch seedlings. The method comprises the following steps: firstly, shaking and cleaning a Tween sterile aqueous solution, and then soaking in a hypertonic solution; secondly, soaking in a transformation culture medium; and thirdly, shaking and washing the mixture by using a mixed solution of mannitol and dithiothreitol, and then, sucking dry water to plant the mixture into soil. The method lays a foundation for more effectively analyzing the birch stress-resistant gene and the function research of the promoter.
Description
Technical Field
The invention relates to a method for transferring exogenous genes into white birch seedlings.
Background
White birch (Betula platyphylla disks.) is a wide variety in the northern temperate zone, grows rapidly, has strong adaptability and stress resistance, and is an ideal biological material for researching the stress resistance mechanism and cloning stress resistance genes. Due to the long breeding period and the slow genetic improvement process of the white birch, the traditional breeding technology cannot meet the requirement of white birch research.
Transient expression refers to a mode of expression of a foreign gene in which an expression product thereof can be detected in a short time after introduction of the foreign gene. The agrobacterium-mediated transient expression method is already applied to a plurality of transgenic plants, and compared with the traditional transgenic process, the agrobacterium-mediated transient expression method has the advantages of short period, safety, high efficiency, simple operation, relatively low cost, high transformation efficiency and the like. But currently, the agrobacterium is only used for mediating adventitious buds formed from leaves, roots, internodes of stems or callus cultured in vitro; culturing the adventitious bud to obtain a complete plant.
Disclosure of Invention
The invention provides a transient expression method for transferring exogenous genes into white birch seedlings.
The transient expression of the exogenous gene transferred into the white birch seedling is realized according to the following steps:
firstly, shaking and cleaning white birch seedlings by using a Tween sterile aqueous solution with the concentration of 0.5ml/L for 2min, and then soaking the white birch seedlings in a hypertonic solution for 10 min;
secondly, putting the birch seedlings soaked in the hypertonic solution into a transformation culture medium, and soaking for 4 hours at the rotating speed of 120 r/min;
thirdly, shaking and washing the infected white birch seedlings for 2 times by using mixed liquor of mannitol and Dithiothreitol (DTT), then sucking dry water by using sterile filter paper, then planting the white birch seedlings into soil, and obtaining transient transformants after covering films and preserving moisture for 3 days, namely realizing transient expression of the exogenous genes transferred into white birch seedlings;
wherein, the hypertonic solution in the first step is 1/4MS culture medium containing 250g/L of sucrose;
in the second step, the transformation medium is 1/2MS + AS 100 MuM + mannitol 100mM + KT 1.5mg/L + NAA 0.5mg/L + sucrose 50g/L + Tween200.1ml/L + Agrobacterium, and the OD value of the transformation medium is 0.6-1.0; (ii) a
In the third step, the concentration of mannitol and dithiothreitol in the mixed solution of mannitol and dithiothreitol is 170mM and 1 mM.
The seedling is a seedling plant obtained by seed propagation and is different from a cutting seedling or a grafting seedling obtained by asexual propagation; the seedling has the characteristics of vigorous growth, developed root system, long service life and the like. The invention aims to provide a method for transferring exogenous genes into white birch seedlings for transient expression, which is used for expressing and analyzing the exogenous genes in the white birch seedlings by constructing a plant overexpression vector under the mediation of agrobacterium. The method adopts the seedling for genetic transformation, has the advantages of short genetic transformation period and high efficiency, and lays a foundation for effectively analyzing the function of the stress-resistant gene of the white birch, analyzing the activity of a promoter, positioning subcellular cells and producing and preparing target protein.
Drawings
FIG. 1 is a schematic diagram of the construction of GUS from the plant overexpression vector pBI121-proBpDREB1B in example 1.
FIG. 2 is an amplification gel electrophoresis image of the proBpDREB1B promoter fragment in example 1.
FIG. 3 is the PCR amplification gel electrophoresis image of the E.coli strain of GUS, pBI121-proBpDREB1B in example 1.
FIG. 4 is a diagram of the double-restriction gel electrophoresis of the GUS plasmid HindIII and XbaI in example 1, pBI121-proBpDREB 1B.
FIG. 5 is the PCR amplification gel electrophoresis image of the Agrobacterium GUS bacterial liquid in pBI121-proBpDREB1B in example 1.
FIG. 6 is a graph showing the GUS staining results of soil-cultured seedlings of white birch (white birch seedlings infected with Agrobacterium transformed with PBI121 empty vector) after GUS staining in the control group in example 1.
FIG. 7 is a graph showing the GUS staining results of the leaves of the birch seedlings after GUS staining in the control group (birch seedlings infected with Agrobacterium transformed with PBI121 empty vector) in example 1.
FIG. 8 is a diagram showing the observation of soil-cultured seedlings of white birch after GUS staining and bleaching in the negative control group (white birch seedlings were infected with Agrobacterium without PBI121 empty vector) in example 1.
FIG. 9 is a graph showing the observation of soil-cultured seedlings of Betula platyphylla after GUS staining and bleaching of transient transformation of the soil-cultured seedlings of Betula platyphylla (seedlings of Betula platyphylla Miq. infected with Agrobacterium transformed with GUS using the plant overexpression vector pBI121-proBpDREB1B:: in example 1).
FIG. 10 is an amplification gel electrophoresis of the proBpbZIP38 promoter fragment of example 2.
FIG. 11 is a PCR amplification gel electrophoresis image of E.coli strain liquid of GUS, pBI121-proBpbZIP38 in example 2.
FIG. 12 is a double-restriction gel electrophoresis of the plasmid HindIII and XbaI of GUS in example 2, pBI121-proBpbZIP 38.
FIG. 13 is a gel electrophoresis image of PCR amplification of Agrobacterium GUS bacterial fluid from pBI121-proBpbZIP38 in example 2.
FIG. 14 is a GUS staining result of soil-cultured seedlings of Betula platyphylla after GUS staining of soil-cultured seedlings of Betula platyphylla transiently transformed in example 2 (seedlings of Betula platyphylla Betula infected with the plant overexpression vector pBI121-proBpbZIP38:: GUS-transformed Agrobacterium).
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the implementation mode realizes the transient expression of the exogenous gene transferred into the white birch seedlings according to the following steps:
firstly, shaking and cleaning white birch seedlings by using a Tween sterile aqueous solution with the concentration of 0.5ml/L for 2min, and then soaking the white birch seedlings in a hypertonic solution for 10 min;
secondly, putting the birch seedlings soaked in the hypertonic solution into a transformation culture medium, and soaking for 4 hours at the rotating speed of 120 r/min;
thirdly, shaking and washing the infected white birch seedlings for 2 times by using mixed liquor of mannitol and DTT, then sucking dry water by using sterile filter paper, then planting the white birch seedlings into soil, and obtaining transient transformants after covering films and preserving moisture for 3 days, namely realizing transient expression of the exogenous genes transferred into white birch seedlings;
wherein, the hypertonic solution in the first step is 1/4MS culture medium containing 250g/L of sucrose;
in the second step, the transformation medium is 1/2MS + AS (acetosyringone) 100 MuM + mannitol 100mM + KT (kinetin) 1.5mg/L + NAA (naphthylacetic acid) 0.5mg/L + sucrose 50g/L + Twen200.1ml/L + Agrobacterium, and the OD value of the transformation medium is 0.6-1.0;
in the third step, the concentration of mannitol and dithiothreitol in the mixed solution of mannitol and dithiothreitol is 170mM and 1 mM.
In the first step of the embodiment, the birch seedlings are put into a Tween sterile aqueous solution to be washed by shaking so as to eliminate the surface tension of explants (birch seedlings).
In the embodiment, the common action of the agrobacterium and the hypertonic solution is used for infecting the white birch seedlings with complete epidermis.
In the third step of the embodiment, the infected white birch seedlings are washed by shaking with the mixed solution of mannitol and DTT to prevent the obtained transient transformants from being soaked by contacting with water again.
In the transformation medium of the embodiment, 1/2MS is used AS a basic medium, 100 mu M of AS (acetosyringone), 100mM of mannitol, 0.5mg/L of KT (kinetin) 1.5mg/L, NAA (naphthylacetic acid), 50g/L of cane sugar, 200.1ml/L of Twen and Agrobacterium liquid are added, and the Agrobacterium liquid is added until the OD value of the transformation medium is 0.6-1.0.
The second embodiment is as follows: the present embodiment differs from the first embodiment in that: the white birch seedlings in the step one are 4-week-old white birch seedlings cultured by soil. Other steps and parameters are the same as those in the first embodiment.
The third concrete implementation mode: the present embodiment is different from the first or second embodiment in that: in the third step, the washing time is 3min and the rotating speed is 120 r/min. Other steps and parameters are the same as in one or both embodiments.
The fourth concrete implementation mode: the present embodiment is different from the first to third embodiments in that: and step two, the agrobacterium in the agrobacterium liquid contains plasmid with a reporter gene. Other steps and parameters are the same as those in one of the first to third embodiments.
The fifth concrete implementation mode: the present embodiment is different from one of the first to fourth embodiments in that: the plasmid is pBI121 plasmid. The other steps and parameters are the same as those in one of the first to fourth embodiments.
The sixth specific implementation mode: the present embodiment is different from one of the first to fifth embodiments in that: the plasmid of agrobacterium contains the bpderb 1B gene promoter or the BpbZIP38 gene promoter. Other steps and parameters are the same as those in one of the first to fifth embodiments.
Example 1
First, the cloning of the birch BpDREB1B gene promoter
The DNA of the white birch is used as a template, a primer (pBI121-BPDREB1B-GUS F: 5'-CCCAAGCTTAAAAGGAGTCTCACTAG-3'; pBI121-BPDREB1B-GUS R: 5'-GCTCTAGAAGTAAAGGATGCTCTTCT-3') is designed according to the gene sequence of the BpDRREB 1B gene promoter, and a BpDRREB 1B gene promoter fragment is obtained by cloning, and is shown as SEQ ID NO: 1 and 814bp in length.
Secondly, the 35S promoter of the vector pBI121 plasmid is directionally replaced by the BpDREB1B gene promoter, the BpDREB1B gene promoter fragment and the pBI121 plasmid containing GUS (β -glucuronidase) gene are subjected to double enzyme digestion by restriction enzymes HindIII and XbaI, and then the plant overexpression vector pBI121-proBpDREB1B is constructed by connection, wherein the GUS (shown in figure 1).
The plant over-expression vector pBI121-proBpDREB1B, GUS, is used for transforming escherichia coli and agrobacterium tumefaciens, and PCR and double enzyme digestion verification (a double enzyme digestion gel electrophoresis picture is shown in figures 2-5) prove that the plant expression vector containing the proBpDREB1B, GUS, is successfully constructed.
Thirdly, successfully transforming the agrobacterium into the culture medium in the second step, and then centrifuging and collecting the agrobacterium.
Fourthly, shaking and cleaning the birch seedlings cultivated by soil with age of 4 weeks with a Tween sterile aqueous solution with the concentration of 0.5ml/L for 2min, and then soaking the birch seedlings in a hypertonic solution for 10 min;
fifthly, putting the birch seedlings soaked in the hypertonic solution into a transformation medium, and soaking for 4 hours at the rotating speed of 120 r/min;
sixthly, shaking and washing the infected white birch seedlings for 2 times by using mixed liquid of mannitol and DTT, wherein the shaking and washing time is 3min each time, and the rotating speed is 120 r/min; then, absorbing water by using sterile filter paper, planting the seedlings in soil, covering a film and keeping moisture for 3 days, and realizing the transient expression of the exogenous gene transferred into the white birch seedlings;
wherein, the hypertonic solution in the fourth step is 1/4MS culture medium containing 250g/L of cane sugar;
in the fifth step, a transformation medium is 1/2MS + AS (acetosyringone) 100 MuM + mannitol 100mM + KT (kinetin) 1.5mg/L + NAA (naphthylacetic acid) 0.5mg/L + sucrose 50g/L + Twen200.1ml/L + Agrobacterium, and the OD value of the transformation medium is 0.6-1.0;
in the step, the concentration of mannitol and dithiothreitol in the mixed solution of the hexamannitol and the dithiothreitol is 170mM, and the concentration of the dithiothreitol is 1 mM.
Experiment of
Carrying out GUS staining on the transient transformation nursery stock obtained in the embodiment (50ml of staining solution is prepared by 20ml of sodium phosphate buffer solution with the concentration of 0.1mol/L, pH value of 7.0, 1ml of EDTA solution with the concentration of 0.5mol/L, 0.5ml of Triton X-100, 42.5mg of potassium ferrocyanide, 33mg of potassium ferricyanide, 5mg of chloramphenicol and 50mgX-Gluc dissolved by 10ml of methanol solution and the balance of water), incubating overnight at 37 ℃, then placing the plant in a decolorizing solution (absolute ethyl alcohol: glacial acetic acid volume ratio of 3:1) to decolorize for 2-4 h, then storing the decolorized plant in ethanol with the volume concentration of 70%, and observing the staining condition; GUS staining is carried out on a control group by adopting a PBI121 empty vector; infecting white birch seedlings with agrobacterium without PBI121 empty vector in a negative control group; the experimental results are shown in FIGS. 6 to 9. In FIGS. 6 and 7, the stained leaves and white birch seedlings infected with the Agrobacterium transformed with the empty vector PBI121 are blue, which shows that the plasmid has been successfully introduced into the birch seedlings mediated by the Agrobacterium according to the method of the present invention. FIG. 8 shows that white birch seedlings are infected by agrobacterium without PBI121 empty vector, and white birch seedlings are cultivated by soil after GUS staining and decoloring; in the figure 9, the plant over-expression vector pBI121-proBpDREB1B shows that white birch seedlings infected by GUS transformed agrobacterium are dyed and decolored by GUS and are locally blue on leaves and roots of white birch seedling soil culture seedlings, so that the reporter gene GUS is specifically expressed in the young leaves and roots under the drive of the BpDREB1B gene promoter, the exogenous reporter gene is successfully transferred into the white birch seedlings and realizes transient expression, and the experimental result also shows that the BpDREB1B gene promoter not only has expression activity, but also has certain tissue expression specificity.
Example 2
First, white birch BpbZIP38 gene promoter clone
The DNA of the white birch is taken as a template, a primer (pBI121-bZIP38-GUS F: 5'-CCCAAGCTTACGGGGTGGATCAAGACCTC-3'; pBI121-bZIP38-GUS R: 5'-GCTCTAGAATTTTGAAAACTTGAAGCAGTG-3') is designed according to the gene sequence of the BpbZIP38 gene promoter, and a BpbZIP38 gene promoter fragment is obtained by cloning, and is shown as SEQ ID NO: 2, the length is 1862 bp.
Secondly, directionally replacing the 35S promoter of the vector pBI121 plasmid by a BpbZIP38 gene promoter, carrying out double digestion on a BpbZIP38 gene promoter fragment and a pBI121 plasmid containing a GUS (β -glucuronidase) gene by using restriction endonucleases HindIII and XbaI, and then connecting to construct a plant over-expression vector pBI121-proBpbZIP38, wherein GUS (shown in figure 1).
The plant over-expression vector pBI121-proBpbZIP38 (GUS) is used for transforming escherichia coli and agrobacterium tumefaciens, and PCR and double enzyme digestion verification (a double enzyme digestion gel electrophoresis picture is shown in figures 10-13) prove that the plant expression vector containing the proBpbZIP38 (GUS) is successfully constructed.
Thirdly, successfully transforming the agrobacterium into the culture medium in the second step, and then centrifuging and collecting the agrobacterium.
Fourthly, shaking and cleaning the birch seedlings cultivated by soil with age of 4 weeks with a Tween sterile aqueous solution with the concentration of 0.5ml/L for 2min, and then soaking the birch seedlings in a hypertonic solution for 10 min;
fifthly, putting the birch seedlings soaked in the hypertonic solution into a transformation medium, and soaking for 4 hours at the rotating speed of 120 r/min;
sixthly, shaking and washing the infected white birch seedlings for 2 times by using mixed liquid of mannitol and DTT, wherein the shaking and washing time is 3min each time, and the rotating speed is 120 r/min; then, absorbing water by using sterile filter paper, planting the seedlings in soil, covering a film and keeping moisture for 3 days, and realizing the transient expression of the exogenous gene transferred into the white birch seedlings;
wherein, the hypertonic solution in the fourth step is 1/4MS culture medium containing 250g/L of cane sugar;
in the fifth step, a transformation medium is 1/2MS + AS (acetosyringone) 100 MuM + mannitol 100mM + KT (kinetin) 1.5mg/L + NAA (naphthylacetic acid) 0.5mg/L + sucrose 50g/L + Twen200.1ml/L + Agrobacterium, and the OD value of the transformation medium is 0.6-1.0;
in the step, the concentration of mannitol and dithiothreitol in the mixed solution of the hexamannitol and the dithiothreitol is 170mM, and the concentration of the dithiothreitol is 1 mM.
Experiment of
GUS staining is carried out on the transient transformation nursery stock obtained in the embodiment (50ml staining solution is prepared by 20ml of sodium phosphate buffer solution with the value of 0.1mol/L, pH being 7.0, 1ml of EDTA solution with the concentration of 0.5mol/L, 0.5ml of Triton X-100, 42.5mg of potassium ferrocyanide, 33mg of potassium ferricyanide, 5mg of chloramphenicol and 50mgX-Gluc being dissolved by 10ml of methanol solution and the balance being water), the mixture is incubated at 37 ℃ overnight, then the plant is placed in a decolorizing solution (absolute ethyl alcohol: glacial acetic acid volume ratio being 3:1) to be decolorized for 2 h-4 h, then the decolorized plant is stored in ethanol with the volume concentration being 70%, the staining condition is observed, the result is shown in figure 14, under the drive of BpbZIP38 gene promoter, the reporter gene GUS can be specifically expressed in the tender leaf and root, the reporter gene is successfully transferred into the white birch seedling and transient expression is realized, the experimental result also shows that the BpbZIP38 gene promoter has expression activity and certain tissue expression specificity.
Claims (3)
1. The transient expression method of the exogenous gene transferred birch seedling is characterized in that the transient expression of the exogenous gene transferred birch seedling is realized according to the following steps:
firstly, shaking and cleaning white birch seedlings by using a Tween sterile aqueous solution with the concentration of 0.5ml/L for 2min, and then soaking the white birch seedlings in a hypertonic solution for 10 min;
secondly, putting the birch seedlings soaked in the hypertonic solution into a transformation culture medium, and soaking for 4 hours at the rotating speed of 120 r/min;
thirdly, shaking and washing the infected white birch seedlings for 2 times by using a mixed solution of mannitol and dithiothreitol, then sucking dry water by using sterile filter paper, then planting the white birch seedlings into soil, and obtaining transient transformants after covering films and preserving moisture for 3 days, namely realizing transient expression of the exogenous genes transferred into white birch seedlings;
wherein, the hypertonic solution in the first step is 1/4MS culture medium containing 250g/L of sucrose;
the transformation medium in the second step is 1/2MS + AS 100 MuM + mannitol 100mM + KT 1.5mg/L + NAA 0.5mg/L + sucrose 50g/L + Tweenen 200.1ml/L + Agrobacterium, the OD value of the transformation medium is 0.6-1.0, the Agrobacterium contains a plasmid with a reporter gene and a gene promoter, the plasmid is pBI121 plasmid, and the gene promoter has a nucleotide sequence of SEQ ID NO: 1 ofBpDREB1BThe gene promoter or nucleotide sequence is SEQ ID NO: 2 ofBpbZIP38A gene promoter;
in the third step, the concentration of mannitol and dithiothreitol in the mixed solution of mannitol and dithiothreitol is 170mM and 1 mM.
2. The transient expression method of exogenous gene transferred into white birch seedling as claimed in claim 1, wherein the white birch seedling in the first step is 4 weeks old white birch seedling.
3. The transient expression method of exogenous gene transferred birch seedling as claimed in claim 1, wherein the washing time is 3min and the rotation speed is 120 r/min.
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| CN101502237A (en) * | 2009-01-24 | 2009-08-12 | 东北林业大学 | Micro-propagation method for white birch with purple leaf |
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| WO2001044459A2 (en) * | 1999-12-15 | 2001-06-21 | Regents Of The University Of Minnesota | Method to enhance agrobacterium-mediated transformation of plants |
| WO2003062434A2 (en) * | 2002-01-22 | 2003-07-31 | Council Of Scientific And Industrial Research | Transgenic tea plants obtained through microprojectile bombardment of leaf explants |
| CN101502237A (en) * | 2009-01-24 | 2009-08-12 | 东北林业大学 | Micro-propagation method for white birch with purple leaf |
| CN103125399A (en) * | 2013-03-20 | 2013-06-05 | 东北林业大学 | Rapid issue culture propagation method for white birch tetraploid |
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