CN110656129A - Genetic transformation method of agrobacterium-mediated coix lacryma-jobi - Google Patents
Genetic transformation method of agrobacterium-mediated coix lacryma-jobi Download PDFInfo
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Abstract
The invention relates to an agrobacterium tumefaciens-mediated genetic transformation method for coix lacryma-jobi. The method uses mature seeds of the coix lacryma-jobi as a genetic transformation receptor after germination accelerating treatment and transverse cutting of shoot apical meristem, and comprises the following specific operation steps: (1) preparation of genetic transformation receptor, (2) culture of agrobacterium, genetic transformation of coix seed, and transplantation and management of transgenic plant. The invention does not need tissue culture technology, does not need aseptic operation except for culturing agrobacterium, has the advantages of simple operation, lower cost, capability of transforming large-fragment DNA, stable heredity, low copy number and the like, and becomes the most widely applied genetic transformation method at present.
Description
Technical Field
The invention belongs to the technical field of plant biotechnology, and particularly relates to a genetic transformation method for mediating coix lacryma-jobi stem tips by utilizing agrobacterium.
Background
Coix lacryma-jobi is a medicinal and edible economic crop of Coix of Gramineae, and has important medicinal value and high nutritive value. In recent years, the industry of the coix seeds in Guizhou province is rapidly developed, and the planting area of the whole province reaches 4 million more hm2It accounts for over 80% of the planting area in China. However, the small white shell of the Xingxing seed of the Job's tears has high stem, easy lodging and disease resistancePoor, etc., which affect the improvement of the yield and restrict the development of the industry.
The genetic transformation is carried out by taking the stem tip meristem of the coix lacryma-jobi as a material, and the agrobacterium-mediated method has the advantages of simple operation, low cost, capability of transforming large-fragment DNA, stable heredity, low copy number and the like, and becomes the most widely applied genetic transformation method at present. At present, a stable genetic transformation system is widely applied to monocotyledons such as corn, wheat and the like, but the application of the stable genetic transformation system to coix is not clearly reported, so that the stable genetic transformation system cannot be improved and bred by modern molecular biology means.
Disclosure of Invention
The invention obtains the transgenic plant by utilizing the agrobacterium-mediated genetic transformation method of the coix stem tip, and provides technical support for researches on genomics and gene functions, germplasm innovation and the like of coix.
A genetic transformation method of coix lacryma-jobi mediated by agrobacterium comprises the following steps:
(1) preparation of genetic transformation receptors: germinating Coicis semen seed, making stem tip grow to 2-3cm, cross-cutting germinated Coicis semen stem tip with scalpel, exposing stem tip meristem, and reserving;
(2) and (3) culturing agrobacterium: inoculating agrobacterium tumefaciens strain LBA4404 and a carrier containing hygromycin resistance genes Hyg and GUS genes to be transferred to a YEP solid culture medium containing kanamycin and rifampicin, and culturing for 2 d; picking single colony to YEP liquid culture medium containing kanamycin and rifampicin, and carrying out shake culture for 16 hours; adding the cultured bacterial liquid into a new YEP liquid culture medium containing kanamycin and rifampicin, and performing shake culture until OD600A value of 0.8 for standby;
(3) genetic transformation of coix: pouring the bacterial liquid into a bottle filled with a genetic transformation receptor until the bacterial liquid is completely submerged, adding acetosyringone and a surfactant Silwet-L77, and carrying out vacuum infiltration treatment for 15min at an osmotic pressure of 15 kpa; pouring out the bacterial liquid, placing the seeds on nutrient soil, covering a layer of nutrient soil, pouring the poured out bacterial liquid on the nutrient soil, and carrying out dark culture for 3d at 28 ℃; the nutrient soil is perlite, wherein the nutrient soil is 1: 4;
(4) transplanting and managing transgenic plants
And (4) canceling the dark culture of the dark-cultured plants, transplanting the living plants to a greenhouse of a transgenic plant demonstration base after one week, and performing conventional fertilizer and water management.
The transverse cutting is the coix seed stem tip which is transversely cut from the position 0.1-0.2cm above the stem ring.
The coix seed germination comprises placing sterilized seeds on a culture tray, covering with absorbent paper to keep moist, sealing the culture tray with preservative film to prevent water evaporation, and placing in a constant temperature incubator at 28 deg.C for dark culture until the seeds germinate.
The disinfection treatment method comprises the following steps: selecting seeds with full grains and bright color, cleaning impurities on the surface by using a detergent, then putting the seeds into boiling water for disinfection for 3-4s, cooling, soaking the seeds in 1% carbendazim for 7-8 hours, taking out the seeds, and cleaning the seeds by using clean water.
The YEP culture medium in the step (2) contains 50mg/L kanamycin and 50mg/L rifampicin; inoculating the strain to a solid culture medium, and culturing in an incubator at 28 ℃ for 2 d; picking single colony to 5ml YEP liquid culture medium, shaking and culturing at 28 deg.C and 180rpm for 16 hr; adding 100ul of cultured bacterial liquid into 100ml YEP liquid culture medium, performing shaking culture at 28 deg.C and 180rpm to OD600The value is 0.8 for standby.
100ul of 100mg/ml Acetosyringone (AS) and 20ul of 200ul of surfactant Silwet-L77 are added in the step (3).
The vector containing the gene to be transferred is a vector PcambiaU1301 carrying hygromycin resistance genes Hyg and GUS genes.
The method also comprises transgenic plant identification, wherein the transgenic plant identification comprises GUS chemical tissue staining detection of a resistant plant and PCR detection of the transgenic plant.
The GUS chemical tissue staining detection method of the resistant plant comprises the steps of after transplanting for two weeks, taking transgenic coix lacryma-jobi leaves and root systems, cutting the leaves and the root systems into 1-2 cm long leaves, placing the leaves and the root systems into a PCR tube, adding 200ul of GUS dye solution into the PCR tube, carrying out vacuum treatment for 5min at 15Kpa, and placing the PCR tube in a constant-temperature incubator at 37 ℃ for overnight reaction. When the leaves and the root system turn blue, absorbing the dye liquor, adding 1ml of 20%, 50%, 70% and 100% ethanol in sequence for decolorization, and decolorizing for 1h each time until the chlorophyll disappears, and photographing and observing.
The PCR detection method of the transgenic plant is to take GUS dye solution to detect the plants with positive leaves and roots, extract DNA by adopting a CTAB method and carry out further PCR detection.
The PCR detection method of the transgenic plant comprises that a 10 mu L PCR system contains 1.0 mu L DNA, 0.2 mu L of each primer, rTaq5.0ul and ddH2O3.6. mu.L. Reaction conditions are as follows: 94 ℃ for 4 min; 30s at 94 ℃; at 54 ℃ for 30 s; 72 ℃ for 1 min; 35 cycles; extending for 5min at 72 ℃; storing at 12 deg.C. After the PCR reaction, 5ul of the amplified product was electrophoresed in 1% agarose gel, observed under a gel imaging system and photographed.
Mature seeds of coix seeds are used as materials, stem tip meristems of the coix seeds are transversely cut after germination accelerating treatment, the mature seeds are used as genetic transformation receptors, bacterial liquid, acetosyringone and a surfactant Silwet-L77 are added, dark cultivation is carried out for 3d after dip dyeing under different conditions, GUS chemical tissue dyeing and PCR identification of transgenic plants are carried out after the plants grow out. Different pressures and different vacuum treatment times are researched in the process, factors influencing the transformation efficiency are verified by combining an orthogonal experiment, and finally the agrobacterium tumefaciens-mediated coix lacryma-jobi stem tip transformation method and optimal transformation conditions are obtained; and (3) directly planting the receptor after genetic transformation in nutrient soil, pouring the genetically transformed bacterial liquid on the nutrient soil, carrying out dark culture at 28 ℃ for 3d, and then directly planting according to a conventional method.
The invention obtains the transgenic plant by utilizing the agrobacterium-mediated genetic transformation method of the coix stem tip, and provides technical support for researches on genomics and gene functions, germplasm innovation and the like of coix.
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FIG. 1 shows the germination of seeds of Coix lacryma-jobi and the cutting of the stem tip of Coix lacryma-jobi.
Wherein, A: selecting plump coix seeds before germination; b: seeds germinated for 3 days; c: the stem tip of the coix lacryma-jobi is transversely cut from the position 0.1-0.2cm above the stem ring.
FIG. 2 genetically transformed plants.
Wherein, A: 3 days after genetic transformation, the dark culture plants are cancelled; b: transplanting the plants 3 days after dark culture is cancelled; c: transgenic plants two weeks after transplantation; d: transplanting the transgenic plants one month later.
FIG. 3GUS staining Coix lacryma-jobi leaves.
Wherein, A: the GUS staining does not show blue wild type coix plant leaves B: GUS stains the blue leaves of the transgenic coix plants.
FIG. 4GUS staining root system of Job's tears.
Wherein, A: the root system B of the coix plant of the blue wild type does not appear after GUS staining: GUS stains the blue root system of the transgenic coix plant.
FIG. 5GUS gene PCR results.
Detailed Description
The present invention will be described in further detail with reference to examples.
YEP culture medium formula: yeast extract 10g/L, peptone 10g/L, sodium chloride 5g/L, pH 7.2, solid added agar 15 g/L.
Example 1 genetic transformation of Coix lacryma-jobi Using Agrobacterium
1.1 seed disinfection: cleaning surface impurities of seeds with detergent, sterilizing in boiling water for 3-4s, taking out, naturally drying, and soaking in 1 ‰ carbendazim for 7-8 hr. Taking out, cleaning, placing on a culture plate, placing in a constant temperature incubator at 28 deg.C, dark culturing until the seed germinates and the stem tip grows to 2-3cm, transversely cutting the germinated Coicis semen stem tip with a scalpel, and exposing its stem tip meristem for use. As shown in fig. 1.
1.2 culture of the bacteria: agrobacterium tumefaciens strain LBA4404, vector PcambiaU1301 vector carrying hygromycin resistance gene Hyg and GUS gene (commercially available) was inoculated onto YEP solid medium containing 50mg/L kanamycin (Kan) and 50mg/L rifampicin (Rif), and cultured in an incubator at 28 ℃ for 2 d; single colonies were picked up to 5ml YEP (containing 50mg/L Kan and 50mg/L Rif) in liquid medium, and after shaking culture at 28 ℃ and 180rpm for 16 hours, 100ul of the single colonies were added to 100ml YEP (containing 50mg/L Kan and 50mg/L Rif) in liquid medium, and shaking culture at 28 ℃ and 180rpm was carried out to OD600The value is 0.8 for standby.
1.3 genetic transformation: the prepared genetic transformation receptors (100 particles/group) are poured into the bacterial liquid until the bacterial liquid is completely submerged, 100ul of 100mg/ml Acetosyringone (AS) and 20ul of 200ul/L surfactant Silwet-L77 are added.
The dyeing was carried out under the conditions of the respective factors (see Table 1). Pouring out the bacterial liquid, placing on a substrate (perlite: nutrient soil is 1: 4), covering a layer of substrate, pouring the impregnated bacterial liquid on the substrate, and performing dark culture at 28 ℃ for 3 d.
TABLE 1 Dip-dyeing of Coix lacryma-jobi under different conditions
1.4 transplanting and managing the transgenic plants: and (4) canceling the dark culture of the dark-cultured plants, watering the dark-cultured plants frequently, transplanting the living plants to a greenhouse of a transgenic plant demonstration base after one week, and performing conventional fertilizer and water management. As shown in fig. 2.
1.5 identification of transgenic plants
1.5.1 GUS chemical tissue staining detection of resistant plants: cutting the transgenic coix leaf and root system into 1-2 cm long, placing in a PCR tube, vacuum treating at 15Kpa for 5min, adding GUS dye solution 100ul into the PCR tube, and placing in a constant temperature incubator at 37 deg.C for overnight reaction. Sucking the dye solution, adding 1ml of 20%, 50%, 70% and 100% ethanol in sequence for decolorization for 1h each time until chlorophyll disappears, and taking a picture for observation. The results showed that in table 1, 165 survivors in total, and 11 positive plants were identified in total by GUS staining treatment, specifically: under the vacuum osmotic pressure of 15Kpa, when the vacuum processing time is respectively 8min, 10min and 15min, 100 genetic transformation acceptors/group are adopted, 20 plants, 25 plants and 30 plants respectively survive, the surviving plants are subjected to GUS dyeing, the dyeing liquor is blue, 0 plant, 1 plant and 5 plants respectively exist, and the survival rate and the GUS dyeing rate are respectively 20%, 25%, 30%, 0%, 4.00% and 16.67%; under the vacuum osmotic pressure of 18Kpa, when the vacuum processing time is respectively 8min, 10min and 15min, 100 genetic transformation acceptors are used per group, the number of the surviving plants is respectively 25, 20 and 15, the surviving plants are subjected to GUS dyeing, the dyeing liquor is blue, 2 plants, 1 plant and 1 plant are respectively, and the survival rate and the GUS dyeing rate are respectively 25%, 20%, 15%, 8.00%, 5.00% and 6.67%; under the vacuum osmotic pressure of 20Kpa, when the vacuum processing time is respectively 8min, 10min and 15min, 100 genetic transformation acceptors are used per group, 15 plants, 10 plants and 5 plants survive, the GUS staining is carried out on the survived plants, the staining solution is blue and respectively comprises 1 plant, 0 plant and 0 plant, and the survival rate and the GUS staining rate are respectively 15%, 10%, 5% and 6.67%, 0% and 0%; as can be seen from the table, the higher the vacuum osmotic pressure is, the longer the vacuum treatment time is, the higher the survival rate of the plant and the GUS staining rate is, and after a certain pressure is reached, the survival rate and the GUS staining rate are reduced on the contrary along with the increase of the time. The optimal transformation condition is that the vacuum osmotic pressure is 15Kpa, the survival rate of the plants is 30 percent and the GUS staining rate is 16.67 percent when the vacuum treatment time is 15 min. See fig. 3 and 4.
1.5.2 PCR detection of transgenic plants
1.5.2.1 CTAB method for extracting DNA:
1. to a 2ml centrifuge tube, add 1ml CTAB extraction buffer, 65 degrees C preheating.
2. Grinding fresh leaves of 0.1-0.2g Coicis semen positive plant until cells are broken, maintaining in 65 deg.C water bath for 1h, and mixing by reversing every 10 min.
3. 13200rpm, centrifuge for 7min, absorb 800ul of supernatant into a new centrifuge tube, add equal volume of chloroform/phenol (1:1), mix by gentle inversion for 2-3 times.
4. 13200rpm, 5min, transfer supernatant 700ul in new centrifuge tube, add equal volume of chloroform (precool), gently reverse mixing.
5. 13200rpm, centrifuge for 5min, transfer the supernatant 600ul into a new centrifuge tube, add equal volume of chloroform (precool), mix by gentle inversion.
6. 13200rpm, centrifuging for 5min, transferring the supernatant 500ul into a new centrifuge tube, adding 0.6-0.8 times of isopropanol, slightly reversing and mixing, and standing at-20 ℃ for 30 min.
7. 10000rpm, centrifugation for 10min, supernatant removal, addition of 1ml of 75% alcohol, and repetition for 2 times.
8. Removing supernatant, naturally drying, and dissolving in 50-100ul ddH2O-RNAase for use.
1.5.2.2 GUS gene PCR detection
And (4) taking a GUS dye solution to detect positive plants, and carrying out PCR detection on GUS genes. 10 uL PCR system containing 1.0 uL DNA, 0.2 uL each of primers, 5.0ul rTaq, ddH2O3.6. mu.L. Reaction conditions are as follows: 94 ℃ for 4 min; 30s at 94 ℃; at 54 ℃ for 30 s; 72 ℃ for 1 min; 35 cycles; extending for 5min at 72 ℃; storing at 12 deg.C. After the PCR reaction, 5ul of the amplified product was electrophoresed in 1% agarose gel, observed under a gel imaging system and photographed. PCR identification is carried out on 11 positive plants obtained by GUS staining treatment of 9 experimental groups, and the result shows that: in the PCR detection of GUS gene of 11 coix plants which are positive in GUS dye liquor detection, 9 groups of experimental groups obtain 9 transgenic plants in total, and the transgenic plants are identified to account for 81.80 percent of the positive plants. As shown in fig. 5.
Claims (10)
1. A genetic transformation method of coix lacryma-jobi mediated by agrobacterium comprises the following steps:
(1) preparation of genetic transformation receptors: germinating Coicis semen, cutting the germinated Coicis semen stem tip with scalpel, and exposing its stem tip meristem for use;
(2) and (3) culturing agrobacterium: inoculating agrobacterium tumefaciens strain LBA4404 and a vector containing a gene to be transferred to a YEP solid culture medium containing kanamycin and rifampicin, and culturing for 2 d; picking single colony to YEP liquid culture medium containing kanamycin and rifampicin, and carrying out shake culture for 16 hours; adding the cultured bacterial liquid into a new YEP liquid culture medium containing kanamycin and rifampicin, and performing shake culture until OD600A value of 0.8 for standby;
(3) genetic transformation of coix: pouring the bacterial liquid into a bottle filled with a genetic transformation receptor until the bacterial liquid is completely submerged, adding acetosyringone and a surfactant Silwet-L77, and carrying out vacuum infiltration treatment for 15min at an osmotic pressure of 15 kpa; pouring out the bacterial liquid, placing the seeds on nutrient soil, covering a layer of nutrient soil, pouring the poured out bacterial liquid on the nutrient soil, and carrying out dark culture for 3d at 28 ℃;
(4) transplanting and managing the transgenic plants: and (4) canceling the dark culture of the dark-cultured plants, transplanting the living plants to a greenhouse of a transgenic plant demonstration base after one week, and performing conventional fertilizer and water management.
2. The method of claim 1, wherein the transection is a coixseed stem tip transected from 0.1-0.2cm above the stem annulus.
3. The method of claim 1, wherein the coix seed germination comprises placing sterilized seeds on a culture tray, covering with absorbent paper to keep wet, sealing the culture tray with preservative film to prevent water evaporation, and placing in a constant temperature incubator at 28 ℃ for dark culture until seeds germinate.
4. The method of claim 3, the sterilization process method: selecting seeds with full grains and bright color, cleaning impurities on the surface by using a detergent, then putting the seeds into boiling water for disinfection for 3-4s, cooling, soaking the seeds in 1% carbendazim for 7-8 hours, taking out the seeds, and cleaning the seeds by using clean water.
5. The method according to claim 1, wherein the YEP medium of step (2) contains 50mg/L kanamycin and 50mg/L rifampicin; inoculating the strain to a solid culture medium, and culturing in an incubator at 28 ℃ for 2 d; picking single colony to 5ml YEP liquid culture medium, shaking and culturing at 28 deg.C and 180rpm for 16 hr; adding 100ul of cultured bacterial solution into 100ml YEP liquid culture medium, performing shaking culture at 28 deg.C and 180rpm to OD600The value is 0.8 for standby.
6. The process of claim 1, wherein in step (3) 100ul of 100mg/ml Acetosyringone (AS) and 20ul of 200ul of surfactant Silwet-L77 are added.
7. The method according to claim 1, wherein the vector containing the gene to be transferred is a vector PcambiaU1301 carrying hygromycin resistance gene Hyg and GUS gene.
8. The method of claim 1, further comprising the steps of (5) transgenic plant identification comprising GUS chemical tissue staining detection of resistant plants and transgenic plant PCR detection;
the GUS chemical tissue staining detection method of the resistant plant comprises the steps of after transplanting for two weeks, shearing leaves and root systems of coix lacryma-jobi in a seedling stage, putting the leaves and the root systems into a PCR tube, adding GUS staining solution, pouring off the staining solution after the leaves and the root systems turn blue, adding ethanol for eluting until chlorophyll is completely removed, and taking a picture for observation;
the PCR detection method of the transgenic plant is to take GUS dye solution to detect the plants with positive leaves and roots, extract DNA by adopting a CTAB method and carry out further PCR detection.
9. The method as claimed in claim 8, wherein the GUS chemical tissue staining detection method for the resistant plants comprises cutting leaves and roots of transgenic coix lacryma-jobi into 1-2 cm long, placing in a PCR tube, vacuum treating at 15Kpa for 5min, adding 200ul GUS staining solution into the PCR tube, and placing in a constant temperature incubator at 37 ℃ for overnight reaction. Sucking the dye solution, adding 1ml of 20%, 50%, 70% and 100% ethanol in sequence for decolorization, and decolorizing for 1h each time until chlorophyll disappears, and taking a picture for observation.
10. The method of claim 8, wherein the PCR detection method for the transgenic plant is that a 10 μ L PCR system contains 1.0 μ L DNA, 0.2 μ L each of primers, 5.0ul rTaq and 3.6 μ L ddH2O3. Reaction conditions are as follows: 94 ℃ for 4 min; 30s at 94 ℃; at 54 ℃ for 30 s; 72 ℃ for 1 min; 35 cycles; extending for 5min at 72 ℃; storing at 12 deg.C. After the PCR reaction, 5ul of the amplified product was electrophoresed in 1% agarose gel, observed under a gel imaging system and photographed.
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