CN111118055A - Method for establishing transgenic system of high-sugar variety beet - Google Patents
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Abstract
The invention discloses a method for establishing a transgenic system of a high-sugar variety beet, which comprises the steps of beet seed germination, beet seedling growth, agrobacterium infection, gene identification or resistance screening and the like. The invention adopts a transgenic beet method, provides technical support for improving the important alkaloid content of the beet, improving the ornamental quality of the beet and the basic research that the economic value is related to and depends on the transgenic technology, and has important reference significance for establishing transgenic methods of other plants of the beet.
Description
Technical Field
The invention relates to a method for establishing a beet transgenic system.
Background
Sugar beet (Beta vulgaris) is a group of herbaceous plants with high economic and ornamental value. Belongs to a biennial herb of the genus beta of the family Chenopodiaceae, the aerial part of which is basal leaves with large leaves and the root of which is cone-shaped to spindle-shaped and juicy. The stem is upright, the basal leaves are rectangular and round, the upper part of the stem is long, the stem is wrinkled and uneven, the lower part of the stem is provided with a thick and protruded leaf vein which is full-edged or slightly wavy, the stem is thick, the stem and the leaf are grown mutually, the stem and the leaf are small, the flower cluster is in a cluster shape, the flower is in a strip shape or in a narrow rectangular and round shape, and the upper part of the fruiting body is slightly fleshy. The seeds are biconvex, reddish brown, bloom in 5-6 months and bear fruits in 7 months.
Native to the western and southern coasts of europe, transplanted from sweden to spain, is widely cultivated, highly variable, and divided into several subspecies, varieties, and varieties.
Sugar beet is also a major sugar source in addition to sugar cane. Beet is commonly cooked or salted in the united states, and russian beet pottage is the traditional beet soup in eastern europe. Sugar beet is the most important commercial type, and fodder beet and leaf beet are cultivated as in most crops.
Bright in color, unique in flower posture, leaves after flowers and the like, and is widely applied to afforestation of garden grounds and production of fresh cut flowers. And the special alkaloid contained in the beet also has important pharmacological value. Research shows that betaine has the functions of resisting cancer, diminishing inflammation and inhibiting cholinesterase, and has been widely used in clinical treatment. However, the content of important alkaloids in the beet is very low, which cannot meet the expanding demand, and the wild beet resources are seriously damaged by excessive collection. Therefore, increasing the content of important alkaloids in beet plants is a problem that needs to be solved urgently in the current beet development and utilization. The beet is a species with the widest distribution range and the strongest adaptability in the beet plants in China, but belongs to self-incompatible species and cannot improve the quality by a conventional hybridization method; and other beet plants propagated by the seeds need at least 2-3 years from the seedling to the flowering, and the cross breeding period is very long. Therefore, the establishment of the beet transgenic technology system not only is a necessary way for improving the beet, but also has the advantage of high efficiency for improving other beet plants, and can greatly shorten the breeding process. Although the techniques of sugar beet tissue culture and rapid propagation are mature and callus induction is reported, the transgenic method of sugar beet is not reported, and the transgenic method of the sugar beet is not reported in the genus of sugar beet.
Disclosure of Invention
The invention aims to provide a method for establishing a transgenic system of a high-sugar variety beet cultivated by an agrobacterium-mediated method.
The technical solution of the invention is as follows:
a method for establishing a transgenic system of a high-sugar variety beet is characterized by comprising the following steps: comprises the following steps:
A. germinating beet seeds:
selecting 60 beet seeds harvested in the current year, placing the beet seeds in a 250ml conical flask, adding tap water to immerse the beet seeds, placing the beet seeds in a shaking table at 37 ℃ for culturing for 24 hours, washing the beet seeds with running water for 24 hours, sowing the beet seeds in a culture bottle containing moist gauze, placing the culture bottle in a culture room at 22 ℃ and with illumination duration of 16 hours per night 8 hours for culturing;
B. growth of beet seedlings:
when the beet seeds germinate buds, the number of days for growth is recorded, and the beet seeds are used for experiments when growing for 10 days;
C. infection of agrobacterium:
a. expanded culture of Agrobacterium
1) Preparing LB solid and liquid culture medium containing kanamycin; the kanamycin is used at the concentration of 50 mg/L;
2) in a workbench, the preserved strain is scratched on an LB solid culture medium containing kanamycin by using an inoculating loop and cultured for 48 hours at 28 ℃ in the dark;
3) taking out the bacterial plate, picking a single bacterial colony by using a sterilized gun head, placing the single bacterial colony in 20ml of LB liquid culture medium containing kanamycin, and shaking the bacterial colony at the speed of 220rpm at the temperature of 28 ℃ for 24 hours;
4) pouring the shaken bacterial liquid into 200ml of LB liquid culture medium containing kanamycin, shaking at 28 ℃ and 220rpm for 8-12 h; volume ratio: v culture medium V bacterial liquid =100: 1;
b. agrobacterium infection of sugar beet
1) Cutting one cotyledon and terminal bud of the young beet seedling with cotyledon, and pouring the cut beet into a beaker;
2) adding a certain amount of AS with the preservation concentration of 0.5mol/l into the bacteria liquid shaken in the step a 4) to ensure that the final concentration of AS is 0.1mmol/l, then pouring the bacteria liquid into a beaker, and shaking uniformly, wherein the bacteria liquid is 100ml of bacteria liquid plus 20ul of AS generally;
3) putting the beaker into a vacuum bottle, and vacuumizing for 20 min;
4) preparing a box, and laying a layer of filter paper soaked in water on the bottom of the box;
5) step 3), after vacuum treatment, taking out the beet seedlings in the beaker, putting the beet seedlings in a box paved with filter paper, and then shielding the roots with paper;
6) sealing the box with preservative film, then completely covering the box with black bag, and culturing in dark for 1 day in 25 deg.C incubator;
c. transplanting of beet after dip dyeing
1) Mixing the components in a mass ratio of 1: 1, sterilizing the nutrient soil and vermiculite mixed according to the proportion under high pressure, then putting the mixture into a tray, and pouring water into the tray for soaking;
2) planting the beet seedlings cultured in the dark into nutrient soil, and exposing cotyledons;
3) sealing with preservative film, placing in a greenhouse, culturing for 3 days, and uncovering the film after three days;
D. gene identification or resistance screening
After the membrane is uncovered, identifying after new cotyledon grows out; the vector is a recombinant vector containing green fluorescence, and during identification, leaves are taken for fluorescence observation and PCR identification; furthermore, the callus growth was also observed by placing explants sterile on resistant callus medium containing kanamycin.
E. PCR detection of transgenic sugar beet
And extracting DNA from the obtained transgenic beet plant, designing GFP gene primer amplification, carrying out electrophoresis detection, and identifying the transgenic plant.
The Agrobacterium used for the padding in step C may be any Agrobacterium tumefaciens, such as EHA 105.
The invention adopts a transgenic beet method, provides technical support for improving the important alkaloid content of the beet, improving the ornamental quality of the beet and the basic research that the economic value is related to and depends on the transgenic technology, and has important reference significance for establishing transgenic methods of other plants of the beet.
The invention will produce the following positive effects: (1) accelerating the transfer of the excellent exogenous gene to the beet; (2) provides a quick and effective method for improving the content, sugar content and ecological environment value of important betaine; (3) provides a new idea for the production of important betaine.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a schematic representation of the germination of sugar beet seeds.
FIG. 2 is a schematic illustration of the growth of sugar beet seedlings.
FIG. 3 is a schematic representation of the culture after transfection of transgenic sugar beets.
FIG. 4 is a schematic representation of fluorescence observation of transgenic plant seedlings.
FIG. 5 is a schematic diagram showing the results of PCR amplification detection of transgenic sugar beet.
Detailed Description
Example 1: germination and growth of sugar beet seeds
1) Selecting 60 beet seeds harvested in the current year, placing in a 250ml conical flask, adding tap water to immerse, culturing in a shaking table at 37 ℃ for 16h, washing with running water for 24h, sowing in a culture flask containing moist gauze, and placing in a culture room at 22 ℃ and with illumination time of 16h/8h for culturing, as shown in figure 1.
2) When the beet seeds germinate buds, the growth days are recorded, and the beet seeds can be used for experiments when growing about 10 days.
See fig. 2.
Example 2: transformation of transgenic sugar beet
a. Expanded culture of Agrobacterium
① LB solid and liquid culture media containing kanamycin with a concentration of 50 mg/L;
② in the working table, the preserved strain is cut into plates by inoculating loop in the solid culture medium containing kanamycin, and cultured for 48h at 28 ℃ in the dark;
③ taking out the bacterial plate, picking out single bacterial colony with sterilized yellow gun head, placing in 20ml LB liquid culture medium containing kanamycin, shaking at 28 deg.C and 220rpm for 24 h;
④ pouring the shaken bacterial liquid into 200ml LB liquid culture medium containing kanamycin, shaking at 28 deg.C and 220rpm for 8-12h (note: volume ratio: V culture medium: V bacterial liquid =100: 1);
b. agrobacterium dip-dyeing of sugar beet
① cutting one cotyledon and terminal bud of young beet with scalpel, and pouring the cut beet into beaker;
② adding AS (acetosyringone) with preservation concentration of 0.5mol/l into the shaken agrobacterium to make the final concentration of AS 0.1mmol/l, then pouring the bacterial liquid into a beaker, generally 100ml bacterial liquid +20ul AS, shaking up;
③ placing the beaker in a vacuum bottle, and vacuumizing for 20min
④ A box is prepared, a layer of filter paper soaked in water is laid on the bottom of the box,
⑤ 20min vacuum, taking out the soaked beet seedling, placing into a box with filter paper, and covering the root with paper;
⑥ sealing the box with preservative film, then completely covering the box with black bag, culturing in 25 deg.C incubator in dark for 1 day;
c. transplanting of beet after dip dyeing
① sterilizing the nutrient soil and vermiculite mixed at a mass ratio of 1: 1, placing into a tray, and soaking in appropriate water;
② planting the seedling of beet in the dark in the nutrient soil to expose the cotyledon;
③ sealing with plastic wrap, placing in a greenhouse, culturing for 3 days, and uncovering the plastic wrap after three days;
gene identification or resistance screening
After the membrane is removed, the new cotyledon is identified after growing out. The vector used in the experiment is a recombinant vector containing green fluorescence, so that leaves can be taken for fluorescence observation and PCR identification during identification. Furthermore, the callus growth can be observed by placing the explant on a resistant callus culture medium which also has resistance to Carna
Implementation 3: transgenic sugar beet detection
1. Fluorescence detection of transgenic beet young and tender leaves
The freshly grown fresh leaves obtained in example 2 were subjected to fluorescence detection, which was performed as follows: fresh leaf back epidermal tissue is taken and placed on a glass slide, and sterile water is dripped for carrying out fluorescence detection.
2. PCR detection of transgenic sugar beet
Detecting fresh leaves by using a PCR method, designing a primer according to a GFP gene sequence carried on a vector, wherein the size of a theoretical amplification product is 516 bp, and the primer sequence is as follows:
primer 1 (forward primer): 5' -ATCCCGCCGGGAATGGTGATTACCGA
Primer 2 (downstream primer): 5' -GTCGTGCACCATCAGCACGTTATCGA
Extracting total DNA of fresh beet leaves by SDS method, using the total DNA as template, and carrying out PCR amplification on the DNA of transgenic plant under the guide of primer 1 and primer 2, wherein the PCR reaction conditions are as follows: 94 ℃, 5min, -94 ℃, 30 s-58 ℃, 30 s-72 ℃, 30 s-30 cycles-72 ℃ and 10 min.
After the reaction is finished, 1% agarose gel electrophoresis detection is carried out on the PCR product, the detection result is shown in figure 5 (Lane M: DNA marker DL2000, Lanes 1-4: different plants to be detected, Lane 5: non-transgenic control, Lane 6: negative control), and the positive transgenic plant with 516 bp DNA fragment can be amplified.
Claims (3)
1. A method for establishing a transgenic system of a high-sugar variety beet is characterized by comprising the following steps: comprises the following steps:
A. germinating beet seeds:
selecting 60 beet seeds harvested in the current year, placing the beet seeds in a 250ml conical flask, adding tap water to immerse the beet seeds, placing the beet seeds in a shaking table at 37 ℃ for culturing for 24 hours, washing the beet seeds with running water for 24 hours, sowing the beet seeds in a culture bottle containing moist gauze, placing the culture bottle in a culture room at 22 ℃ and with illumination duration of 16 hours per night 8 hours for culturing;
B. growth of beet seedlings:
when the beet seeds germinate buds, the number of days for growth is recorded, and the beet seeds are used for experiments when growing for 10 days;
C. infection of agrobacterium:
a. expanded culture of Agrobacterium
1) Preparing LB solid and liquid culture medium containing kanamycin; the kanamycin is used at the concentration of 50 mg/L;
2) in a workbench, the preserved strain is scratched on an LB solid culture medium containing kanamycin by using an inoculating loop and cultured for 48 hours at 28 ℃ in the dark;
3) taking out the bacterial plate, picking a single bacterial colony by using a sterilized gun head, placing the single bacterial colony in 20ml of LB liquid culture medium containing kanamycin, and shaking the bacterial colony at the speed of 220rpm at the temperature of 28 ℃ for 24 hours;
4) pouring the shaken bacterial liquid into 200ml of LB liquid culture medium containing kanamycin, shaking at 28 ℃ and 220rpm for 8-12 h; volume ratio: v culture medium V bacterial liquid =100: 1;
b. agrobacterium infection of sugar beet
1) Cutting one cotyledon and terminal bud of the young beet seedling with cotyledon, and pouring the cut beet into a beaker;
2) adding a certain amount of AS with the preservation concentration of 0.5mol/l into the bacteria liquid shaken in the step a 4) to ensure that the final concentration of AS is 0.1mmol/l, then pouring the bacteria liquid into a beaker, and shaking uniformly, wherein the bacteria liquid is 100ml of bacteria liquid plus 20ul of AS generally;
3) putting the beaker into a vacuum bottle, and vacuumizing for 20 min;
4) preparing a box, and laying a layer of filter paper soaked in water on the bottom of the box;
5) step 3), after vacuum treatment, taking out the beet seedlings in the beaker, putting the beet seedlings in a box paved with filter paper, and then shielding the roots with paper;
6) sealing the box with preservative film, then completely covering the box with black bag, and culturing in dark for 1 day in 25 deg.C incubator;
c. transplanting of beet after dip dyeing
1) Mixing the components in a mass ratio of 1: 1, sterilizing the nutrient soil and vermiculite mixed according to the proportion under high pressure, then putting the mixture into a tray, and pouring water into the tray for soaking;
2) planting the beet seedlings cultured in the dark into nutrient soil, and exposing cotyledons;
3) sealing with preservative film, placing in a greenhouse, culturing for 3 days, and uncovering the film after three days;
D. gene identification or resistance screening
After the membrane is uncovered, identifying after new cotyledon grows out; the vector is a recombinant vector containing green fluorescence, and during identification, leaves are taken for fluorescence observation and PCR identification; furthermore, the callus growth was also observed by placing explants sterile on resistant callus medium containing kanamycin.
2. The method of claim 1, wherein the transgenic line of high sugar beet variety comprises: the agrobacterium used for the padding in step C was EHA 105.
3. The method for creating a transgenic line of high-sugar beet as claimed in claim 1 or 2, wherein the transgenic line comprises: also included are transgenic sugar beet assays:
1) fluorescence detection of transgenic beet young and tender leaves
And D, performing fluorescence detection on the fresh leaves newly grown after the treatment of the step C, wherein the fluorescence detection step is as follows: placing the leaf back epidermal tissue of a fresh leaf on a glass slide, and dropping a sterile water-pressed sheet for fluorescence detection;
2) PCR detection of transgenic sugar beet
Detecting fresh leaves by using a PCR method, designing a primer according to a GFP gene sequence carried on a carrier, wherein the size of a theoretical amplification product is 516 bp, and the primer sequence is as follows:
primer 1 (forward primer): 5' -ATCCCGCCGGGAATGGTGATTACCGA
Primer 2 (downstream primer): 5' -GTCGTGCACCATCAGCACGTTATCGA
Extracting total DNA of fresh beet leaves by SDS method, using the total DNA as template, and carrying out PCR amplification on the DNA of transgenic plant under the guide of primer 1 and primer 2, wherein the PCR reaction conditions are as follows: 94 ℃, 5min, 94 ℃, 30 s-58 ℃, 30 s-72 ℃, 30 s-30 cycles-72 ℃, 10 min;
after the reaction is finished, 1% agarose gel electrophoresis detection is carried out on the PCR product, and a positive transgenic plant with the DNA fragment of 516 bp is amplified.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116445537A (en) * | 2023-06-19 | 2023-07-18 | 海南大学三亚南繁研究院 | Genetic transformation method of dragon fruit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116445537A (en) * | 2023-06-19 | 2023-07-18 | 海南大学三亚南繁研究院 | Genetic transformation method of dragon fruit |
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