Genetic transformation method of radish
The injection of Agrobacterium infectious solution carrying AtLEC2 gene by low-temperature culture and micro-injection method solved the problem that the radish genetic transformation system depends on tissue culture, achieved efficient radish genetic transformation, improved the conversion rate and promoted radish molecular biology Research.
Patent Information
- Application Number
- CN202411349711.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2024-12-27
AI Technical Summary
The existing genetic transformation system of radish relies on tissue culture, is inefficient and difficult to achieve efficient gene transformation.
Low-temperature culture and microinjection method were used to inject Agrobacterium infestation solution carrying the target gene into the apical meristem of radish seedlings. Transgenic plants were verified by PCR detection, and an efficient radish genetic transformation system that did not rely on tissue culture was established.
The genetic transformation of radish with a high conversion rate (up to 82%) was achieved, which shortened the generation time of transgenic plants, and promoted the molecular biology research and genetic breeding of radish.
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Figure CN119193665A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant transgenic technology, and more specifically to a genetic transformation method for radish. Background Art
[0002] Radish, also known as radish and radish, is a biennial herbaceous plant of the genus Raphanus in the family Cruciferae. It is a popular vegetable with a long history of cultivation in China. As the main edible organ, the fleshy root of radish is rich in various nutrients, including carbohydrates, vitamins, inorganic salts and mustard oil. Radish can be used to make soup, pickles, and oil. In addition to being a food ingredient, radish also has a long history of medicinal use. According to relevant historical records, the medicinal history of radish has exceeded 1,500 years. The Compendium of Materia Medica describes radish as follows: it tastes spicy and sweet, is cool in nature, enters the lung and stomach meridians, is a good food therapy product, can treat or assist in the treatment of many diseases, and is called "the most beneficial vegetable". Among them, the Latin name of radish is Raphanus sativus L.
[0003] LEAFYCOTYLEDON gene is abbreviated as LEC gene. There are two types of LEC gene, LEC1 and LEC2. Scientists have found that its mutation in Arabidopsis can cause embryonic death or abnormal development. LEC2 gene encodes a transcription factor that can bind to DNA sequences and regulate the expression of other genes. The expression of LEC2 gene is regulated by many factors, including light, hormones, nutrition, etc. During the development of Arabidopsis, LEC2 gene directly or indirectly regulates the expression of a large number of genes related to seed development, mainly expressed in endosperm and ovules. Its expression level in endosperm directly affects the size and morphology of seeds. The protein encoded by it can regulate the seed development and the synthesis of nutrients in plants. LEC2 gene plays a key role in embryonic development. Its main functions include controlling the transformation of endosperm, the synthesis of embryonic nutrients and the accumulation of oils. In the early stage of embryonic development, LEC2 gene can regulate the expression of Seed Storage Protein gene and promote the accumulation of protein in endosperm. At the same time, the LEC2 gene can also regulate the expression of multiple key genes in the lipid metabolism pathway, such as Acetyl-CoaCarboxylase, Ketoacyl-Acyl Carrier Protein SynthaseⅡ, etc., thereby promoting the synthesis and accumulation of lipids. In addition to its role in embryonic development, the LEC2 gene also plays an important role in the seedling and mature stages of plants. In the seedling stage, the LEC2 gene can regulate the germination of dormant seeds and the growth and development of embryos; in the mature stage, the LEC2 gene can promote the accumulation and metabolism of lipids in plants. Among them, the full name of LEC is Leafy Cotyledon. The full name of LEC1 is LeafyCotyledon 1. The full name of LEC2 is Leafy Cotyledon 2. The Latin name of Arabidopsis thaliana is Arabidopsisthaliana. The abbreviation of Seed Storage Protein is SSP. The abbreviation of Acetyl-Coa Carboxylase is ACC. The abbreviation of Ketoacyl-Acyl CarrierProtein SynthaseⅡ is KASⅡ.
[0004] Radish is a common vegetable crop with important economic and medicinal value. According to a large number of studies, radish is a plant that is extremely difficult to regenerate. In recent years, many researchers have done a lot of research on the establishment of a radish genetic transformation system, but there is no mature radish genetic transformation system so far. At present, the radish genetic transformation system relies on tissue culture. Therefore, it is necessary to develop a genetic transformation system that does not rely on tissue culture. Summary of the invention
[0005] The invention provides a radish genetic transformation method, which is used to solve the problem of radish genetic transformation system relying on tissue culture in the prior art.
[0006] In a first aspect, the present invention provides a method for genetic transformation of radish, comprising the following steps:
[0007] Obtain robust radish seedlings by low-temperature cultivation: After the radish seeds germinate, they are cultured at a low temperature of 2.5℃ to 5.5℃ for 9 to 16 days, and then vernalized to obtain radish plants in the seedling stage.
[0008] Agrobacterium carrying the target gene is cultured, centrifuged and resuspended to obtain Agrobacterium infection solution.
[0009] The Agrobacterium infection solution is injected into the apical meristem of radish plants at the seedling stage, cultured to the seedling stage, and rapidly identified by PCR to obtain T0 generation transgenic plants.
[0010] The invention provides a radish genetic transformation method, which establishes an efficient radish genetic transformation system. The method provided by the invention does not rely on tissue culture and genotype, and can solve the problem of radish genetic transformation system relying on tissue culture in the prior art.
[0011] As a possible implementation manner, the culture conditions for culturing the radish seeds until germination are: culturing at 25° C. to 28° C. for 1 to 2 days.
[0012] As a possible implementation method, the low-temperature culture conditions are: low-temperature culture at 3°C to 5°C for 9 to 16 days.
[0013] As a possible implementation method, the culture condition for radish seed germination is 25° C. to 28° C. for 1 to 2 days.
[0014] As a possible implementation method, the OD of the Agrobacterium infection solution is 600 It is 0.1~0.5.
[0015] As a possible implementation, the injection dosage of the Agrobacterium infection solution is 0.2 mL to 1 mL per plant.
[0016] As a possible implementation, a micro syringe is used for injection, and the needle specification of the micro syringe is 0.2×15-1.0×15 TWLB.
[0017] In a second aspect, the present invention claims protection for the transgenic radish prepared in combination with the first aspect.
[0018] As a possible implementation manner, the transgenic radish includes the AtLEC2 gene, and the nucleotide sequence of the AtLEC2 gene is shown in SEQ ID No.1.
[0019] As a possible implementation manner, the transgenic radish is of T0 generation or a progeny produced by self-pollination or hybridization of T0 generation.
[0020] The invention clones the LEC2 gene from Arabidopsis thaliana, constructs the pCAMBIA1302-35s-GFP-AtLEC2 expression vector, injects the Agrobacterium infection solution carrying the AtLEC2 gene into the apical meristem of radish seedlings using a micro-injector, obtains transgenic radish, and establishes an efficient radish genetic transformation system. The invention further detects the transgenic plants by PCR and calculates the transformation rate, performs fluorescence quantitative PCR on the transgenic positive plants and the control, respectively, confirms whether the transgenic is successful, and proves that the transformation system provided by the invention obtains transgenic plants in the T0 generation, and the transformation rate is high.
[0021] The present invention adopts the injection method to provide a radish genetic transformation system that is independent of tissue culture and genotype.
[0022] The invention provides a radish genetic transformation method with a transformation rate as high as 82%, which effectively improves the success rate of radish transgenic.
[0023] The present invention can inject more bacterial liquid and improve the conversion rate through low temperature treatment, and promote the radish seedlings to bloom and bear fruits early through vernalization treatment, thereby shortening the generation time of transgenic plants.
[0024] The invention provides a simple, rapid, convenient and tissue culture-independent radish genetic transformation method, which is beneficial to promoting radish molecular biology research and genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0026] Figure 1 A schematic diagram of a radish genetic transformation method provided by an embodiment of the present invention; wherein: Figure 1 a in the figure represents germination of radish seeds; Figure 1 b in the figure is a robust radish seedling obtained after low temperature culture; Figure 1 c in the figure is the infection solution of Agrobacterium carrying the target gene, which is injected into the apical meristem of radish seedlings; Figure 1 The d in the figure indicates dark culture after injection; the plants were cultured until the fleshy root swelling stage and T0 generation transgenic plants were obtained by PCR detection.
[0027] Figure 2 The PCR results of the first group of repeatedly treated radish plants provided in the embodiment of the present invention, wherein M represents DNAMaker 2000bp, CK represents the control group, and 1-50 represents the experimental group.
[0028] Figure 3 The PCR results of the second group of repeatedly treated radish plants provided in the embodiment of the present invention, wherein M represents DNAMaker 2000bp, CK represents the control group, and 1-50 represents the experimental group.
[0029] Figure 4 The PCR results of the third group of repeatedly treated radish plants provided in the embodiment of the present invention, wherein M represents DNAMaker 2000bp, CK represents the control group, and 1-50 represents the experimental group.
[0030] Figure 5 The real-time fluorescence quantitative PCR test results of the transgenic positive plants provided in the embodiments of the present invention show that the expression levels of the AtLEC2 gene in different parts of the transgenic plants are higher than those of the non-transgenic control plants. DETAILED DESCRIPTION
[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0032] In order to solve the problem of low T0 generation transformation rate in the radish genetic transformation system in the prior art, this embodiment provides a radish genetic transformation method.
[0033] Figure 1 The schematic diagram of the radish genetic transformation method provided by the embodiment of the present invention is shown as an example. Figure 1 As shown, including: Figure 1 a in the figure represents germination of radish seeds; Figure 1 b in the figure is a robust radish seedling obtained after low temperature culture; Figure 1 c in the figure is the infection solution of Agrobacterium carrying the target gene, which is injected into the apical meristem of radish seedlings; Figure 1 The d in the figure indicates dark culture after injection; the plants were cultured until the fleshy root swelling stage and T0 generation transgenic plants were obtained by PCR detection.
[0034] The invention clones the LEC2 gene from Arabidopsis thaliana, constructs the pCAMBIA1302-35s-GFP-AtLEC2 expression vector, injects the Agrobacterium infection solution carrying the AtLEC2 gene into the apical meristem of radish seedlings using a micro-injector, obtains transgenic radish, and establishes an efficient radish genetic transformation system. The invention further detects the transgenic plants by PCR and calculates the transformation rate, performs fluorescence quantitative PCR on the transgenic positive plants and the control, analyzes the expression amount of the target gene in different parts of the radish, and proves that the transformation system provided by the invention obtains transgenic plants in the T0 generation, and the transformation rate is high.
[0035] The test material selected in the embodiment of the present invention is the white radish numbered 'B11' provided by the radish research group of Yangtze Normal University.
[0036] The technical solution of the present invention will be further described below in conjunction with specific embodiments.
[0037] Example 1
[0038] This example provides a transgenic experiment of apical meristem.
[0039] The AtLEC2 gene was constructed into a vector to obtain the pCAMBIA1302-35s-GFP-AtLEC2 expression vector, which was synthesized by Broadline Corporation. The nucleotide sequence of the AtLEC2 gene is shown in SEQ ID No. 1:
[0040]
[0041] The vector pCAMBIAL1302-35s-GFP-AtLEC2 used in the experiment was synthesized by Shaanxi Broad Company. A small amount of GV3103 Agrobacterium solution was taken on the clean bench. GV3103 Agrobacterium was purchased from Shanghai Shenggong Biological Co., Ltd. and diluted to 10 -3 Then, streak or spread on YEP solid medium supplemented with antibiotic Kan, and incubate in the dark at 28℃ for 48h for activation. Pick a single clone and transfer it to 200mL YEP liquid medium containing 50mg / L antibiotic Kan. Shake and incubate at 200rpm and 28℃ for about 20h until OD 600 =0.8, centrifuge at room temperature and 4000 rpm for 10 min, collect the precipitate. Resuspend the cells in 1 / 2MS medium supplemented with 200 μmol / L acetosyringone in a clean bench, and adjust the concentration of the resuspension to OD 600 The value is 0.2, and the Agrobacterium infection solution is obtained.
[0042] The formula of the YEP liquid culture medium is: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, pH=7.0, and the solvent is water.
[0043] The English abbreviation of acetosyringone is AS.
[0044] Soak the radish seeds in warm water for 12 hours, sterilize them in 75% alcohol by volume for 30 seconds, and wash them three times with sterile water; soak them in 2% sodium hypochlorite by volume for 10 minutes, and wash them three times with sterile water. After drying, plant them in sterile soil and water them appropriately to keep the soil well hydrated.
[0045] When the seeds grow to the germination stage, that is, the period from seed germination to the first pair of true leaves germinating, the growth morphology is as follows: Figure 1 As shown; 153 plants with the same growth conditions were selected and randomly divided into 3 replicates, of which 50 plants in each replicate were used as the experimental group and 1 plant as the control group. The Agrobacterium infection solution was injected into the apical meristem of the experimental group plants at a dose of 0.2 mL / plant using a micro-syringe with a needle specification of 0.45×15TWLB, and the YEP liquid culture medium was injected into the apical meristem of each plant in the control group at the same dose to obtain the treated plants.
[0046] Among them, the micro syringe was purchased from Anhui Kangtai Medical Instruments.
[0047] Example 2
[0048] This example provides a genetic modification experiment of radish apical meristem.
[0049] The plants obtained in Example 1 were covered with black bags, cultured in the dark for 3 days, the bags were removed, and cultured in a constant temperature culture room at 22° C. until the seedling stage, when the radish had 5 true leaves.
[0050] Prepare DNA extraction buffer: weigh 1.2114g Tris and dissolve it in 100mL ultrapure water, adjust the pH to 8.0 with HCl to obtain 0.1M Tris-HCL; weigh 2.922g NaCl and dissolve it in 100mL ultrapure water to obtain 0.5M NaCl; weigh 0.7542g EDTA-Na2 and dissolve it in 100mL ultrapure water to obtain 50mM EDTA-Na2; 10mM β-mercaptoethanol; mix well and sterilize. 15 mg of the first true leaf of the seedling plant was taken and placed in a 1.5 mL centrifuge tube, and ground with a grinding rod until the leaf became a pulp; 600 μL of DNA extraction buffer was added and the grinding rod was washed, mixed, and incubated in a 65°C water bath for 10 min, inverted twice during the water bath; 350 μL of phenol-chloroform was added and shaken, placed in a centrifuge and centrifuged at 10000 r / min for 10 minutes, 500 μL of supernatant was aspirated into a 1.5 mL centrifuge tube containing 400 μL of isopropanol, allowed to stand at room temperature for 5 min, centrifuged at 10000 r / min for 5 min, the supernatant was discarded, and the precipitate was washed twice with 250 μL of 75% by volume ethanol, dried, and 20 μL of ddH2O was added to dissolve the DNA for later use.
[0051] The isopropanol is pre-cooled in a -20°C refrigerator.
[0052] 1% agarose gel was prepared with 1×TBE buffer for later use, and PCR identification was performed using DNA of the experimental group plants and the control group plants as templates. The PCR reaction system was 20 μL: 1 μL DNA, 1 μL GFP-1F primer, 1 μL GFP-1R primer, 10 μL 2×taqMasterMix enzyme, and 7 μL ddH2O. The nucleotide sequence of the GFP-1F primer was as shown in SEQ ID No.2: 5'-GAGGGATACGTGCAGGAG-3', and the nucleotide sequence of the GFP-1R primer was as shown in SEQ ID No.3: 5'-GATGTTGTGGCGGGTCTT-3'. The reaction procedure was as follows: 94°C for 3 min, 94°C for 30 s, 57°C for 30 s, 72°C for 30 s, 33 cycles, and 72°C for 5 min. After the reaction is completed, 5 μL of DNA and 1 μL of 5× loading buffer are mixed and added to 1% agarose gel electrophoresis to detect the results. The conversion rate is calculated according to the formula "conversion rate = number of transgenic plants / total number of tested plants", and the average value of 3 replicates is taken. Figures 2 to 4 And the results are shown in Table 1.
[0053] Table 1 Statistics of radish conversion rate
[0054]
[0055] Depend on Figure 2 to Figure 4 As shown in Table 1, there was no GFP marker gene band in the control group, while a 325 bp fragment of GFP marker gene was amplified in the three repeated experimental groups, among which the conversion rates of repeat I, repeat II, and repeat III were 70.0%, 84.0%, and 92.0%.
[0056] Example 3
[0057] This example provides the expression of the AtLEC2 gene in various tissues of seedling plants.
[0058] RNA was extracted from the roots, stems, leaves and flowers of transgenic radish seedlings using a plant RNA rapid extraction kit and stored at -80°C. RNA was reverse transcribed into cDNA using the following reverse transcription system: 3 μL RNA, 4 μL All-in-one 5×RT Master Mix, 13 μL ddH2O; reaction procedure: 37°C for 30 min, 60°C for 10 min. cDNA was detected by 2% agarose gel electrophoresis and stored at -20°C. Fluorescence quantitative PCR system, 10 μL: cDNA 1 μL, 2×SYBP Green RealtimePCRMasterMix 5 μL, internal reference primer RsRPⅡ-F 1 μL, internal reference primer RsRPⅡ-R 1 μL, ddH2O 2 μL, wherein the nucleotide sequence of the internal reference primer RsRPⅡ-F is shown in SEQ ID No.4: 5'-ATCACGCTAAATGGTCTCCT-3', the nucleotide sequence of the internal reference primer RsRPⅡ-R is shown in SEQ ID No.5: 5'-GCTGCTCTCAATCAAGTCAATC-3'; reaction procedure: hold stage: 50℃2min, 95℃2min; PCR stage (40×): 95℃15s, 55℃15s, 72℃30s; Meltcurve stage: 60℃1min, 95℃1s. Figure 5 The results are shown in Table 2. The expression differences in each organ were tested by t test, and the results are shown in Table 3.
[0059] Table 2 Relative expression levels of AtLEC2 gene in different organs of radish
[0060] root stem leaf flower Plants in the experimental group 1.1182 1.8623 1.1482 1.1701 Control group plants 1.0011 1.5155 1.0233 0.8637
[0061] Table 3 Results of t-test on expression differences in different organs of radish
[0062] sum of squares Degrees of Freedom Mean Square F-number p-value A Factor 0.1002 1 0.1002 13.9390 0.0335 B Factor 0.6093 3 0.2031 28.2620 0.0106 error 0.0216 3 0.0072 / /
[0063] Note: Factor A refers to whether the AtLEC2 gene was transferred, and factor B refers to different organs.
[0064] Depend on Figure 5 As shown in Table 2, the AtLEC2 gene is expressed in the roots, stems, leaves, and flowers of the experimental group plants and the control group plants, with the highest expression level in the stems of the experimental group, and the expression levels in various organs of the experimental group plants are higher than those in the corresponding organs of the control group plants. This shows that Example 1 has a very high transformation rate for radish seedlings and can be used as an efficient and simple transformation method for radish genetic transformation.
[0065] As shown in Table 3, the expression levels of AtLEC2 gene in the experimental group and the control group were significantly different, and the expression levels in different parts of the plants treated with the same method were significantly different, which proved that the radish genetic transformation method established in the present invention is reliable.
[0066] It can be seen from the above examples that the present invention does not use tissue culture technology in the process of implementing radish gene genetic transformation. Three repeated examples respectively use three genotypes of radish as materials, all of which have high genetic transformation efficiency. Therefore, the radish genetic transformation method provided by the present invention does not rely on tissue culture and genotype, and can solve the problem of relying on tissue culture in the radish genetic transformation system in the prior art.
[0067] The invention provides a simple, rapid, convenient and tissue culture-independent radish genetic transformation method, which is beneficial to promoting radish molecular biology research and genetic breeding.
[0068] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.
[0069] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.
Claims
1. A method for genetic transformation of radish, characterized in that: The steps include: After the radish seeds germinate, they are cultured at a low temperature of 2.5°C to 5.5°C for 9 to 16 days, and then vernalized to obtain radish plants at the seedling stage; Cultivate Agrobacterium carrying the target gene, resuspend after centrifugation, and obtain Agrobacterium infection solution; The Agrobacterium infection solution is injected into the apical meristem of the radish plant at the seedling stage, and cultured to the seedling stage to obtain T0 generation transgenic plants.
2. The method according to claim 1, characterized in that The culture conditions for culturing the radish seeds until germination are: culturing at 25° C. to 28° C. for 1 to 2 days.
3. The method according to claim 1, characterized in that The conditions for low-temperature culture are: low-temperature culture at 3℃~5℃ for 9 days to 16 days.
4. The method according to claim 1, characterized in that The culture conditions for radish seed germination are 25℃~28℃ for 1 day to 2 days.
5. The method according to claim 1, characterized in that The OD of the Agrobacterium infection solution 600 It is 0.1-0.
5.
6. The method according to claim 1, characterized in that The injection dosage of the Agrobacterium infection solution is 0.2 mL / plant to 1 mL / plant.
7. The method according to claim 1, characterized in that The injection is performed using a micro syringe, the needle of which has a specification of 0.2×15TWLB to 1.0×15TWLB.
8. The transgenic radish prepared by the method according to any one of claims 1 to 7.
9. The transgenic radish according to claim 8, characterized in that: The transgenic radish is T0 generation or the offspring produced by self-pollination or hybridization of T0 generation.