CN111575311A - Cotton gene editing method based on gene gun mediation and application - Google Patents
Cotton gene editing method based on gene gun mediation and application Download PDFInfo
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- CN111575311A CN111575311A CN202010295546.XA CN202010295546A CN111575311A CN 111575311 A CN111575311 A CN 111575311A CN 202010295546 A CN202010295546 A CN 202010295546A CN 111575311 A CN111575311 A CN 111575311A
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a cotton gene editing method based on gene gun mediation and application, and belongs to the technical field of genetic engineering. The method obtains embryonic callus through culturing hypocotyl stem segments, transforms the embedded gene editing plasmid DNA into cotton embryonic callus by using a gene gun, and obtains gene editing seedlings through recovery culture, screening culture and somatic embryo differentiation. The gene gun transformed embryogenic callus regeneration method provided by the invention can shorten the cotton genetic transformation period, improve the transformant efficiency, save time and workload, and has important application prospects in cotton gene function research and breeding practice.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a gene gun-mediated cotton gene editing method and application. The invention utilizes plant tissue culture technology to obtain cotton embryogenic callus from cotton hypocotyl stem segments. The embryogenic callus is transformed by a gene gun, and gene editing offspring can be obtained after 3-4 months through screening and somatic cell regeneration cultivation.
Background
Cotton is a natural fiber source and is an important economic crop in China. Since the gene engineering technology appeared in the eighties of the last century, through the development of mechanism and technical research for more than thirty years, the gene engineering plays a significant role in cotton breeding. In the late nineties of the last century, cotton bollworm pests are outbreak in large areas, which causes the global cotton yield and planting area to be greatly reduced, and even threatens the survival of the cotton industry once. The cultivation and commercial application of the transgenic Bt insect-resistant cotton play an important role in cotton production. Bt cotton refers to transgenic cotton which is resistant to cotton bollworm and is obtained by introducing insecticidal gene of Bacillus thuringiensis (Bacillus thuringiensis) into cotton, and helps cotton farmers overcome cotton bollworm disasters. Although commercial planting of transgenic cotton varieties has been open for nearly 30 years. However, the successful application of the gene is limited to Cry1A, Cry1c, EPSPS and other genes. The yield, the fiber quality, the disease resistance, the stress resistance and the like of cotton are always breeding targets for breeding new varieties, and related mechanism and mechanism analysis also have good research foundation. Benne hypersensitivity et al reviewed the research progress of cotton stress resistance-related genes and suggested that genetic engineering techniques could be used to successfully transfer cotton endogenous or exogenous resistance-related genes into cotton and obtain transgenic materials with significantly improved corresponding phenotypes, but these works are currently limited to laboratory studies and are not really applied to breeding practices (benne hypersensitivity et al, 2012). In recent years, a great deal of gene information is released after the sequencing of different reference genomes of cotton is completed, and a good foundation and an information platform are laid for deep mechanism research and deep excavation of genome information (Zhang et al, 2015; Hu et al, 2019).
The CRISPR-Cas system is widely present in bacteria and archaea, is an adaptive immune reaction system when viruses and plasmids invade, realizes a recognition function by using base complementary pairing between guide RNA and target DNA, and depends on a multifunctional Cas protein mediated cleavage effect (Carroll, 2012). Gene editing techniques are widely used in plant research, not only for gene knockout, but also for site-specific insertion, replacement, chromosomal recombination, and multigene knockout (Dang et al, 2015). Compared with the traditional transgenic method, the main advantages of the genome editing technology are that the site on the genome is targeted and operated by modifying DNA binding protein or guide RNA, the integration or deletion of the target gene can be more accurately realized, and the gene can be successfully applied to model plants and various crops (Kim et al, 2016; Chen et al, 2019).
The genetic transformation method of cotton mainly comprises a pollen tube channel method, agrobacterium-mediated genetic transformation and gene gun bombardment transformation, and the three methods have advantages and disadvantages respectively. The pollen tube channel method is proposed by Mr. Zhou Guangyu of molecular plant breeders in China, has the advantages of no restriction of genotypes, no need of establishing an in vitro regeneration system, capability of being directly carried out in a field, simple and convenient operation and shortened breeding time. The method makes an important contribution to cotton transgenic breeding in China, and transgenic cotton materials with transgenic Bt genes, which are insect-resistant, disease-resistant, herbicide-resistant, yield-increasing and high-quality, are bred by the method (Zhou Guang et al, 1988; Xiexian et al, 1991; Lizhongwang et al, 2012). The limiting factors of this approach are: 1) the DNA integration mechanism is not clear; 2) only suitable for integration between a parent and "donor DNA" that possess a significant phenotype, easily distinguishable; 3) the influence of external environment is large, the method can be carried out only in the flowering period, and the injection part is positioned in a fuzzy manner; 4) to low power (Zhou Guangyu et al, 1988; kombucha et al, 2006). Agrobacterium-mediated transformation is the most commonly used method for genetic transformation of cotton. Umbeck et al (1987) reported for the first time a technical system for Agrobacterium-mediated genetic transformation and regeneration of cotton hypocotyls. Firoozabady et al reported a regeneration system for Agrobacterium transformed cotton cotyledons. Subraniam et al (2001) studied the influencing factors of Agrobacterium strains, receptor genotypes, acetosyringone, co-culture temperature, callus size at the beginning of the screening phase and embryogenic callus culture mode, etc. in the transformation and regeneration of cotton, and formed a complete system. The Jinshuangman and the like (2017) improve the cotton conversion efficiency and the regeneration frequency by screening and domesticating the receptor Jin 668. The cotton gene gun transformation system was first reported in 1988. McCabe et al (1988) continued to culture plants after bombardment with a gene gun using hypocotyl sections of sterile cotton embryos as explants. Finer et al (1990) reported methods for transformation and regeneration of embryogenic cells in cotton suspension by particle gun bombardment. Chlan et al (1995) published a biolistic transformation system that screened Coker312 lines for positive transformed cells at a dose of 50mg/L kanamycin. Of the 35 tested plants, 31 positive for the NPT gene were indicated, while the previous Agrobacterium transformation system took 10-12 months to obtain regenerated plants, which were transplanted into soil three months after bombardment (Chlan et al, 1995).
The optimization of the cotton gene gun system mainly lies in the selection of proper transformed explants, improves the transformation efficiency, shortens the transformation period and can ensure the genetic stability. Rech et al (2008) obtained transgenic cotton using the hypocotyl of a cotton seed embryo as the bombarding explant. The aseptic seeds are first soaked in aseptic water to promote the elongation of the embryonic axis. After the embryonic axis is exposed, the embryonic axis is separated and used as a gene gun to bombard the explant. After bombardment, screening and cultivation are carried out, and seedlings transferred to soil can be obtained in about 2 months (Rech et al, 2008). Kanniah systematically summarized and optimized the gene gun bombardment transformation and regeneration system with meristematic tissue as explant and suspension embryogenic cell line as explant, respectively. The meristem is used as explant for transforming and regenerating system, and has the advantages of greatly shortened transformation and regeneration period, fast transplanting of transformed seedling to soil within one week after bombardment. The disadvantages are that: 1) labor and time are required to isolate the appropriate meristem; 2) the chimera phenomenon is serious; 3) molecular detection needs to wait until the next generation (Rajasekaran,2013 a). The suspended embryonic cell line as the gene gun bombarded explant has the advantages that: 1) the embryonic cell line can be stored for a long time and can start transformation at any time. 2) The conversion rate can reach 4 percent. The disadvantages are that: 1) suspension culture causes potential reproductive and developmental damage to cells, and abnormal seedlings and sterility sometimes occur; 2) the operation of suspension culture is troublesome (Rajasekaran,2013 b).
The invention relates to a gene gun-mediated cotton gene editing method, which is based on a hormone-free induced embryonic callus differentiation technology, utilizes gene gun bombardment, and obtains regenerated seedlings through recovery culture, screening culture and regeneration culture. The transformation of different vectors proves that the method has the advantages of high transformation efficiency, short regeneration period, normal growth and development of transgenic plants and the like, and plays an important role in the research of cotton gene functions and breeding practice.
Disclosure of Invention
The invention aims to provide a cotton gene editing method based on gene gun mediation and application thereof. The method obtains the embryonic callus of the cotton hypocotyl stem section by plant tissue culture, converts the embedded gene editing plasmid DNA into cotton embryonic callus by gene gun bombardment, and obtains the gene editing seedling by recovery culture, screening culture and somatic embryo differentiation.
The purpose of the invention is realized by the following technical scheme:
a cotton gene editing method based on gene gun mediation, which comprises the following steps:
1) obtaining cotton embryogenic callus: placing the aseptic seedling hypocotyl stem section cultured on the seedling culture medium on an embryonic callus induction culture medium for embryonic callus induction culture, subculturing once every 22-35 days, and replacing a new culture medium until embryonic callus appears; selecting newly induced embryogenic callus, subculturing in a new culture medium, and selecting embryogenic callus with consistent callus state as explant bombarded by gene gun when the embryogenic callus is transformed into rice-yellow rice-shaped callus;
2) bombardment with a gene gun: culturing the embryogenic callus on a hypertonic culture medium and then performing gene gun transformation; the process of the gene gun transformation comprises the following steps: sucking 25 mu L of thoroughly vortexed gold powder suspension with the concentration of 60mg/mL, sucking 10 mu L of sterile ultrapure water, adding 6 mu L of gene editing plasmid DNA with the concentration of 500ng/uL, sucking, beating and uniformly mixing; adding 50 μ L of 2.5M calcium chloride dihydrate, adding 20 μ L of 3mg/mL protamine, rapidly mixing, vortexing, and centrifuging (preferably rapidly mixing, vortexing for 3min, centrifuging for 30s) to remove supernatant; adding 200 μ L of anhydrous ethanol, vortexing to suspend gold powder particles, centrifuging, and removing supernatant; repeating the steps once; adding 40 mu L of absolute ethyl alcohol to fully suspend the gold powder particles to obtain an embedded gold powder suspension; bombarding 0.2g embryogenic callus by using 10 mu L embedded gold powder suspension liquid for gene gun bombardment each gun;
3) and (3) recovery culture: continuing dark culture on a hypertonic culture medium after gene gun transformation;
4) screening and culturing: dividing the callus after recovery culture into a plurality of cell groups, carefully subculturing on a screening culture medium, culturing until the growth of the resistant callus can be obviously seen, and continuously subculturing the callus with good growth vigor;
5) inducing somatic embryo differentiation culture: after culturing on a screening culture medium, selecting the well-growing callus to a differentiation culture medium for inducing somatic cell differentiation for differentiation culture, and subculturing each cell group in a culture bottle;
6) seedling cultivation, hardening and transplanting: culturing the plantlets obtained in the step 5) on a basic culture medium, then cleaning a root culture medium with clear water, carrying out water culture until new roots grow out, and transplanting and culturing the plantlets to finish the whole growth period;
7) molecular detection and gene editing efficiency detection: when a new leaf grows out from a transplanted plant, taking a leaf to extract total DNA for PCR detection, and designing a primer by using a U6 promoter and a U6 terminator to verify whether sgRNA is successfully transformed; verifying the transformation result of the Cas9 protein by using a Cas9 specific primer; and designing an upstream primer and a downstream primer at the 5 'end and the 3' end of the editing site respectively, carrying out PCR amplification by taking total DNA of leaves as a template, purifying and connecting a PCR product with a T vector, transforming the competence of escherichia coli, and selecting a monoclonal bacterial liquid to carry out sequencing verification of gene editing.
As a preferred technical scheme:
the basic culture medium comprises: macroelement mother liquor (50 mL. L)-1) + ferric salt mother liquor (5 mL. L)-1) + microelement mother liquor (5 mL. L)-1) + organic mother liquor (5 mL. L)-1) + glucose (30 g.L)-1) + plant gel (3.0 g.L)-1)+MgCl2(1.0g·L-1)。
The seedling culture medium: macroelement mother liquor (25 mL. L)-1) + plant gel (7 g.L)-1) Adjusting the pH value to 5.8;
the callus induction culture medium comprises: minimal medium +1.9 g.L-1Potassium nitrate, the pH value is adjusted to 5.8;
the hypertonic culture medium: minimal Medium + sorbitol (72.88 g. L)-1) Adjusting the pH value to 5.8;
the screening culture medium: minimal medium +1.9 g.L-1Potassium nitrate +50 mg. L-1Kanamycin, and the pH value is adjusted to 5.8;
the differentiation culture medium: minimal Medium-NH4NO3(not containing NH)4NO3The minimal medium) +0.5 g.L-1Asparagine +1.0 g.L-1Glutamyl ammonium, pH 6.0.
Further preferably, the tissue culture conditions of the culture of the aseptic seedling and the embryogenic callus in the step (1), the differentiation culture of the somatic embryo in the step (5) and the culture of the regenerated seedling in the step (6) are as follows: the temperature is 28 +/-2 ℃, the light cycle is 16h/8h day/night, the air humidity is 40% -60%, and the illumination intensity is 12000-15000 Lx.
Further preferably, in the step (2), the vacuum pump and the power switch of the gene gun are turned on, the gene gun can be transformed by a PDS 1000/He gene gun of Bio-Rad company or other similar equipment, and the bombardment conditions of the gene gun bombardment are as follows: helium pressure 1150psi, target distance 6cm bombardment position.
Further preferably, the sterile seedlings in the step (1) are sterile seedlings with the seedling age of 7 d; the length of the stem section is 1.0-1.5 cm; subculturing every 25-30 days.
Further preferably, in step (2), the embryogenic callus is placed on hypertonic medium for 4h for gene gun transformation, and 0.2g of embryogenic callus is bombarded at one time.
Further preferably, the gene gun transformation in step (3) is followed by culturing on hypertonic medium for 16h in the dark at 28 ℃.
Further preferably, in step (4), the callus after recovery culture is divided into 10-15 cell groups with the diameter not more than 3mm, carefully subcultured on a screening medium, after 4 weeks, the growth of resistant callus can be obviously seen, and the well-growing callus is subcultured for another 4 weeks.
Preferably, the plantlets obtained in the step 5) are transferred to a basic culture medium, cultured to 4-5 true leaves, the root culture medium is cleaned by clear water, water culture is carried out for 2-3 weeks until new roots grow out, the plantlets are transplanted into paper cups containing vermiculite and matrix, the paper cups are transplanted into a big pot after 10-20 days, and the outdoor growth is completed in the whole growth period.
The application of the method in cotton gene editing.
The invention has the advantages that:
(1) embryogenic callus cells were obtained without hormone induction. The traditional cotton embryonic callus induction system depends on the action of 2,4-D and KT, and 2,4-D has certain toxicity to plant cells, the hypocotyl stem section is cultured under a hormone-free culture condition, embryonic callus can appear in 2-3 months, the somatic embryo is obviously larger than that induced by hormone, the malformed seedling rate is low, the seedling is more robust and easy to survive, and the T0 generation seed setting rate is high.
(2) Efficient transformation can help to improve gene editing efficiency. The biolistic transformation methods provided herein produce large numbers of transient transformation products that do not integrate into the plant genome but are still capable of editing, providing higher editing efficiencies than smaller numbers of stable transformation systems. In addition, the hypertonic culture technology of the callus cells can improve the transformation efficiency of the gene gun.
(3) Shortens the cotton genetic transformation period and saves time and workload. Compared with the method using stem segments as explants, the method for obtaining the regeneration seedlings by culturing the gene gun bombardment embryonic callus cells provided by the invention omits the previous embryonic callus induction work. In the method in which the stem is an explant, the callus induced by each stem is identified as an independent transformation event, and therefore, a large number of stem pieces need to be cultured to obtain the number of independent transformation events required for the study. The cotton belongs to a plant which is difficult to differentiate, the time for inducing the embryogenic callus is longer, and the step of screening and callus induction is needed in a transformation mode that a stem section is an explant, so that the time for obtaining the embryogenic callus is longer, and the workload is very large. The method provided by the invention is characterized in that a large amount of embryogenic calli are rapidly obtained in the early stage without hormone induction, 10-15 independent transformation events are obtained by taking 0.2g of embryogenic calli as a bombardment object, and the workload and the time cost of callus culture are greatly saved.
Drawings
FIG. 1 shows the genetic transformation and regeneration process of cotton gene gun.
FIG. 2pKSE401 vector map.
FIG. 3 position of sgRNAs on the GhGL2 and GhGL3 genes.
Fig. 4 PCR detection results of sgRNA fragment and Cas9 gene in gene editing progeny. A shows the PCR detection results of sgRNA1, sgRNA2 and sgRNA3 fragments in offspring, M shows DNA Marker of 5K, P shows sgRNA-pKSE401 plasmid, WT is a receptor control, and 3-11 and the like are different sgRNA-pKSE401 transgenic T0 generation individuals respectively.
FIG. 5 Gene edits the sequencing results of T0 generation plants. Editing conditions of GhGl2 and GhGl3 genes in T0 generation plants of cotton. The black background white font label is PAM locus, the gray background white font label is target sequence, the gray background black font label is gene editing result, -2 represents deletion of 2 basic groups.
FIG. 6 phenotype of wild type and gene editing generation T0. A is WT receptor control, B-H is the leaf phenotype of different individuals of sgRNA-pKSE401 transgenic T0 generation.
FIG. 7T0 generation gene editing plant gene expression and gossypol content detection results. Panel A shows the results of detection of the expression level of the target gene in the gene-edited progeny. And B is the gossypol content detection result in the gene editing progeny.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The media components involved in the tissue culture process were as follows:
basic culture medium: a large number of yuanMother liquor (50 mL. L)-1) + ferric salt mother liquor (5 mL. L)-1) + microelement mother liquor (5 mL. L)-1) + organic mother liquor (5 mL. L)-1) + glucose (30 g.L)-1) + plant gel (3.0 g.L)-1)+MgCl2(1.0g·L-1)；
Seedling culture medium: macroelement mother liquor (25 mL. L)-1) + plant gel (7 g.L)-1) Adjusting the pH value to 5.8;
callus induction medium: minimal medium +1.9 g.L-1Potassium nitrate, the pH value is adjusted to 5.8;
hypertonic culture medium: minimal Medium + sorbitol (72.88 g. L)-1) Adjusting the pH value to 5.8;
screening a culture medium: minimal medium +1.9 g.L-1Potassium nitrate +50 mg. L-1Kanamycin, and the pH value is adjusted to 5.8;
differentiation medium: minimal Medium-NH4NO3+ asparagine (0.5 g.L)-1) + Glutamine (1.0 g.L)-1) The pH was 6.0.
TABLE 1 stock solution formula Table for minimal Media (MSB)
Example 1: optimizing an induction system of the embryogenic callus;
cutting hypocotyl of 7d seedling-age aseptic seedling cultured in seedling culture medium into stem segments of 1.0-1.5cm length, placing the stem segments in embryogenic callus induction culture medium for embryogenic callus induction culture, subculturing for 4 weeks, and replacing with new culture medium until embryogenic callus appears. Selecting newly induced embryogenic callus for subculture in a new culture medium, after about 2 weeks, selecting embryogenic callus with consistent callus state as an explant bombarded by a gene gun, and bombarding 0.2g of embryogenic callus at one time;
the tissue culture conditions for culturing the aseptic seedlings and the embryonic calluses are as follows: the temperature is 28 +/-2 ℃, the light cycle is 16h/8h day/night, the air humidity is 40% -60%, and the illumination intensity is 12000-15000 Lx.
Example 2: constructing an sgRNA-pKSE401 vector;
login tohttp：//crispr.hzau.edu.cn/CRISPR/The website inputs a target gene sequence into a corresponding sequence box, searches high score targets and evaluates the off-target condition; after selecting a target sequence, designing a primer according to the following rules:
oligo-F: 5 '-ATTGNNNNNNNNNNNNNNNNNNN-3'; (N is the first 19nt sequence of the PAM sequence)
oligo-R: 5 '-AAACNNNNNNNNNNNNNNNNNNN-3'; (N is the reverse complement of the 19nt sequence before the PAM sequence)
Mixing the above two oligonucleotide chains (each 100. mu. mol/L), heating at 95 ℃ for 5min, then cooling to room temperature to form a double-stranded insert, and constructing a vector according to the following system (see Table 2); the reaction conditions are as follows: 5h at 37 ℃, 5min at 50 ℃ and 10min at 80 ℃; and transforming the 5 mu L of mixed solution into escherichia coli competence, selecting positive clones on an LB plate containing 50mg/L of kanamycin, and verifying the vector through colony PCR identification and sequencing.
Table 2 construction system of sgRNA-pKSE401 vector
In this study, for example, the sgRNA sequences of genes GhGL2 and GhGL3 homologous to genes related to cotton gland formation are edited as follows:
sgRNA1-F：：5’ATTgGTGGGTATACTCCATTTTC 3’
sgRNA1-R：：5’AAACGAAAATGGAGTATACCCAC 3’
sgRNA2-F：：5’ATTgATATCATCATGGAAGACTG 3’
sgRNA2-R：：5’AAACCAGTCTTCCATGATGATAT 3’
sgRNA3-F：：5’ATTgCGATGTGACTGATTATGAG 3’
sgRNA3-R：：5’AAACCTCATAATCAGTCACATCG 3’
example 3: establishing a gene gun transformation method system;
1) preparation of plasmid DNA: and (3) streaking and activating the stored sgRNA-pKSE401 positive escherichia coli liquid, picking a single clone, shaking a small amount of the liquid, then sucking a small amount of the liquid, carrying out amplification culture, and extracting the plasmid by using a plasmid miniextraction kit (magenta, Guangzhou).
2) Bombardment conditions of a gene gun: embryogenic callus was subcultured on hyperosmotic medium for 4h for biolistic transformation. Embedding step of gene editing plasmid DNA: preparing 60mg/mL gold powder; aspirate 25. mu.L of the vortexed gold powder suspension, aspirate 10. mu.L of sterile ultrapure water, add 6. mu.l (3. mu.g) of gene-editing plasmid DNA (500ng/uL), aspirate and mix well. Adding 50 μ L2.5M calcium chloride dihydrate, adding 20 μ L of 3mg/mL protamine, quickly mixing, vortexing for 3min, centrifuging for 30s, removing supernatant, adding 200 μ L anhydrous ethanol, vortexing to suspend gold powder particles, centrifuging, and removing supernatant. This step was repeated once. 40 mul of absolute ethyl alcohol is added to fully suspend the gold powder particles. 10ul of the embedded gold powder suspension was used per gun. Turning on a vacuum pump and a power switch of a gene gun, wherein the gene gun is converted by adopting a PDS 1000/He gene gun of Bio-Rad company under the bombardment conditions that: helium pressure 1150psi, target distance 6cm bombardment position;
3) and (3) recovery culture: after gene gun transformation, dark culture is continuously carried out on a hypertonic culture medium for 16h, and the culture temperature is 28 ℃;
4) screening and culturing: dividing the callus after recovery culture into 10-15 cell groups with the diameter not more than 3mm, carefully subculturing on a screening culture medium, obviously showing the growth of resistant callus after 4 weeks, continuously subculturing the callus with good growth vigor, and culturing for 4 weeks.
5) Inducing somatic embryo differentiation culture: after 2 cycles (8 weeks) of culture on the selection medium, the growing calli were picked on a differentiation medium for somatic cell induction differentiation culture. Each cell population was subcultured in one flask.
6) Seedling cultivation, hardening and transplanting: inserting the plantlets obtained in the step 5) into a basic culture medium, culturing to 4-5 true leaves, cleaning the root culture medium with clear water, carrying out water culture for 2-3 weeks until new roots grow out, transplanting the seedlings into paper cups containing vermiculite and matrix, transplanting the seedlings into large pots after about 2 weeks, and finishing the whole growth period after outdoor growth.
The tissue culture conditions of screening culture, somatic embryo differentiation culture and regeneration seedling culture are as follows: the temperature is 28 +/-2 ℃, the light cycle is 16h/8h day/night, the air humidity is 40% -60%, and the illumination intensity is 12000-15000 Lx.
Example 4: transformation efficiency test and gene editing efficiency test
And (3) when 2-3 new leaves grow out from the transplanted plant, taking the leaves to extract the total DNA for PCR detection. Primers are designed by using a U6 promoter and a U6 terminator to verify whether sgRNA is successfully transformed, and a transformation result of a Cas9 protein is verified by using a Cas9 specific primer. The PCR assay system used 2 × Rapid Taq Master Mix (Vazyme Co., Ltd.) as shown in Table 3 below; the amplification program is 95 ℃ for 3min, 95 ℃ for 15s, 60 ℃ for 15s, 72 ℃ for 15s (the extension time of Cas9 fragment amplification is 30s), 30cycles, 72 ℃ for 7min, and 4 ℃ for 5 min.
Respectively designing an upstream primer and a downstream primer at the 5 'end and the 3' end of an editing site by taking total DNA of leaves as a template, carrying out PCR amplification, purifying and connecting a PCR product with a T vector, transforming the competence of escherichia coli, and selecting monoclonal bacteria liquid for sequencing.
The detection of the conversion efficiency shows that: the conversion rates of all three sgRNA vectors reached more than 90% (fig. 4). The editing efficiency test shows that the genes GhGL2 and GhGL3 are edited simultaneously by taking T0 plant 2-9-5 of sgRNA2 as an example (FIG. 5).
TABLE 3 PCR detection system for sgRNA target sequences and Cas9
The primer sequences are as follows:
U6-F：5’TGTCCCAGGATTAGAATGATTAGGC 3’
U6-R：5’CCCCAGAAATTGAACGCCGAAGAAC 3’
Cas9-F：5’GATTAAGTTCAGGGGCCATTTC 3’
Cas9-R：5’CCTCATTCTCCTCGTTGTCCAG 3’
PCR-sgRNA1-F：5’GTCGGTAATCTTCTTCGTCTTCTCTT 3’
PCR-sgRNA1-R：5’TCAGCCTATTGATGGATT 3’
PCR-sgRNA2-F：5’GTCGGTAATGTGAGCGTGTTAGAGAA3’
PCR-sgRNA2-R：5’ATGGGCAGGTTGATTTGA3’
PCR-sgRNA3-F：5’GTCGGTAAAGGAAAAGGTCGGGAAAA3’
PCR-sgRNA3-R：5’TCTAACACGCTCACACTG 3’
example 5: detection of target gene expression level and gossypol content
1) Detection of expression level of target gene: RNA is extracted from 2-3 tender leaves, an RNA extraction kit (provided by BioFlux company) is selected for RNA extraction, then cDNA is obtained through reverse transcription (HiScript Q-RT Supermix for qPCR enzyme purchased from Vazyme company), qPCR is carried out by adopting a fluorescence quantitative PCR instrument of Bio-Rad company, and the result is calculated and analyzed. The results show that the average expression level of the target gene in the gene editing progeny is significantly reduced, and the reduction range can reach below 10% of the control expression level (A in FIG. 7).
2) And (3) determination of gossypol content: accurately weighing 1.00g of sample to be detected, grinding with liquid nitrogen, adding 10mL of acetone, extracting at room temperature for 60min, centrifuging at 25 deg.C for 10min at 3000r/min, repeatedly extracting for 2 times, and mixing the supernatants. After lyophilization, the mixture was dissolved in 3mL of acetonitrile-0.2% phosphoric acid (85: 15 v/v), filtered through a 0.45 μm organic filter and subjected to Ultra Performance Liquid Chromatography (UPLC). Ultra-high performance liquid chromatograph, model: UltiMate 3000, manufacturer: thermo corporation, usa; a chromatographic column: ACQUITY UPLC HSST3(2.1 mm. times.100 mm. times.1.8 μm); flow rate: 0.2 ml/min; time: 10 min; a (0.2% phosphoric acid): b (acetonitrile) ═ 15: 85 parts by weight; column temperature: 30 ℃, wavelength: 235nm, and the sample injection amount is 2 mu L. The gossypol content measurement result shows that the gossypol content in the gene editing progeny is obviously reduced, and the content of some single plants is low enough to fail to reach the detection level (B in figure 7).
Claims (10)
1. A cotton gene editing method based on gene gun mediation is characterized by comprising the following steps:
1) obtaining cotton embryogenic callus: placing the aseptic seedling hypocotyl stem section cultured on the seedling culture medium on an embryonic callus induction culture medium for embryonic callus induction culture, subculturing once every 22-35 days, and replacing a new culture medium until embryonic callus appears; selecting newly induced embryogenic callus, subculturing in a new culture medium, and selecting embryogenic callus with consistent callus state as explant bombarded by gene gun when the embryogenic callus is transformed into rice-yellow rice-shaped callus;
2) bombardment with a gene gun: culturing the embryogenic callus on a hypertonic culture medium and then performing gene gun transformation; the process of the gene gun transformation comprises the following steps: sucking 25 mu L of thoroughly vortexed gold powder suspension with the concentration of 60mg/mL, sucking 10 mu L of sterile ultrapure water, adding 6 mu L of gene editing plasmid DNA with the concentration of 500ng/uL, sucking, beating and uniformly mixing; adding 50 μ L of 2.5M calcium chloride dihydrate, adding 20 μ L of 3mg/mL protamine, rapidly mixing, vortexing, and centrifuging to remove supernatant; adding 200 μ L of anhydrous ethanol, vortexing to suspend gold powder particles, centrifuging, and removing supernatant; repeating the steps once; adding 40 mu L of absolute ethyl alcohol to fully suspend the gold powder particles to obtain an embedded gold powder suspension; each gun uses 10 mu L of embedded gold powder suspension liquid to carry out gene gun bombardment;
3) and (3) recovery culture: continuing dark culture on a hypertonic culture medium after gene gun transformation;
4) screening and culturing: dividing the callus after recovery culture into a plurality of cell groups, carefully subculturing on a screening culture medium, culturing until the growth of the resistant callus can be obviously seen, and continuously subculturing the callus with good growth vigor;
5) inducing somatic embryo differentiation culture: after culturing on a screening culture medium, selecting the well-growing callus to a differentiation culture medium for inducing somatic cell differentiation for differentiation culture, and subculturing each cell group in a culture bottle;
6) seedling cultivation, hardening and transplanting: culturing the plantlets obtained in the step 5) on a basic culture medium, then cleaning a root culture medium with clear water, carrying out water culture until new roots grow out, and transplanting and culturing the plantlets to finish the whole growth period;
7) molecular detection and gene editing efficiency detection: when a new leaf grows out from a transplanted plant, taking a leaf to extract total DNA for PCR detection, and designing a primer by using a U6 promoter and a U6 terminator to verify whether sgRNA is successfully transformed; verifying the transformation result of the Cas9 protein by using a Cas9 specific primer; and designing an upstream primer and a downstream primer at the 5 'end and the 3' end of the editing site respectively, carrying out PCR amplification by taking total DNA of leaves as a template, purifying and connecting a PCR product with a T vector, transforming the competence of escherichia coli, and selecting a monoclonal bacterial liquid to carry out sequencing verification of gene editing.
2. The method of claim 1,
the basic culture medium comprises: macroelement mother liquor (50 mL. L)-1) + ferric salt mother liquor (5 mL. L)-1) + microelement mother liquor (5 mL. L)-1) + organic mother liquor (5 mL. L)-1) + glucose (30 g.L)-1) + plant gel (3.0 g.L)-1)+MgCl2(1.0g·L-1)；
The seedling culture medium: macroelement mother liquor (25 mL. L)-1) + plant gel (7 g.L)-1) Adjusting the pH value to 5.8;
the callus induction culture medium comprises: minimal medium +1.9 g.L-1Potassium nitrate, the pH value is adjusted to 5.8;
the hypertonic culture medium: minimal Medium + sorbitol (72.88 g. L)-1) Adjusting the pH value to 5.8;
the screening culture medium: minimal medium +1.9 g.L-1Potassium nitrate +50 mg. L-1Kanamycin, and the pH value is adjusted to 5.8;
the differentiation culture medium: minimal Medium-NH4NO3+0.5g·L-1Asparagine +1.0 g.L-1Glutamyl ammonium, pH 6.0.
3. The method according to claim 1, wherein the tissue culture conditions for the culture of the sterile plantlets and the embryogenic callus in the step (1), the differentiation culture of the somatic embryos in the step (5) and the culture of the regenerated plantlets in the step (6) are as follows: the temperature is 28 +/-2 ℃, the light cycle is 16h/8h day/night, the air humidity is 40% -60%, and the illumination intensity is 12000-15000 Lx.
4. The method according to claim 1, wherein the bombardment conditions of the gene gun bombardment in the step (2): helium pressure 1150psi, target distance 6cm bombardment position.
5. The method as claimed in claim 1, wherein the sterile seedlings in the step (1) are sterile seedlings with 7d seedling age; the length of the stem section is 1.0-1.5 cm; subculturing every 25-30 days.
6. The method according to claim 1, wherein the embryogenic callus is cultured in step (2) on hypertonic medium for 4h for biolistic transformation with one bombardment of 0.2g embryogenic callus.
7. The method of claim 1, wherein the gene gun transformation in step (3) is followed by culturing in the dark on hypertonic medium for 16h at 28 ℃.
8. The method according to claim 1, wherein in step (4), the callus after recovery culture is divided into 10-15 cell groups with the diameter not more than 3mm, carefully subcultured on the screening medium, after 4 weeks the growth of resistant callus is evident, and the well-developed callus is subcultured for another 4 weeks.
9. The method as claimed in claim 1, wherein the plantlets obtained in step 5) are transferred on a basic culture medium, cultured to 4-5 true leaves, the root culture medium is cleaned by clear water, water culture is carried out for 2-3 weeks until new roots grow out, the plantlets are transplanted in paper cups containing vermiculite and matrix, the plantlets are transplanted in a large pot for 10-20 days, and outdoor growth is completed for the whole growth period.
10. Use of the method of any one of claims 1 to 9 in cotton gene editing.
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| CN115380721A (en) * | 2022-10-09 | 2022-11-25 | 湖北省农业科学院经济作物研究所 | Efficient cotton transgenic plant grafting technology |
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