CN110982820A - Gene editing method of tobacco haploid - Google Patents
Gene editing method of tobacco haploid Download PDFInfo
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Abstract
The invention discloses a gene editing method of a tobacco haploid, which is characterized by comprising the following steps: constructing a CRISPR/Cas9 expression plasmid with a target gene SgRNA, transferring the CRISPR/Cas9 expression plasmid into agrobacterium tumefaciens, and carrying out gene editing by mediating and transforming a tobacco haploid plant through the agrobacterium tumefaciens to obtain the tobacco haploid plant after target gene editing; the method can effectively edit the target gene in the tobacco haploid and is suitable for market popularization and application.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to a tobacco haploid gene editing method.
Background
Gene editing, artificial modification of nuclease appearing in recent years can greatly improve the efficiency of homologous recombination, and realize accurate and directional modification of genome. The homing nuclease, zinc finger nuclease and TALEN nuclease are successfully applied to plant genetic engineering, and the genome editing technology based on the CRISPR/Cas system, which is developed recently, has the characteristics of high efficiency, convenience and the like. The application of the artificial nucleases presents better prospect for the development of plant genetic engineering; CRISPR/Cas (Clustered modulated intercarried palindromic repeats/CRISPR-associated protein) tandem short palindromic repeats are a gene site-directed editing technology newly discovered in recent years, are important tools for clarifying gene functions in genome analysis, and are an epoch-making major breakthrough in plant breeding. The CRISPR system is related to an immune system for bacteria to resist invasion of exogenous genetic materials, at present, the application is most CRISPR/Cas9, a specific exogenous DNA sequence can be cut only by using a nuclease Cas9 and a mature crRNA (CRISPR-derived RNA) and tracrRNA (trans-activating RNA) complex, the technology realizes successful editing in multiple plants such as arabidopsis thaliana, tobacco, rice and the like, the feasibility of the gene editing technology is proved, and the effects of stress resistance, fruit maturation delay, herbicide tolerance increase, disease resistance and the like of the plants are achieved.
Tobacco haploid is obtained by in vitro culture of tobacco anther. The tobacco anther culture refers to a process of culturing anthers, which develop pollen to a certain stage, on a synthetic culture medium, inducing pollen grains to change the development process, so that the pollen grains do not form gametes, divide like somatic cells, form callus, and further differentiate and finally develop into complete plants. Since the haploid plant is obtained by carrying out isolated culture on the datura stramonium anther for the first time abroad, research work in the aspect of anther culture is carried out in succession in many countries, so that the technology is continuously mature in breeding work. The current research on tobacco anther culture is mature, and the method has important significance on breeding practice.
The germination rates of pollen embryoid bodies inoculated at different times also vary. The optimum period for tobacco anther culture should be the full bloom period of normal, disease-free plants. It has been shown that most plants are cultured ex vivo with pollen at the mononuclear stage more easily than successfully. The method selects the buds with the same length as the corolla and the calyx, and the pollen in the anther is just in the period of the edge of the mononuclear to carry out the culture to obtain the embryo with the best effect, which is the most suitable period for the tobacco anther culture. In order to increase the germination rate of embryoid bodies, it is generally necessary to pre-treat them before anther inoculation. The pretreatment is helpful for cell dedifferentiation and can improve the isolated culture reaction capability of the microspore. The pretreatment measure of the tobacco anther is low-temperature induction, the treatment temperature is generally 0-9 ℃, and the tobacco anther is pretreated before inoculation, so that the germination induction rate of embryoid can be obviously improved.
In recent years, a gene editing technology represented by CRISPR/Cas9 has gradually become an important tool for plant genetic improvement, and is very successful in application to diploid plants such as arabidopsis, rice and corn, but for cultivated tobacco of allopetraploid, gene editing is performed, the editing efficiency is lower than that of diploid plants, haploid plants are obtained by tobacco anther culture, and mutant materials obtained by gene editing can improve the gene editing efficiency, simultaneously, homozygote materials are obtained at an accelerated speed, and a theoretical basis is provided for combined breeding of gene editing and haploid.
Disclosure of Invention
Aiming at the defects of low efficiency of allopolyploid gene editing, long time for obtaining homozygote materials and the like in the prior art, the invention provides a tobacco haploid gene editing method, which comprises the steps of constructing CRISPR/Cas9 expression plasmid with target gene SgRNA, transferring the CRISPR/Cas9 expression plasmid into agrobacterium tumefaciens, and obtaining the tobacco haploid plant after target gene editing through agrobacterium tumefaciens mediated transformation and gene editing of the tobacco haploid plant.
The tobacco haploid gene editing method comprises the following steps:
(1) pretreatment of tobacco anther: in the full-bloom stage, selecting buds with the same length as the corolla and the calyx on the disease-free plant, putting the buds into a kraft paper bag, and finally putting the buds into a refrigerator at 4 ℃ for pretreatment for 3-7 days;
(2) sterilizing the tobacco anther: sterilizing the flower buds pretreated in the step (1), firstly sterilizing the surfaces of the flower buds by using 75% alcohol, and then cleaning the flower buds for 3-4 times by using sterile water;
(3) tobacco anther isolated culture: stripping sepals from the disinfected flower buds obtained in the step (2), inoculating anthers into a culture dish for culture, wherein the culture medium is an MS culture medium containing activated carbon, and pollen grains are differentiated to grow into seedlings; the culture conditions of the anthers are as follows: the culture temperature is 25 +/-1 ℃, and the illumination intensity is 30-50 mu mol/(m)2S) culturing for 45-60d under the condition of 16h/d of illumination time;
(4) strengthening the tobacco seedlings: separating the seedlings obtained in the step (3) by using scissors or a scalpel, and then culturing the seedlings in a culture bottle containing an activated carbon MS culture medium under the culture conditions that: temperature of 25 ±)Illumination intensity of 30-50 mu mol/(m) at 1 DEG C2S) culturing for 30-50 days under the condition of 16h/d of illumination time;
(5) tobacco seedling identification and leaf disc preparation: identifying the haploid of the seedling obtained in the step (4) by using the size of the leaf stomata and a stomata guard cell chloroplast counting method; for the plants determined to be haploids, a puncher or a scalpel is utilized to cut and obtain a tobacco leaf disc;
(6) preparing a bacterial liquid of agrobacterium: transferring CRISPR/Cas9 expression plasmid with PDS gene SgRNA into LBA4404 agrobacterium, culturing the transformed agrobacterium on YEB solid culture medium containing corresponding antibiotics under the culture condition of 28 ℃ in the dark for 2-3 d; selecting Agrobacterium containing the target plasmid, performing dark culture at 28 deg.C in YEB liquid culture medium containing corresponding antibiotics, performing liquid oscillation amplification culture at ratio of 1:50, and culturing until the bacterial liquid concentration is OD600=0.5-0.8, collecting thallus, and suspending thallus to OD with MS liquid culture medium600And = 0.6-0.8, to be used in subsequent experiments.
(7) And (3) infection and co-culture, namely immersing the tobacco leaf disc in the step (5) into the suspension bacterial liquid in the step (6) for 10min, taking out the leaf disc, sucking the bacterial liquid on sterilized filter paper, and placing the leaf disc on an MS co-culture medium for culture under the culture conditions that: culturing at 28 deg.C in dark for 3 d;
(8) and (4) performing differentiation culture, namely taking out the leaf disc co-cultured for 3d in the step (7), culturing the leaf disc on an MS differentiation culture medium containing corresponding antibiotics until a differentiation bud is formed, replacing the differentiation culture medium every 7-10d for 5-6 times, wherein the conditions are as follows: culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m)2S), culturing at 25 ℃ for 8h/d in the dark, and culturing for 45-60d together;
(9) and (3) culturing the differentiated bud, namely cutting the callus formed by the differentiated bud in the step (8), and culturing on an MS culture medium containing corresponding antibiotics until the differentiated bud on the callus grows to be 2-4cm high, wherein the culture conditions are as follows: culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m)2S), culturing at 25 ℃ for 8h/d in the dark, and culturing for 8-14d together;
(10) and (3) performing rooting culture on the regenerated plant, cutting the differentiated bud in the step (9), inserting the cut differentiated bud into an MS culture medium containing corresponding antibiotics to perform rooting culture, wherein the culture conditions are as follows: culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m)2S), culturing at 25 ℃ for 8h/d in the dark, and culturing for 7-10d together;
(11) and (4) detecting the result of the regenerated plant, sampling and detecting the plant obtained in the step (10), and then analyzing the detection result.
In the steps (3) and (4), the MS culture medium containing the activated carbon is MS minimal medium added with 30g/L of sucrose, 4g/L of agar and 0.05g/L of activated carbon, and the pH value of the culture medium is 5.7.
The preparation method of the YEB culture medium in the step (6) is as follows: beef extract powder 5g/L, peptone 5g/L, sucrose 5g/L, yeast extract 1g/L, MgSO4·7H2O0.5 g/L, and agar 15g/L is added to the solid medium. The YEB medium used for culturing transformed Agrobacterium LBA4404 was supplemented with streptomycin 50mg/L, kanamycin 50mg/L, and rifampicin 50 mg/L.
The preparation method of the MS co-culture medium in the step (7) comprises the following steps: 0.2mg/L NAA naphthalene acetate and 2 mg/L6-benzyladenine 6-BA were added to the MS medium.
The preparation method of the MS culture medium containing the antibiotics in the steps (8), (9) and (10) is as follows: the differentiation culture medium is prepared by adding 500mg/L carbenicillin and 50mg/L kanamycin into MS co-culture medium; the culture medium for differentiated bud culture and rooting culture is MS minimal medium supplemented with 500mg/L carbenicillin and 50mg/L kanamycin.
And (11) sampling the transformed plants, extracting DNA, performing sequencing detection, and finally analyzing the detection result.
1L of MS culture medium can be poured into 30 100mm culture dishes, 10 150mm culture dishes and 15 300mL culture bottles.
Compared with the prior art, the invention has the beneficial effects that:
the invention aims at the tobacco haploid, and constructs a tobacco haploid gene editing method by utilizing a gene editing method and through an agrobacterium tumefaciens-mediated tobacco transformation technology.
Drawings
FIG. 1 is a diagram showing the results of sequencing; in the figure: PDS-00 denotes a PDS reference sequence; PDS-A01 and PDS-A02 represent 2 haploid plants subjected to gene editing respectively; underlined sequences indicate the SgRNA sequence of the PDS gene, and the short-lined regions indicate insertions or deletions, respectively, compared to the reference sequence.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
Example 1: the embodiment is a tobacco haploid transformation method based on agrobacterium tumefaciens mediation, which comprises the following steps:
step 1, pretreatment of tobacco anther: in the full-bloom stage, selecting buds with the same length as a corolla and a calyx on a disease-free safflower plant, putting the buds into a kraft paper bag, and finally putting the buds into a refrigerator at 4 ℃ for pretreatment for 5 days;
step 2, sterilizing the tobacco anther: disinfecting the flower buds pretreated in the step 1, firstly disinfecting the surfaces of the flower buds by using 75% alcohol, specifically wiping the surfaces of the flower buds by using alcohol cotton soaked in the 75% alcohol for disinfection, then cleaning the flower buds for 3 times by using sterile water, and finally placing the flower buds on sterile filter paper to absorb the moisture on the surfaces of the flower buds;
step 3, tobacco anther in vitro culture: stripping sepals from the disinfected buds obtained in the step 2, inoculating anthers into a culture dish for culture, wherein the culture medium is an MS culture medium containing activated carbon, and the illumination intensity is 30-50 mu mol/(m) at the culture temperature of 25 +/-1 DEG C2S) culturing for 50 days under the condition of illumination time of 16 hours/day, and differentiating pollen grains into seedlings; the MS culture medium containing the activated carbon is MS minimal medium plus 30 g-Adding 4g/L of agar and 0.05g/L of activated carbon into L sucrose, wherein the pH value of the culture medium is 5.7;
step 4, strengthening tobacco seedlings: separating the seedlings obtained in step 3 with scissors or scalpel, separating into individual plants, culturing in culture flask containing activated carbon MS culture medium at 25 + -1 deg.C under illumination intensity of 30-50 μmol/(m)2S) culturing for 40 days under the condition of 16 hours/day of illumination, and growing seedlings into explants which can be used in subsequent experiments; the culture medium is identical to that of step 3;
step 5, identifying tobacco seedlings and preparing leaf discs: identifying the explant plant obtained in the step 4 by using the leaf stomata size and a stomata guard cell chloroplast counting method; taking tobacco leaves, tearing off the lower epidermis of the leaves, dyeing by using 1% I-KI, performing microscopic examination under a microscope, and cutting the identified haploid plants by using a scalpel to obtain a tobacco leaf disc;
step 6: preparation of a bacterial liquid of agrobacterium LBA 4404: the CRISPR/Cas9 expression plasmid with the PDS gene SgRNA is transferred into the LBA4404 agrobacterium-infected state by an electric shock conversion method (the sequence of the SgRNA is GCTGCATGGAAAGATGATGATGG); the specific operation of electric shock transformation is that firstly LBA4404 agrobacterium is subjected to competence, an electric revolving cup is placed on ice, and the electric revolving cup is precooled after the competence is melted; secondly, adding 2-3 mu L of target plasmid into the competence, slightly mixing uniformly, immediately inserting into ice, quickly moving the competence-plasmid mixture into an electric shock cup by using a 200 mu L gun head, and covering a cup cover; then starting the electrotransfer instrument, and setting parameters: c =25 muF, PC =200 ohm, V =2400V, take out the electric shock cup from the ice, wipe the outer bottom of the electric shock cup with absorbent paper, quickly put into an electric rotary tank, start electric shock, quickly insert into the ice after electric shock is completed, add 1mL of non-antibiotic YEB liquid, transfer into a sterile EP tube, and shake-culture at 28 ℃ for 2-3 h. Finally, centrifugation is carried out at 6000rpm for 1min to collect thalli, about 100 mu L of supernatant is left and mixed uniformly, then the bacterial liquid is coated on YEB solid culture medium containing 50mg/L streptomycin, 50mg/L kanamycin and 50mg/L rifampicin, dark culture is carried out for 3d under the condition of 28 ℃, agrobacterium containing the target plasmid is picked up and put into a culture medium containing 50mg/L streptomycin and 50mg/L canathenCulturing in YEB liquid culture medium containing mycin and 50mg/L rifampicin at 28 deg.C in dark, performing liquid shake culture at a ratio of 1:50 until the bacterial liquid concentration is OD600=0.6, collecting the cells, and finally suspending the cells to OD using MS liquid medium600=0.8, to be used in subsequent experiments; the YEB culture medium is beef extract powder 5g/L, peptone 5g/L, sucrose 5g/L, yeast extract 1g/L, magnesium sulfate heptahydrate 0.5g/L, agar 15g/L, sterilized at 121 ℃ for 15min, poured into a culture dish of 100mm after sterilization for 30mL, and cooled for later use; the MS liquid culture medium is prepared by adding sucrose 30g/L into MS basic culture medium, adjusting pH to 5.7, sterilizing at 121 deg.C for 15min, and cooling;
and 7, infecting and co-culturing, namely immersing the tobacco leaf disc prepared in the step 5 into the agrobacterium tumefaciens suspension bacterial liquid containing the target plasmid in the step 6 for 10min, taking out the leaf disc, sucking the bacterial liquid on sterilized filter paper, and culturing on an MS co-culture medium containing 0.2mg/L of naphthylacetic acid NAA and 2mg/L of 6-benzyladenine 6-BA, wherein the culture conditions are as follows: culturing at 28 deg.C in dark for 3 days;
step 8, differential culture, taking out the leaf disc co-cultured for 3d in the step 7, and culturing the leaf disc on an MS differential medium containing 500mg/L carbenicillin, 50mg/L kanamycin, 0.2mg/L NAA naphthalene acetic acid and 2 mg/L6-benzyl adenine 6-BA, wherein the culture conditions are as follows: culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m)2S), culturing in the dark at 25 ℃ for 8h/d for 45-60 days until a differentiated bud is formed, and replacing the differentiation culture medium for 5-6 times every 7-10 d;
and 9, culturing the differentiated bud, namely cutting the callus formed by the differentiated bud in the step 8, culturing the cut callus on an MS culture medium containing 500mg/L carbenicillin and 50mg/L kanamycin until the differentiated bud on the callus grows to be 2-4cm high, wherein the culture conditions are as follows: culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m)2S), culturing in dark at 25 ℃ for 8h/d for 12 days;
step 10, performing rooting culture on the regenerated plant, cutting the differentiated bud in the step 9, inserting the cut differentiated bud into an MS culture medium containing 500mg/L carbenicillin and 50mg/L kanamycin, and performing rooting culture under the culture conditions that: light culture at 28 deg.CCulturing for 16h/d, and the illumination intensity is 30-50 mu mol/(m)2S) culturing in dark at 25 ℃ for 8h/d and 7-10d to obtain tobacco safflower large golden haploid regeneration plants of PDS genes mediated and transformed by LBA4404 agrobacterium;
step 11, detecting regenerated plants, namely sampling and detecting the plants obtained in the step 10, selecting haploid plants transformed by gene editing for detection, and analyzing detection data; analyzing the data by utilizing a SeqMan program, finding a transformed haploid plant by utilizing gene editing, analyzing a PDS gene and a target position, and finding that the PDS gene of the transformed haploid plant has deletion or insertion allele editing; the analysis result shows that the deletion of the base T occurs in the target gene position of the PDS-A01 plant, and the insertion editing of the base A occurs in the target gene position of the PDS-A02 plant, and the specific result is shown in figure 1.
In this example, the tobacco safflower large golden haploid of PDS gene is transformed by LBA4404 agrobacterium-mediated transformation, and a regenerated plant can be obtained.
As can be seen from the above experiment, the LBA4404 Agrobacterium with the editing PDS gene can obtain a regeneration plant after transforming the tobacco Honghua large golden haploid plant. Therefore, a method for tobacco haploid gene editing is successfully constructed.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
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Claims (5)
1. A method for editing a tobacco haploid gene, which is characterized by comprising the following steps: constructing a CRISPR/Cas9 expression plasmid with a target gene SgRNA, transferring the CRISPR/Cas9 expression plasmid into agrobacterium tumefaciens, and carrying out gene editing by mediating and transforming a tobacco haploid plant through the agrobacterium tumefaciens to obtain the tobacco haploid plant after target gene editing.
2. The tobacco haploid gene editing method according to claim 1, which comprises the following specific steps:
(1) in the full-bloom stage of tobacco, selecting buds with the same length as the corolla and the calyx on disease-free tobacco plants, putting the buds into a kraft paper bag, and placing the buds in a refrigerator at 4 ℃ for pretreatment for 3 to 7 days;
(2) sterilizing the pretreated flower bud, stripping calyx from the sterilized flower bud, inoculating anther into MS culture medium containing activated carbon, and irradiating at 25 + -1 deg.C with illumination intensity of 30-50 μmol/(m)2S) cultivation under 4After 5-60 days, pollen grains are differentiated and grown into seedlings; separating the seedling, transferring into MS culture medium containing activated carbon, and irradiating at 25 + -1 deg.C with illumination intensity of 30-50 μmol/(m)2S) culturing strong seedlings for 30-50 days with illumination time of 16h each day; identifying haploids of seedlings obtained by strengthening seedlings by using a leaf stomata size and stomata guard cell chloroplast counting method, and cutting plants determined to be haploids by using a puncher or a scalpel to obtain tobacco haploid leaf discs;
(3) constructing CRISPR/Cas9 expression plasmid with target gene SgRNA, transferring the successfully constructed CRISPR/Cas9 expression plasmid with target gene SgRNA into LBA4404 agrobacterium, and performing amplification culture on the successfully converted agrobacterium to obtain OD6000.6-0.8 of agrobacterium liquid;
(4) putting the tobacco haploid leaf disc in the step (2) into the agrobacterium liquid in the step (3), taking out the sucked dry liquid after 10-15min, and putting the sucked dry liquid on an MS co-culture medium for co-culture for 3 days; taking out, culturing on MS differentiation culture medium containing antibiotics until differentiation buds are formed, and replacing the differentiation culture medium every 7-10 times; cutting off callus formed by existing differentiated bud, and culturing differentiated bud on MS culture medium containing antibiotic until the differentiated bud grows to 2-4cm high on callus; cutting off the differentiated bud, and inserting the cut differentiated bud into an MS culture medium containing antibiotics to perform rooting culture; and (5) successfully detecting and editing by sequencing, namely obtaining the tobacco haploid plant after gene editing.
3. The method for tobacco haploid gene editing according to claim 1, wherein: the differentiation culture conditions comprise illumination culture at 28 deg.C for 16h/d, and illumination intensity of 30-50 μmol/(m)2S), culturing at 25 ℃ for 8h/d in the dark, and culturing for 45-60 d.
4. The method for tobacco haploid gene editing according to claim 1, wherein: the culture conditions of the differentiated bud comprise illumination culture at 28 deg.C for 16h/d, and illumination intensity of 30-50 μmol/(m)2S), culturing at 25 ℃ for 8h/d in the dark, and culturing for 8-14 d.
5. The method for tobacco haploid gene editing according to claim 1, wherein: the rooting culture conditions are that the culture is carried out under the illumination of 28 ℃ for 16h/d, and the illumination intensity is 30-50 mu mol/(m)2S), culturing at 25 ℃ for 8h/d in the dark, and culturing for 7-10 d.
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| Publication number | Priority date | Publication date | Assignee | Title |
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Cited By (3)
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| CN113498738A (en) * | 2021-07-16 | 2021-10-15 | 云南省烟草农业科学研究院 | Method for creating new interspecific allopolyploid germplasm of tobacco by utilizing horizontal genome transfer |
| CN116941529A (en) * | 2023-07-26 | 2023-10-27 | 湖北省烟草科学研究院 | Tobacco haploid plant doubling and rapid propagation integrated method |
| CN116941529B (en) * | 2023-07-26 | 2024-05-17 | 湖北省烟草科学研究院 | Tobacco haploid plant doubling and rapid propagation integrated method |
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