CN110257406B - codon-Plant-modified Plant Nme2Cas9 gene and application thereof - Google Patents
codon-Plant-modified Plant Nme2Cas9 gene and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biotechnology and Plant genetic engineering, and discloses a codon-modified Plant Nme2Cas9 gene and application thereof. The codon-bothered modified Plant Nme2Cas9 gene has an amino acid sequence shown in SEQ ID No: 1. The Plant Nme2Cas9 gene subjected to codon Plant transformation provided by the invention is obtained by transformation based on a model crop rice codon, namely on the premise of maintaining the sequence of coded amino acids unchanged, the original codon is replaced by the codon screened out from codons related to the rice gene by an inventor to obtain the Plant Nme2Cas9 gene subjected to Plant transformation, and the Plant Nme2Cas9 gene is obtained by chemical synthesis. The gene of the invention can obviously improve the shearing efficiency.
Description
Technical Field
The present invention relates to biotechnology and plant genetic engineering technology. Specifically, the invention relates to a codon-modified Plant Nme2Cas9 gene, an expression cassette, an expression vector, a targeting vector, a transgenic cell containing the codon-modified Plant Nme2Cas9 gene and application of the gene and the vector.
Background
The CRISPR-nuclease technology is a eukaryotic specific site gene editing technology developed in recent years. There are two major classes of nucleases currently found in relation to this technology, and those commonly used in gene editing technology come from the second class. The second class of CRISPR-nucleases is mainly of three types: TypeII, TypeV, and TypeVI. The NmeCas9 belongs to a TypeII-C type, and the main action mode is to specifically cut a DNA double strand with the help of sgRNA and PAM, introduce double strand break of the DNA and edit a specific site. Wherein the NmeCas9 is further divided into Nme1Cas9, Nme2Cas9 and Nme3Cas 9. Cell experiments have demonstrated that Nme1Cas9 and Nme2Cas9 are able to function as nucleases. And the Nme2Cas9 has the following advantages compared with Nme1Cas 9: (1) can cut DNA double strand with high efficiency; (2) PAM of N4CC can be relied on for target recognition, and the editable range of the CRISPR system is expanded.
However, the Nme2Cas9 protein used in cell and animal experiments is separated from neisseria meningitidis, and prokaryotes and eukaryotes have different codon preference and base composition. For example, monocotyledons such as rice have stronger codon preference and higher GC content than those of bacteria, dicotyledons and the like. Therefore, the direct use of the Nme2Cas9 which is not artificially and optimally designed influences the expression efficiency in eukaryotic cells, thereby influencing the cutting efficiency of DNA double strands. In addition, since the Nme2Cas9 is derived from bacteria, it may adversely affect the genome of the eukaryotic transformation receptor and may raise concerns about its safety.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a Plant Nme2Cas9 gene subjected to codon Plant transformation and application thereof in Plant genome editing,
in order to achieve the above object, one aspect of the present invention provides a codon-botanically modified Plant Nme2Cas9 gene, wherein the codon-botanically modified Plant Nme2Cas9 gene has the nucleotide sequence shown in SEQ ID No: 1.
In a second aspect, the invention provides an expression cassette containing the Plant Nme2Cas9 gene subjected to codon Plant transformation.
In a third aspect, the invention provides an expression vector, wherein the expression vector is inserted with the Plant Nme2Cas9 gene subjected to codon Plant transformation or the expression cassette.
The fourth aspect of the invention provides a targeting vector, which is inserted with the above-mentioned codon-bothered Plant Nme2Cas9 gene or the above-mentioned expression cassette and target site sequence.
The fifth aspect of the invention provides a transgenic cell, wherein the transgenic cell is transferred with the Plant Nme2Cas9 gene subjected to codon Plant transformation, the expression cassette, the expression vector or the targeting vector.
In a sixth aspect, the invention provides a codon-botanically modified Plant Nme2Cas9 gene as described above, an expression cassette as described above, an expression vector as described above, a targeting vector as described above, or a transgenic cell as described above for use in Plant genome editing, wherein the Plant genome editing comprises splicing a Plant genome to obtain a transgenic Plant or Plant part comprising a mutation site.
In a seventh aspect, the present invention provides a method for obtaining a transgenic rice or rice part containing a mutation site, the method comprising:
(1) transforming the targeting vector into agrobacterium tumefaciens to obtain a transgenic cell of the agrobacterium tumefaciens;
(2) inducing the rice embryo on a callus induction culture medium to obtain a secondary callus;
(3) contacting and infecting the transgenic cell of the agrobacterium tumefaciens and the secondary callus, and co-culturing the infected secondary callus in an agrobacterium tumefaciens culture medium to obtain a co-cultured secondary callus;
(4) and sequentially screening, seedling and rooting the secondary callus.
The Plant Nme2Cas9 gene with the codon transformed in a vegetative way is obtained by transforming a model crop rice codon, namely replacing the original codon by the codon screened out from the codon related to the rice gene by the inventor on the premise of maintaining the coded amino acid sequence unchanged to obtain the Plant Nme2Cas9 gene which is transformed in a vegetative way, and then carrying out chemical synthesis. The obtained Plant Nme2Cas9 base which is transformed in a Plant way is integrated into an expression vector, a corresponding targeting vector is constructed on the basis, and then Plant specific gene editing is realized through Plant genetic transformation, so that the shearing efficiency can be obviously improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a diagram of a PHUN7B11 (with plant Nme2Cas9 inserted) vector plasmid.
FIG. 2 is a PCR detection electrophoretogram of transgenic plants; wherein the amplified fragment is part of a plant Nme2Cas9 gene, and the size of the fragment is 697 bp; m is DL2 kbmarker; NC is negative control; 1-11 are randomly selected transgenic plants.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For numerical ranges, each range between its endpoints and individual point values, and each individual point value can be combined with each other to give one or more new numerical ranges, and such numerical ranges should be construed as specifically disclosed herein.
In a first aspect, the invention provides a codon-botanically-modified Plant Nme2Cas9 gene, wherein the codon-botanically-modified Plant Nme2Cas9 gene has a nucleotide sequence shown in SEQ ID No: 1.
The invention also needs to explain that the invention "has SEQ ID No: 1 "and does not mean any nucleic acid molecule comprising a nucleotide sequence other than that shown in SEQ ID No: 1, but refers to a nucleotide or nucleotide sequence other than the nucleotide sequence shown in SEQ ID No: 1 or to achieve the nucleotide sequence shown in SEQ ID No: 1, and the like, for example, an enzyme cleavage site, a marker gene, a selection gene, a promoter, an enhancer, and the like, which do not affect the function of the nucleic acid sequence or the nucleotide sequence. Thus, the "peptide having the amino acid sequence of SEQ ID No: 1 refers to a nucleotide sequence having the sequence shown in SEQ ID No: 1, but still achieves the nucleotide sequence shown in SEQ ID No: 1 in sequence with the nucleotide sequence function.
According to a specific embodiment of the invention, the nucleotide sequence of the plant Nme2Cas9 gene is shown as SEQ ID No: 1 is shown.
According to the invention, both ends of the plant Nme2Cas9 gene can be connected with NotI/SacI enzyme cutting sites, for example, the 5 'end of the plant Nme2Cas9 gene can be connected with the NotI enzyme cutting sites, and the 3' end can be connected with the SacI enzyme cutting sites.
According to the invention, in order to enable the plant Nme2Cas9 gene provided by the invention to enter a cell nucleus more efficiently, a plurality of nuclear entry signals are preferably connected in series at the 3' end of the plant Nme2Cas9 gene, for example, 3 NLS signals can be provided, and the nucleic acid sequence of the nuclear entry signals is shown as SEQ ID No: 2, respectively.
According to a preferred embodiment of the invention, the 5 'end of the plant Nme2Cas9 gene is connected with a NotI enzyme cutting site, and the 3' end is connected with a SacI enzyme cutting site and 3 NLS signals, wherein the 3 NLS signals are connected with the upstream of the SacI enzyme cutting site, and the specific sequence is as shown in SEQ ID No: 3, respectively.
In a second aspect, the invention also provides an expression cassette containing the codon-botanically-modified Plant Nme2Cas9 gene as described above.
In a third aspect, the invention also provides an expression vector, wherein the expression vector is inserted with the Plant Nme2Cas9 gene subjected to codon Plant transformation or the expression cassette.
According to the invention, the construction method of the expression vector can be carried out according to the conventional method in the field, for example, the same restriction enzyme is used for carrying out enzyme digestion on the plant Nme2Cas9 gene and the vector to be inserted, and then the ligase is used for connecting the plant Nme2Cas9 gene to the vector, so as to obtain the expression vector.
The restriction enzyme can be specifically selected according to the enzyme cutting site introduced into the plant Nme2Cas9 gene, and for example, the restriction enzyme can be NotI/SacI restriction enzyme.
The ligase may be any ligase conventionally used in the art which can ligate two nucleic acid fragments, and may be, for example, T4 ligase.
Among them, the vector may be various vectors conventionally used in the art, and preferably, the vector may be pHUN600, pHUN611, PUC57-AMP, pHUN400 or pHUN 900. According to a preferred embodiment of the invention, the vector is pHUN600, further, the pHUN600 vector and the plant Nme2Cas9 gene can be cut by NotI/SacI enzyme cutting sites and recovered, and then the plant Nme2Cas9 gene is connected to the pHUN600 vector by T4 ligase, so as to obtain the plant expression vector pHUN-plant Nme2Cas9 (abbreviated as pHUN7B 11).
In a fourth aspect, the invention also provides a targeting vector, which is inserted with the above-mentioned codon-bothering modified Plant Nme2Cas9 gene or the above-mentioned expression cassette and target site sequence.
According to the invention, the target site sequence can be determined according to the sequence which needs to be subjected to genome editing in practice, but the target site sequence is in the form of 5 '- (N) X-NNCC-3', wherein X is a main sequence and NNCC is a characteristic sequence. According to a specific embodiment of the present invention, the target site sequence is SEQ ID No: 8 (nucleotide sequence at position 1381-1402 in the rice PDS gene (LOC _ Os03g 0184000))GGCACCATGATATTTGCCATGCCAAA, the underlined part is the TGCC part in the 5 '- (N) X-NNCC-3' structure).
According to the invention, the targeting vector can be obtained by simple annealing and enzyme digestion ligation on the basis of an expression vector.
In a fifth aspect, the invention also provides a transgenic cell, wherein the transgenic cell is transferred with the codon-botanically-modified Plant Nme2Cas9 gene, the expression cassette, the expression vector or the targeting vector.
According to the present invention, the selection of the cell can be determined according to the stage of gene targeting, specifically, for example, when the amplification of the plant Nme2Cas9 gene is required to be achieved before the construction of the targeting vector, escherichia coli can be used as the host cell, and when the targeting vector is required to be transformed into a plant cell, Agrobacterium tumefaciens (Agrobacterium tumefaciens) can be used as the host cell.
In a sixth aspect, the invention also provides the use of the codon-botanically modified Plant Nme2Cas9 gene, the expression cassette, the expression vector, the targeting vector or the transgenic cell as described above in Plant genome editing, wherein the Plant genome editing comprises shearing the Plant genome to obtain a transgenic Plant or Plant part containing a mutation site.
The plant Nme2Cas9 gene subjected to codon plant transformation can be used for completing the shearing of DNA double chains in a plant body, and a transgenic plant or a plant part with a mutation site is obtained under the action of a self-repairing system.
According to the present invention, the plant is preferably a monocotyledon, more preferably rice, further preferably japonica rice, and still further preferably japonica rice nippon.
In a seventh aspect, the present invention also provides a method of obtaining a transgenic rice or rice part containing a mutation site, the method comprising:
(1) transforming the targeting vector into agrobacterium tumefaciens to obtain transgenic cells of the agrobacterium tumefaciens;
(2) inducing the rice embryo on a callus induction culture medium to obtain a secondary callus;
(3) contacting and infecting the transgenic cell of the agrobacterium tumefaciens and the secondary callus, and then co-culturing the infected secondary callus in an agrobacterium tumefaciens culture medium to obtain a co-cultured secondary callus;
(4) and sequentially screening the secondary callus, differentiating into seedlings and rooting.
According to the present invention, in step (1), the Agrobacterium tumefaciens may be Agrobacterium tumefaciens conventionally used in the art, for example, Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 strain may be preserved in the Rice institute of agricultural sciences, Anhui province.
The method for transforming Agrobacterium tumefaciens with the targeting vector can be performed according to a conventional technique in the art, and can be, for example, a freeze-thaw method. After transformation, Agrobacterium tumefaciens containing the targeting vector can be streaked on an LB plate containing 40-60mg/L kanamycin (see Table 1 for composition), dark-cultured at 25-35 ℃, and after 20-30h, the activated Agrobacterium tumefaciens is inoculated onto a fresh LB plate containing 40-60mg/L kanamycin by using a sterile inoculating loop, activated for the second time, and dark-cultured at 25-35 ℃ overnight. Agrobacterium suspension medium (see Table 1 for components) was added to a sterile centrifuge tube, Agrobacterium tumefaciens activated 2 times was scraped off with an inoculating loop, OD660(Optical density660nm, absorbance at 660 nm) was adjusted to about 0.10-0.25, and the tube was allowed to stand at room temperature for more than 30 min.
In step (2), the rice embryo can be obtained by a method conventional in the art, for example, removing the husk from the mature rice seed, sterilizing, and separating the embryo. Specifically, the mature rice seeds are hulled, the seeds with normal appearance and clean and without mildew stains are selected, and the seeds are washed and disinfected by alcohol and sodium hypochlorite solution in sequence. Then, the sodium hypochlorite is poured off, the solution is washed by sterile water until the smell of the sodium hypochlorite does not exist, finally, the sterile water is added, the solution is soaked overnight, and the embryo is separated along the aleurone layer by a surgical blade.
The callus induction medium may be a callus induction medium conventionally used in the art, and is specifically shown in table 1. The scutellum of the isolated embryos were placed upside down on callus induction medium and cultured in dark at 28-32 ℃ to induce callus.
The induction time is based on the appearance of spherical, rough and light yellow secondary callus, and then the pre-culture operation can be carried out, namely the secondary callus is transferred to a new callus induction culture medium, and the pre-culture is continued for 3 to 8 days at the temperature of between 28 and 32 ℃ in a dark environment. After the pre-culture is finished, collecting the small particles with good state and vigorous division into a sterile centrifuge tube by using a spoon for infecting agrobacterium tumefaciens.
In the step (3), the method of contacting and infecting the transgenic cell of Agrobacterium tumefaciens with the secondary callus may comprise: and (3) adding the agrobacterium tumefaciens suspension prepared in the step (1) into the callus obtained in the step (2), and soaking for 10-20min, wherein the suspension is gently shaken at intervals to realize the contact and infection.
Wherein the co-culturing step may comprise: and pouring out the liquid (dripping the liquid as far as possible) after the soaking is finished, sucking the redundant agrobacterium liquid on the surface of the callus, and drying by sterile air. Placing sterile filter paper on a sterile culture dish pad, adding a proper amount of Agrobacterium tumefaciens suspension culture medium, uniformly dispersing the callus on the filter paper, and culturing for 45-50h in the dark at 20-25 ℃.
In step (4), the screening may include pre-screening and formal screening, i.e., the co-cultured callus is spread on a pre-screening medium (see Table 1 for composition), and cultured in the dark at 25-32 ℃ for 4-6 days. After the pre-screening culture is finished, transferring the callus onto a screening culture medium (the components are shown in table 1), culturing in the dark at 25-32 ℃, and after 2-3 weeks, the resistant callus obviously grows and can be subjected to differentiation and regeneration operation.
Wherein, the specific steps of differentiating into seedlings can comprise: several fresh small particles with good growth state are selected from each independent transformant and transferred to a differentiation regeneration medium (the components are shown in table 1) for differentiation into seedlings. Culturing at 25-30 deg.C under illumination for 12-18h, 6-10h dark, and 3000-6000lx light intensity.
Wherein, the specific steps of rooting may comprise: when the seedlings differentiated from the resistant callus grow to about 2cm, well-grown seedlings are taken from each independent transformant and transferred to a rooting medium (the components are shown in the table 1), the seedlings are cultured under illumination at 25-30 ℃, the illumination period is 12-18h, the illumination time is 6-10h, the darkness is 6000lx, and the light intensity is 3000-. After rooting, selecting seedlings with developed root systems, washing the culture medium with water, and transplanting the seedlings into soil.
According to the present invention, various media used as above may be media conventionally used in the art, and preferably, the media listed in table 1 may be used, wherein the configuration of the media used may be as follows: yongbo Duan, Chenguang Zhai, Hao Li, Juan Li, Wenqian Mei et al, an effective and high-throughput protocol for Agrobacterium mediated transformation based on phosphorus hormone immunoassay porous selection in Japonica node (Oryza sativa L.). Plant cell reports,2012,31: 1611-.
TABLE 1
Note: "macroelement N6" means that the macroelement N6 contains [ NO3 -]/[NH4 +]＝40mM/10mM。
The present invention will be described in detail below by way of examples.
The operations in the following detailed description are performed by conventional operations commonly used in the art, unless otherwise specifically indicated. The skilled person can easily derive teachings on such routine procedures from the prior art, for example with reference to the textbooks Sambrook and David Russell, molecular μ lar Cloning: A Laboratory Manual,3rd ed., Vols1, 2; charles New Stewart, Alisher Touraev, vitay Citovsky and Tzvi Tzfira, Plant Transformation Technologies, and the like. The raw materials, reagents, materials and the like used in the following examples are all commercially available products unless otherwise specified.
Example 1
This example is used to illustrate the construction of a plant targeting vector containing the plant Nme2Cas9 gene
1. The Suzhou Jinzhi Biotechnology Ltd is entrusted to synthesize SEQ ID No: 3 (comprising a sequence shown in SEQ ID No: 1, the 15 'end of the SEQ ID No: is connected with NotI enzyme cutting site, the 3' end is connected with 3 NLS signals (shown in SEQ ID No: 2) and SacI enzyme cutting site), is connected to a PUC57-AMP vector to form a PUC57-AMP-plant NMe2Cas9 vector, and is loaded into an escherichia coli XL-blue strain.
2. Extracting plasmids from the Escherichia coli XL-blue containing the PUC57-AMP-plant Nme2Cas9 vector by using an Axygen plasmid extraction kit, carrying out enzyme digestion by using NotI/SacI, and recovering a plant Nme2Cas9 fragment. At the same time, pHUN600 is linearized by NotI/SacI enzyme, pHUN600 is recovered, the plant Nme2Cas9 fragment and pHUN600 fragment are connected by T4 ligase (purchased from TaKaRa), and plant expression vector pHUN600-plant Nme2Cas9 (figure 1) is obtained and named as pHUN7B 11.
3. Selecting the nucleotide sequence at position 1381-1402 in the rice PDS gene (LOC _ Os03g0184000)GGCACCATGATATTTGCCATGCCAAA (underlined part is 5 '- (N) X-NNCC-3' structure TGCC part) as targeting site. The target site sequence was fused to pHUN7B11 to form pHUN7B 11-PDS. The plant expression vector was transferred to Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 strain (stored by the Rice research institute of agricultural sciences, Anhui) by freeze-thawing for genetic transformation.
Example 2
This example illustrates the genetic transformation of rice using pHUN7B11-PDS as a targeting vector and the acquisition of mutants.
1. Induction and preculture of mature embryo calli
Removing hull from mature seed of Nipponbare (preserved by Rice institute of agricultural sciences, Anhui province), selecting seed with normal appearance, cleanness and no mildew spot, shaking with 70% alcohol for 90sec, and pouring off alcohol; then 50% sodium hypochlorite solution containing Tween20 (the effective chlorine concentration of the stock solution is more than 4%, wherein the 50% sodium hypochlorite solution is the solution obtained by diluting the stock solution by 1 time, 1 drop of Tween20 is added to each 100 ml), and the seeds are washed and shaken on a shaking table for 45min (180 r/min). Pouring out sodium hypochlorite, washing with sterile water for 5-10 times until no smell of sodium hypochlorite exists, adding sterile water, and soaking at 30 deg.C overnight. Embryos were separated along the aleurone layer with scalpel blade, scutellum up placed on callus induction medium (see table 1 for ingredients) and cultured in dark at 30 ℃ to induce callus at 12 grains/dish.
Spherical, rough and light yellow secondary callus appears after two weeks, and the preculture operation can be carried out, that is, the secondary callus is transferred to a new callus induction culture medium and precultured for 5 days at 30 ℃ in dark. After the pre-culture is finished, collecting the small particles with good state and vigorous division into a 50mL sterile centrifuge tube by using a spoon for agrobacterium infection.
2. Culture and suspension preparation of Agrobacterium strains
The Agrobacterium strain EHA105 containing the pHUN7B11-PDS vector was streaked on LB solid medium containing 50mg/L kanamycin (see Table 1), cultured in the dark at 28 ℃ for 24 hours, and then the activated Agrobacterium was inoculated with a sterile inoculating loop onto a fresh LB plate containing 50mg/L kanamycin for a second activation and cultured in the dark at 28 ℃ overnight. 20-30mL of Agrobacterium suspension medium (see Table 1 for components) was added to a 50mL sterile centrifuge tube, the Agrobacterium after 2-time activation was scraped off with an inoculating loop, OD660(Optical density660nm, absorbance at 660 nm) was adjusted to about 0.10-0.25, and the mixture was allowed to stand at room temperature for 30min or more.
3. Infection and Co-cultivation
To the prepared callus (see step 1), the Agrobacterium suspension was added and soaked for 15min with occasional gentle shaking. After soaking, pouring off the liquid (dripping the liquid as far as possible), sucking the redundant agrobacterium liquid on the surface of the callus by using sterile filter paper, and drying the callus by using sterile wind in a super clean bench. Three pieces of sterile filter paper are placed on a disposable sterile culture dish pad with the size of 100 multiplied by 25mm, 2.5mL of agrobacterium suspension culture medium is added, the callus after being sucked dry is evenly dispersed on the filter paper, and the mixture is cultured in the dark at the temperature of 23 ℃ for 48 h.
4. Pre-screening and screening cultures
After the end of the co-culture, the co-cultured calli were spread evenly on the pre-selection medium (see Table 1 for composition) and cultured in the dark at 30 ℃ for 5 days. After the pre-screening culture was completed, the calli were transferred to a screening medium (see table 1 for composition), and 25 calli were inoculated to each plate under two culture conditions, one: culturing at 30 deg.C in dark for 45 days; the other is dark culture at 37 ℃ for 12 hours and 30 ℃ for 12 hours, and the culture is continuously carried out for 45 days at the temperature in a cycle; after the culture is finished, the resistant callus grows obviously and can be subjected to differentiation and regeneration operation.
5. Differentiation and regeneration
2-3 fresh small particles with good growth status were selected from each independent transformant and transferred to differentiation regeneration medium (see Table 1 for composition). Each culture dish was inoculated with 5 independent transformants. Culturing at 28 ℃ under illumination with 16h illumination and 8h darkness illumination and 3000-6000lx light intensity.
6. Rooting and transplanting
When the bud differentiated from the resistant callus grows to about 2cm, only one well-grown seedling is taken from each independent transformant and is transferred to a rooting medium (the components are shown in the table 1), the seedling is cultured under the light of 28 ℃, the light period is 16h, the light is 8h and the dark, and the light intensity is 3000-. After two weeks, 42 seedlings with developed root systems were selected, washed with water to remove the medium, and transplanted into soil.
7. Molecular characterization
Before transplanting, a rice leaf sample is taken, and DNA is subjected to small extraction by a CTAB method. The resulting genomic DNA samples were used for PCR analysis. The PCR primer for amplifying the codon-modified plant Nme2Cas9 is SEQ ID No: 4 (5'-GGCCTACCACGCGATCTCCAGG-3') and SEQ ID No: 5 (5'-GTAGCCCTTCTCGTTGAGCCGC-3'), resulting in a fragment of 697bp in length. Genomic DNA was first held at 95 ℃ for 5 minutes, then subjected to 32 cycles: 94 ℃ for 45 seconds, 56 ℃ for 45 seconds, 72 ℃ for 45 seconds, and finally extension at 72 ℃ for 10 minutes. 11 transgenic plants are randomly selected and identified to be positive, and the positive rate reaches 100 percent (see figure 2).
All of the 42 transgenic plants were subjected to leaf DNA extraction, and the resulting genomic DNA samples were used for PCR analysis. The PCR primer for amplifying the PDS gene segment is SEQ ID No: 6 (5'-ACATATATGAATATGACAGATA-3') and SEQ ID No: 7 (5'-AACTTCACCTTCTCTGGCCAA-3'), resulting in a fragment of 553bp in length. Genomic DNA was first held at 95 ℃ for 5 minutes, then subjected to 32 cycles: 30 seconds at 94 ℃, 30 seconds at 60 ℃, 30 seconds at 72 ℃ and finally 10 minutes at 72 ℃. The PCR product was sequenced. The results were aligned to the wild type sequence. 20 of the 42 transgenic plants tested underwent point mutation; the mutation efficiency was 47.6%. The modified plant Nme2Cas9 can cut the rice genome and has high mutation efficiency. Similarly, a targeting vector is constructed by using the original Plant Nme2Cas9 gene, and rice genetic transformation is carried out, but a positive editing Plant is not obtained. The codon-optimized Plant Nme2Cas9 is shown to achieve effective gene editing in plants under optimized culture conditions.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
SEQUENCE LISTING
<110> institute of Rice research of agricultural science institute of Anhui province
<120> codon-modified Plant Nme2Cas9 gene and application thereof
<130> HFI00864-NYSD
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 3249
<212> DNA
<213> codon-modified Plant Nme2Cas9 gene
<400> 1
atggcggcgt tcaagcctaa cccaatcaac tacatcctcg gtctggacat cggcatcgca 60
tcagtgggct gggccatggt cgagatcgac gaggaggaga atccaatcag gctgattgat 120
ctcggcgtcc gggtgttcga gagggcggag gtgcctaaga caggcgactc actggcaatg 180
gccaggaggc tggccaggtc tgtccggagg ctcacaagga ggagggccca caggctcctg 240
agggccagga ggctcctgaa gagggaggga gtgctccagg ccgcggactt cgatgagaat 300
ggcctgatca agtccctccc aaacacaccg tggcagctca gggctgccgc cctggatagg 360
aagctgaccc cgctggagtg gtccgccgtg ctcctgcacc tcattaagca tcgcggctac 420
ctgagccagc ggaagaacga gggagagaca gccgacaagg agctgggcgc gctcctgaag 480
ggagtcgcca acaatgccca tgcgctccag accggcgatt tcaggacacc ggccgagctg 540
gcgctgaata agttcgagaa ggagtccggc cacatccgga accagagggg cgactactcc 600
cataccttca gccgcaagga cctccaggcc gagctgattc tcctgttcga gaagcagaag 660
gagttcggca atccacatgt gtctggcggc ctcaaggagg gaatcgagac actcctgatg 720
actcagaggc cagcactctc aggcgacgcc gtccagaaga tgctgggaca ttgcaccttc 780
gagcctgccg agccaaaggc cgccaagaac acctacacag cggagaggtt catttggctg 840
acaaagctca acaatctgcg catcctggag cagggctctg agcggccact caccgacaca 900
gagagggcga ccctgatgga tgagccttac cggaagtcca agctcacata cgcacaggcc 960
aggaagctcc tgggcctgga ggacaccgcc ttcttcaagg gcctgcggta cggcaaggat 1020
aatgccgagg cgtccacact catggagatg aaggcctacc acgcgatctc cagggccctg 1080
gagaaggagg gcctgaagga caagaagagc ccgctcaacc tgtccagcga gcttcaggat 1140
gagattggca ccgcgttctc actgttcaag accgacgagg atattaccgg aaggctcaag 1200
gacagggtgc agccagagat tctggaggcc ctcctgaagc acatctcctt cgataagttc 1260
gtgcagatta gcctgaaggc gctcaggcgc atcgtccctc tcatggagca gggcaagcgc 1320
tacgatgagg cctgcgcgga gatctacggc gatcattacg gcaagaagaa cacagaggag 1380
aagatctacc tcccgccaat tccagccgac gagatcagga atcctgtggt cctgcgcgcc 1440
ctctcacagg ccaggaaggt catcaacggc gtggtccggc gctacggctc tcctgcgcgc 1500
atccatattg agacagcccg ggaggtcggc aagtccttca aggacaggaa ggagattgag 1560
aagcgccagg aggagaatcg caaggatcgg gagaaggctg ccgccaagtt ccgcgagtac 1620
ttccctaact tcgtgggcga gccgaagagc aaggacatcc tcaagctgcg gctctacgag 1680
cagcagcacg gcaagtgcct ctactcaggc aaggagatta atctggtgcg gctcaacgag 1740
aagggctacg tcgagatcga tcatgcgctg ccattctcca ggacctggga cgatagcttc 1800
aacaataagg tcctggtgct cggcagcgag aaccagaata agggcaatca gacaccttac 1860
gagtacttca atggcaagga taactcaagg gagtggcagg agttcaaggc ccgcgtggag 1920
acatccaggt tcccacgctc taagaagcag cgcatcctcc tccagaagtt cgacgaggat 1980
ggcttcaagg agtgcaacct caatgacacc cgctacgtga accgcttcct gtgccagttc 2040
gtcgccgatc acattctgct gacaggcaag ggcaagcgca gggtgttcgc gtctaatggc 2100
cagattacaa acctcctgag gggattctgg ggcctcagga aggtcagggc ggagaatgac 2160
cggcaccatg cgctcgatgc cgtggtggtg gcctgctcca cagtggccat gcagcagaag 2220
atcacacgct tcgtccggta caaggagatg aacgccttcg acggcaagac cattgataag 2280
gagacaggca aggtgctcca ccagaagaca catttcccac agccttggga gttcttcgcc 2340
caggaggtca tgatcagggt cttcggcaag ccagacggca agcctgagtt cgaggaggcg 2400
gatacaccag agaagctccg cacactcctg gcagagaagc tgtcatctcg gccagaggcg 2460
gtgcatgagt atgtgacccc gctcttcgtg tcacgggcgc caaataggaa gatgtctggc 2520
gcccacaagg acacactgcg ctcagcgaag cggttcgtca agcataacga gaagatttct 2580
gtgaagcgcg tctggctcac cgagatcaag ctggccgacc tggagaacat ggtgaattac 2640
aagaacggcc gggagatcga gctgtacgag gccctcaagg ccaggctgga ggcgtacggc 2700
ggaaatgcca agcaggcgtt cgacccgaag gataacccat tctacaagaa gggcggccag 2760
ctggtgaagg ccgtcagggt ggagaagacc caggagagcg gcgtcctcct gaataagaag 2820
aacgcctaca caattgcgga caacggcgat atggtccgcg tggacgtctt ctgcaaggtg 2880
gataagaagg gcaagaatca gtacttcatc gtgcctatct acgcctggca ggtcgcggag 2940
aacatcctcc cggacattga ttgcaagggc tacaggattg acgattcata cacattctgc 3000
ttctctctcc acaagtacga cctgatcgcc ttccagaagg atgagaagag caaggtcgag 3060
ttcgcgtact acattaattg cgactccagc aacggcaggt tctacctcgc ctggcatgat 3120
aagggctcca aggagcagca gttccgcatt agcacccaga atctggtgct catccagaag 3180
taccaggtca acgagctggg caaggagatc aggccttgcc gcctgaagaa gaggcctccg 3240
gtgcgctaa 3249
<210> 2
<211> 99
<212> DNA
<213> 3 NLS signals connected in series
<400> 2
tccggcggct ccccgaagaa gaagaggaag gtgtccggcg gtagtccaaa gaagaagagg 60
aaggtgtcgg gaggtagccc aaagaagaag aggaaggtt 99
<210> 3
<211> 3362
<212> DNA
<213> Plant Nme2Cas9+ enzyme cleavage site +3 NLS signals obtained by codon Plant transformation
<400> 3
gcggccgcat ggcggcgttc aagcctaacc caatcaacta catcctcggt ctggacatcg 60
gcatcgcatc agtgggctgg gccatggtcg agatcgacga ggaggagaat ccaatcaggc 120
tgattgatct cggcgtccgg gtgttcgaga gggcggaggt gcctaagaca ggcgactcac 180
tggcaatggc caggaggctg gccaggtctg tccggaggct cacaaggagg agggcccaca 240
ggctcctgag ggccaggagg ctcctgaaga gggagggagt gctccaggcc gcggacttcg 300
atgagaatgg cctgatcaag tccctcccaa acacaccgtg gcagctcagg gctgccgccc 360
tggataggaa gctgaccccg ctggagtggt ccgccgtgct cctgcacctc attaagcatc 420
gcggctacct gagccagcgg aagaacgagg gagagacagc cgacaaggag ctgggcgcgc 480
tcctgaaggg agtcgccaac aatgcccatg cgctccagac cggcgatttc aggacaccgg 540
ccgagctggc gctgaataag ttcgagaagg agtccggcca catccggaac cagaggggcg 600
actactccca taccttcagc cgcaaggacc tccaggccga gctgattctc ctgttcgaga 660
agcagaagga gttcggcaat ccacatgtgt ctggcggcct caaggaggga atcgagacac 720
tcctgatgac tcagaggcca gcactctcag gcgacgccgt ccagaagatg ctgggacatt 780
gcaccttcga gcctgccgag ccaaaggccg ccaagaacac ctacacagcg gagaggttca 840
tttggctgac aaagctcaac aatctgcgca tcctggagca gggctctgag cggccactca 900
ccgacacaga gagggcgacc ctgatggatg agccttaccg gaagtccaag ctcacatacg 960
cacaggccag gaagctcctg ggcctggagg acaccgcctt cttcaagggc ctgcggtacg 1020
gcaaggataa tgccgaggcg tccacactca tggagatgaa ggcctaccac gcgatctcca 1080
gggccctgga gaaggagggc ctgaaggaca agaagagccc gctcaacctg tccagcgagc 1140
ttcaggatga gattggcacc gcgttctcac tgttcaagac cgacgaggat attaccggaa 1200
ggctcaagga cagggtgcag ccagagattc tggaggccct cctgaagcac atctccttcg 1260
ataagttcgt gcagattagc ctgaaggcgc tcaggcgcat cgtccctctc atggagcagg 1320
gcaagcgcta cgatgaggcc tgcgcggaga tctacggcga tcattacggc aagaagaaca 1380
cagaggagaa gatctacctc ccgccaattc cagccgacga gatcaggaat cctgtggtcc 1440
tgcgcgccct ctcacaggcc aggaaggtca tcaacggcgt ggtccggcgc tacggctctc 1500
ctgcgcgcat ccatattgag acagcccggg aggtcggcaa gtccttcaag gacaggaagg 1560
agattgagaa gcgccaggag gagaatcgca aggatcggga gaaggctgcc gccaagttcc 1620
gcgagtactt ccctaacttc gtgggcgagc cgaagagcaa ggacatcctc aagctgcggc 1680
tctacgagca gcagcacggc aagtgcctct actcaggcaa ggagattaat ctggtgcggc 1740
tcaacgagaa gggctacgtc gagatcgatc atgcgctgcc attctccagg acctgggacg 1800
atagcttcaa caataaggtc ctggtgctcg gcagcgagaa ccagaataag ggcaatcaga 1860
caccttacga gtacttcaat ggcaaggata actcaaggga gtggcaggag ttcaaggccc 1920
gcgtggagac atccaggttc ccacgctcta agaagcagcg catcctcctc cagaagttcg 1980
acgaggatgg cttcaaggag tgcaacctca atgacacccg ctacgtgaac cgcttcctgt 2040
gccagttcgt cgccgatcac attctgctga caggcaaggg caagcgcagg gtgttcgcgt 2100
ctaatggcca gattacaaac ctcctgaggg gattctgggg cctcaggaag gtcagggcgg 2160
agaatgaccg gcaccatgcg ctcgatgccg tggtggtggc ctgctccaca gtggccatgc 2220
agcagaagat cacacgcttc gtccggtaca aggagatgaa cgccttcgac ggcaagacca 2280
ttgataagga gacaggcaag gtgctccacc agaagacaca tttcccacag ccttgggagt 2340
tcttcgccca ggaggtcatg atcagggtct tcggcaagcc agacggcaag cctgagttcg 2400
aggaggcgga tacaccagag aagctccgca cactcctggc agagaagctg tcatctcggc 2460
cagaggcggt gcatgagtat gtgaccccgc tcttcgtgtc acgggcgcca aataggaaga 2520
tgtctggcgc ccacaaggac acactgcgct cagcgaagcg gttcgtcaag cataacgaga 2580
agatttctgt gaagcgcgtc tggctcaccg agatcaagct ggccgacctg gagaacatgg 2640
tgaattacaa gaacggccgg gagatcgagc tgtacgaggc cctcaaggcc aggctggagg 2700
cgtacggcgg aaatgccaag caggcgttcg acccgaagga taacccattc tacaagaagg 2760
gcggccagct ggtgaaggcc gtcagggtgg agaagaccca ggagagcggc gtcctcctga 2820
ataagaagaa cgcctacaca attgcggaca acggcgatat ggtccgcgtg gacgtcttct 2880
gcaaggtgga taagaagggc aagaatcagt acttcatcgt gcctatctac gcctggcagg 2940
tcgcggagaa catcctcccg gacattgatt gcaagggcta caggattgac gattcataca 3000
cattctgctt ctctctccac aagtacgacc tgatcgcctt ccagaaggat gagaagagca 3060
aggtcgagtt cgcgtactac attaattgcg actccagcaa cggcaggttc tacctcgcct 3120
ggcatgataa gggctccaag gagcagcagt tccgcattag cacccagaat ctggtgctca 3180
tccagaagta ccaggtcaac gagctgggca aggagatcag gccttgccgc ctgaagaaga 3240
ggcctccggt gcgctaatcc ggcggctccc cgaagaagaa gaggaaggtg tccggcggta 3300
gtccaaagaa gaagaggaag gtgtcgggag gtagcccaaa gaagaagagg aaggttgagc 3360
tc 3362
<210> 4
<211> 22
<212> DNA
<213> PCR upstream primer for amplifying plant Nme2Cas9 subjected to codon phytochemical modification
<400> 4
ggcctaccac gcgatctcca gg 22
<210> 5
<211> 22
<212> DNA
<213> PCR downstream primer for amplifying codon-botanically-modified plant Nme2Cas9
<400> 5
gtagcccttc tcgttgagcc gc 22
<210> 6
<211> 22
<212> DNA
<213> PCR upstream primer for amplifying PDS Gene fragment
<400> 6
acatatatga atatgacaga ta 22
<210> 7
<211> 21
<212> DNA
<213> PCR downstream primer for amplifying PDS gene fragment
<400> 7
aacttcacct tctctggcca a 21
<210> 8
<211> 26
<212> DNA
<213> target site sequence
<400> 8
ggcaccatga tatttgccat gccaaa 26
Claims (9)
1. A codon-Plant-modified Plant Nme2Cas9 gene is characterized in that the nucleotide sequence of the codon-Plant-modified Plant Nme2Cas9 gene is shown as SEQ ID No: 1 is shown.
2. An expression cassette comprising the codon-vegetated Plant Nme2Cas9 gene according to claim 1.
3. An expression vector, wherein the expression vector is inserted with the codon-vegetalized Plant Nme2Cas9 gene of claim 1 or the expression cassette of claim 2.
4. A targeting vector, wherein the Plant Nme2Cas9 gene that is codon-modified according to claim 1 or the expression cassette according to claim 2 is inserted into the targeting vector.
5. A transgenic cell, which is transformed with a Plant Nme2Cas9 gene transformed with the codon according to claim 1, the expression cassette according to claim 2, the expression vector according to claim 3 or the targeting vector according to claim 4, using Escherichia coli and/or Agrobacterium tumefaciens as a host cell.
6. The use of the codon-vegetatively modified Plant Nme2Cas9 gene according to claim 1, the expression cassette according to claim 2, the expression vector according to claim 3, the targeting vector according to claim 4 or the transgenic cell according to claim 5 for the editing of a rice genome, wherein said editing of a rice genome comprises splicing the rice genome to obtain transgenic rice or rice parts containing mutation sites.
7. The use according to claim 6, wherein the rice is japonica rice.
8. The use of claim 6, wherein the rice is japonica Nipponbare.
9. A method for obtaining a transgenic rice or rice part containing a mutation site, comprising:
(1) transforming agrobacterium tumefaciens with the targeting vector of claim 4 to obtain agrobacterium tumefaciens transgenic cells;
(2) inducing the rice embryo on a callus induction culture medium to obtain a secondary callus;
(3) contacting and infecting the transgenic cell of the agrobacterium tumefaciens with the secondary callus, and co-culturing the infected secondary callus to obtain the co-cultured secondary callus;
(4) and sequentially screening, seedling and rooting the secondary callus.
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| WO2017222773A1 (en) * | 2016-06-20 | 2017-12-28 | Pioneer Hi-Bred International, Inc. | Novel cas systems and methods of use |
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