CN105505979A - Method for acquiring aromatic rice strain by targeting Badh2 gene via CRISPR/Cas9 gene editing technology - Google Patents
Method for acquiring aromatic rice strain by targeting Badh2 gene via CRISPR/Cas9 gene editing technology Download PDFInfo
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Abstract
The invention discloses a method for acquiring an aromatic rice strain by targeting a Badh2 gene via CRISPR/Cas9 gene editing technology. The method comprises the following steps: separately targeting sequences, recognizable by Cas9, of every exon and intron of an aromatic gene by using the CRISPR/Cas9 gene editing technology and then cutting a genomic DNA sequence to initiate DNA restoration so as to obtain an afunctional Badh2 gene; with the callus of Indica rice, japonica rice or glutinous rice as a receptor material of genetic transformation and an mature embryo, young ear, ovary or the like as explant, carrying out induction so as to obtain a diploid callus, introducing a targeting vector into cells of the callus by using an Agrobacterium mediated transformation method, screening and identifying positive plants and separating the plants from a T1 colony so as to obtain an aromatic rice strain; and with anther, pollen, unfertilized ovary or the like as explant, carrying out induction so as to obtain a haploid callus, introducing the targeting vector into cells of the callus by using the Agrobacterium mediated transformation method, screening a positive callus, reduplicating the positive callus by using colchicine, carrying out differentiation to form seedlings and identifying genetic transformation positive plants so as to eventually obtain the aromatic rice strain.
Description
Technical field
The present invention relates to gene editing technical field, particularly relate to a kind of method obtaining fragrant rice strain with CRISPR/Cas9 gene editing technology target practice Badh2 gene.
Background technology
Transcriptional activation sample effector nuclease (transcriptionactivator-likeeffectornuclease, TALEN) technology, Zinc finger nuclease (Zinc-fingernuclease, ZFN) and the short palindrome in Regularity interval to repeat (clusteredregulatoryinterspacedshortpalindromicrepeat, CRISPR) technology be the three large technology in current genome editor field.At present, TALEN and ZFN is two kinds of site-directed mutagenesis techniques of development comparative maturity, but these two kinds of technology are comparatively loaded down with trivial details at the nuclease of carrier construction process identifiable design specific site, and each site needs structure two corresponding nucleases.
Summary of the invention
Based on the technical problem that background technology exists, the present invention proposes a kind of method obtaining fragrant rice strain with CRISPR/Cas9 gene editing technology target practice Badh2 gene.
The present invention proposes a kind of method obtaining fragrant rice strain with CRISPR/Cas9 gene editing technology target practice Badh2 gene, comprise the following steps:
S1, utilize CRISPR/Cas9 gene editing technology each exon of scent gene and intron place to be practiced shooting by the sequence that Cas9 identifies respectively, then repair causing DNA after genomic dna sequence cutting, thus produce deletion mutantion, obtain the Badh2 gene of afunction;
S2, using the acceptor material of the callus of japonica rice, long-grained nonglutinous rice and glutinous rice as genetic transformation, by mature embryo, young fringe and ovary etc. for explant induction obtains diploid callus, obtain monoploid callus with flower pesticide, pollen and Unfertilized Ovaries etc. for explant induction, with agrobacterium-mediated transformation, targeting vector is imported callus cell;
S3, imports to targeting vector in the cell of diploid callus, screens positive callus, seedling differentiation, and qualification genetic transformation positive plant, is separated from T1 colony and obtains fragrant rice strain; Imported to by targeting vector in the cell of monoploid callus, screen positive callus, double by colchicine, seedling differentiation, qualification genetic transformation positive plant, finally obtains fragrant rice strain.
Preferably, described gene knockout target spot should be located near initiator codon or the exon region in its downstream, target sequence is generally made up of 19 bases, must be that bases G is as the start signal of transcribing before target sequence, and the PAM sequence of target sequence end must be NGG, and target sequence must have uniqueness.
Preferably, utilize Agrobacterium competence EHA105 to be transformed into Agrobacterium the described vector plasmid DNA built, and take out the object fragment of plasmid checking expression vector.
Preferably, the callus of described japonica rice, long-grained nonglutinous rice and glutinous rice, as the acceptor material of genetic transformation, realizes genetic transformation with agrobacterium-mediated transformation, obtains fragrance rice plant, below for Hubei Province early 17:
(1) with Hubei Province early 17 mature embryos, the callus (diploid) of explant such as children fringe and ovary etc. is acceptor, agrobacterium-mediated transformation is utilized by targeting vector to be transferred in diploid recipient cell, seedling differentiation again after screening, filters out the plant of the band fragrance isozygotied from T1 colony.
(2) early 17 flower pesticide is carried out for material with Hubei Province, the explant such as pollen and Unfertilized Ovaries callus (monoploid) is acceptor material, agrobacterium-mediated transformation is utilized to import in monoploid callus by the targeting vector of scent gene Badh2, after hygromycin selection, colchicine is utilized to double process, be divided into plant again, obtain the zygoid plant of fragrant gene like this.
The CRISPR/Cas9 mentioned in the present invention is third generation gene editing technology, its principle of work is that bacterium is when resisting the foreign DNA such as phage of degraded invasion, under the regulation and control of leader, CRISPR is transcribed into long RNA precursor, then be processed into a series of short ripe crRNA containing conservative tumor-necrosis factor glycoproteins and transcribed spacer, finally identify and be attached on the exogenous DNA array with its complementation and play shearing action.The each crRNA for specific site of CRISPR/Cas9 system only has tens bases, and whole carrier is less.And TALEN and ZFN technological operation is more loaded down with trivial details, cost intensive, particularly TALEN technology needs a large amount of molecular clonings and sequencing procedures in building process, and common laboratory all cannot operate.And CRISPR/Cas9 system only needs to design target practice site, to design two mononucleotide chains, replacing with needing the object fragment of practicing shooting, building corresponding carrier.This technology not only has expansibility, and the process of carrier construction only needs a few step just can complete, simple to operation, common laboratory all can operate, the present invention not only with traditional explant callus for acceptor material, simultaneously also with flower pesticide, the callus (monoploid) of the explants such as Unfertilized Ovaries is acceptor material, the positive plant that can obtain isozygotying will be doubled after gene transformation, compared with the cross breeding method of routine, the method saves manpower and materials and time cost greatly.
Embodiment
Below in conjunction with specific embodiment, further explanation is done to the present invention.
A kind of method obtaining fragrant rice strain with CRISPR/Cas9 gene editing technology target practice Badh2 gene that the present invention proposes, comprises the following steps:
S1, utilize CRISPR/Cas9 gene editing technology each exon of scent gene and intron place to be practiced shooting by the sequence that Cas9 identifies respectively, then repair causing DNA after genomic dna sequence cutting, thus generation deletion mutantion, obtain the Badh2 gene of afunction;
S2, using the acceptor material of the callus of japonica rice, long-grained nonglutinous rice and glutinous rice as genetic transformation, by mature embryo, young fringe and ovary etc. for explant induction obtains diploid callus, obtain monoploid callus with flower pesticide, pollen and Unfertilized Ovaries etc. for explant induction, with agrobacterium-mediated transformation, targeting vector is imported callus cell;
S3, targeting vector is imported diploid callus cell, screening, qualification positive plant, be separated from T1 colony and obtain fragrant rice strain; Imported to by targeting vector in the cell of monoploid callus, screen positive callus, double by colchicine, seedling differentiation, qualification genetic transformation positive plant, finally obtains fragrant rice strain.
In the present invention, the target sequence of described gene editing should be located near initiator codon or in the exon in its downstream, target sequence is generally made up of 19 bases, must be that bases G is as identification start signal before target sequence, and target sequence PAM sequence below must be NGG, and target sequence must have uniqueness; Utilize Agrobacterium competence EHA105 to be transformed into Agrobacterium the described vector plasmid DNA built, and take out the object fragment of plasmid checking expression vector.
In the present invention, the callus of described japonica rice, long-grained nonglutinous rice and glutinous rice, as the acceptor material of genetic transformation, realizes genetic transformation with agrobacterium-mediated transformation, obtains fragrance rice plant, below for Hubei Province early 17:
(1) with Hubei Province early 17 mature embryos, the callus (diploid) of explant such as children fringe and ovary etc. is acceptor, agrobacterium-mediated transformation is utilized by targeting vector to be transferred in diploid recipient cell, seedling differentiation again after screening, filters out the plant of the band fragrance isozygotied from T1 colony.
(2) early 17 flower pesticide is carried out for material with Hubei Province, the explant such as pollen and Unfertilized Ovaries callus (monoploid) is acceptor material, agrobacterium-mediated transformation is utilized to import in monoploid callus by scent gene Badh2 targeting vector, after hygromycin selection, colchicine is utilized to double process, be divided into plant again, obtain fragrant zygoid plant like this.
Genetic transfoumation is also called transgenic technology, it is the new technology that 20 century 70s grow up from molecular genetics basis, it is application DNA recombinant technology, comprehensively have employed physics, chemistry and biology techniques is autotelic foreign gene or DNA fragmentation is imported in the cell of recipient plant.At present, Application comparison mainly contains agrobacterium-mediated transformation and particle bombardment widely, and agrobacterium-mediated transformation is widely used on plant transgenic technology, and the method is structurally complete when importing exogenous dna fragment, transformation efficiency is higher, and technological operation is relatively simple.And Bombardment-Mediated Transformation cost is higher, if when exogenous sequences DNA is larger, be difficult to be shifted by the method, so agrobacterium-mediated transformation is the first-selection of current Genetic Transformation in Higher Plants, as the acceptor material of rice transformation, traditional method is all with mature embryo, and the diploid callus of ovary and young fringe explant is acceptor material, although transformation efficiency is higher, but offspring needs selfing just can isolate positive plant, and time cost is high.
CRISPR/Cas9 gene editing technology is utilized to obtain Badh2 gene in the present invention, as compared to TALEN with ZFN technology, operating process is simple and convenient, cost-saving, common laboratory all can operate, and imports in diploid and haploid cell tissue respectively by scent gene Badh2 targeting vector, compared with the cross-breeding of routine with agrobacterium-mediated transformation, greatly having saved time cost, is the molecular breeding method of the fragrant rice strain of a kind of acquisition newly.
The above; be only the present invention's preferably embodiment; but protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to according to technical scheme of the present invention and inventive concept thereof and replace or change, all should be encompassed within protection scope of the present invention.
Claims (4)
1. obtain a method for fragrant rice strain with CRISPR/Cas9 gene editing technology target practice Badh2 gene, it is characterized in that, comprise the following steps:
S1, utilize CRISPR/Cas9 gene editing technology each exon of scent gene and intron place to be practiced shooting by the sequence that Cas9 identifies respectively, then repair causing DNA after genomic dna sequence cutting, thus produce deletion mutantion, obtain the Badh2 gene of afunction;
S2, using the acceptor material of the callus of japonica rice, long-grained nonglutinous rice and glutinous rice as genetic transformation: be explant induction acquisition diploid callus by mature embryo, young fringe and ovary etc., obtain monoploid callus with flower pesticide, pollen and Unfertilized Ovaries etc. for explant induction, with agrobacterium-mediated transformation, targeting vector is imported callus cell;
S3, imports to targeting vector in the cell of diploid callus, screens positive callus, seedling differentiation, and qualification genetic transformation positive plant, is separated from T1 colony and obtains fragrant rice strain; Imported to by targeting vector in the cell of monoploid callus, screen positive callus, double by colchicine, seedling differentiation, qualification genetic transformation positive plant, finally obtains fragrant rice strain.
2. a kind of method obtaining fragrant rice strain with CRISPR/Cas9 gene editing technology target practice Badh2 gene according to claim 1, it is characterized in that, described gene knockout target sequence should be located near initiator codon or the exon region in its downstream, target sequence is generally made up of 19 bases, must be that bases G is as the start signal of transcribing before target sequence, and target sequence PAM sequence below must be NGG, and target sequence must have uniqueness.
3. a kind of method obtaining fragrant rice strain with CRISPR/Cas9 gene editing technology target practice Badh2 gene according to claim 1, it is characterized in that, utilize Agrobacterium competence EHA105 to be transformed into Agrobacterium the described vector plasmid DNA built, and take out the object fragment of plasmid checking expression vector.
4. a kind of method obtaining fragrant rice strain with CRISPR/Cas9 gene editing technology target practice Badh2 gene according to claim 1, it is characterized in that, the callus of described japonica rice, long-grained nonglutinous rice and glutinous rice is as the acceptor material of genetic transformation, realize genetic transformation with agrobacterium-mediated transformation, obtain fragrance rice plant.
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| CN114127299B (en) * | 2020-06-09 | 2024-01-30 | 山东舜丰生物科技有限公司 | Method for enhancing plant fragrance |
| WO2021249229A1 (en) * | 2020-06-09 | 2021-12-16 | 山东舜丰生物科技有限公司 | Method for enhancing plant aroma |
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