CN110592135A - Method for editing rice aroma gene Badh2 by CRISPR/Cas9 - Google Patents
Method for editing rice aroma gene Badh2 by CRISPR/Cas9 Download PDFInfo
- Publication number
- CN110592135A CN110592135A CN201910900591.0A CN201910900591A CN110592135A CN 110592135 A CN110592135 A CN 110592135A CN 201910900591 A CN201910900591 A CN 201910900591A CN 110592135 A CN110592135 A CN 110592135A
- Authority
- CN
- China
- Prior art keywords
- cas9
- crispr
- rice
- badh2
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for editing rice aroma gene Badh2 by CRISPR/Cas9, which respectively takes the No. 2 exon and No. 7 exon of rice Badh2 gene as target sites, respectively designs conservative specific sequences, and constructs the target sites into an expression vector of CRISPR/Cas9 after Oligo dimer is prepared. The constructed plasmid vector utilizes the primers SG-SR3 and PUV4-R to carry out PCR amplification, the sequence of a PCR product is determined to be constructed into an expression vector, the expression vector is transformed into agrobacterium-infected EHA105, and then rice callus is utilized to carry out genetic transformation. The transgenic mutant line obtained by the method can be separated into homozygous lines without transgenic markers from transgenic offspring, the mutation characteristic can be stably inherited to the offspring, the fragrance components are obviously higher than those of wild type control, and the fragrance character is obviously improved.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, relates to a plant transgenic technology and crop genetic breeding, and particularly relates to a method for editing a rice aroma gene Badh2 by using CRISPR/Cas 9.
Background
The rice is an important grain crop in China, and the fragrant rice is deeply favored by consumers due to the unique fragrance and has higher production and market values. Therefore, the aroma character of rice becomes an important index for measuring the quality of rice, and is an important content of rice quality improvement research, and the discovery and creation of high-quality fragrant rice resources are important ways for developing rice quality improvement research.
Since the 70 s of the last century, many researchers have conducted extensive and intensive studies on the aroma components of scented rice, Yajima et al reported in 1979 that scented rice contains 114 kinds of volatile compounds, Buttery et al found that 2-acetyl-1-pyrroline (2-AP) is a main component of the aroma of scented rice, and 2-AP was detected in various tissues of the scented rice variety except for the root system. The current research shows that the rice fragrance is mainly regulated and controlled by a recessive gene Badh2 on the 8 th chromosome of rice. The gene consists of 15 exons and 14 introns, wherein the 8 th exon encodes an VTELGGKSP structural domain, the 9 th exon encodes a 28 cysteine structural domain, the two conserved domains are respectively BAD1 and BAD2 of beta-acetaldehyde dehydrogenase, and EGCRLGSVVS encoded by the 10 th exon is also well conserved in BAD protein and is found in rice with a non-odor type. It was found that in some varieties of fragrant rice such as Suyunuo, Thailand jasmine type fragrant rice and Indian Pasma base type fragrant rice, translation is terminated prematurely due to a frame shift mutation caused by 8bp base deletion and 3 SNPs difference on the 7 th exon of Badh2 gene; in some fragrant rice varieties in China, 7bp base deletion on the No. 2 exon of the Badh2 gene is found, so that mutation occurs in the Badh2 gene, and fragrance is generated. Since frame shift mutation occurs in exon7 or exon2 of the coding region of the Badh2 gene in oryza sativa, translation of the encoded acetaldehyde dehydrogenase is terminated early, and a domain with a conserved function cannot be translated. Therefore, the dehydrogenation reaction cannot be completed, and thus 2-acetyl-1-pyrroline (2AP) is accumulated to generate fragrance. Zhangli et al identified 86 rice germplasm resources with aroma, with most of the aroma traits controlled by the Badh2 allele. Therefore, the Badh2 gene is a main gene for regulating fragrance of fragrant rice, and DNA genetic information of the gene is changed in an artificial mutation mode, so that a novel fragrant germplasm resource can be created.
At present, the conventional method for identifying the rice fragrance is to directly judge by artificial olfaction or manually judge according to the odor after boiling tissues to be detected in water or KOH solution[8]Alternatively, the flavor can be determined by component detection using a gas chromatograph. The methods need a great deal of manpower, material resources and time in the process of detecting the fragrance, and the detection result is not accurate due to individual difference, so that the utilization of the methods in the breeding process is limited. With the development of biotechnology, the breeding efficiency can be improved and the cultivation of fragrant materials can be accelerated by using methods such as molecular marker-assisted selection, transgene suppression expression, gene editing and the like. By utilizing the molecular characteristics of the Badh2 gene in the aromatic variety, the developed SNP marker can be used for molecular marker-assisted selection of the aromatic gene, thereby improving the breeding efficiency. The expression of the Badh2 gene in a transgenic plant is reduced by an RNAi technology, so that the 2-AP can not be degraded; chen and the like drive the expression of the artificial microRNA in the transgenic plant by using the ubiquitin promoter of corn, so that the expression of the Badh2 gene in the transgenic plant is reduced, and the 2-AP content in the progeny seed is obviously improved. In recent years, with the development and maturation of gene editing technology, it has become one of the effective means to improve target trait genes by using gene editing technology. Transcription activator-like effector nuclease (TALEN) technology, Zinc Finger Nuclease (ZFN) technology, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) are considered as effective gene editing tools. Therefore, the gene editing technology is used for improving the aroma character of the rice, and the breeding process of the fragrant rice can be accelerated.
Disclosure of Invention
The invention utilizes CRISPR/Cas9 gene editing technology to mutate rice aroma gene Badh2, the 2-AP content of the gene editing strain is obviously higher than that of non-transgenic control, and new germplasm resources are provided for the utilization of aroma rice.
The invention is realized by the following technical scheme:
a method for editing rice aroma gene Badh2 by CRISPR/Cas9, comprising the following steps:
1) respectively designing target point joint primers by using the 2 nd exon and 7 th exon target sites of the rice Badh2 gene;
2) preparing target site dimers by using the obtained target site linker primers respectively;
3) constructing target site dimers into a CRISPR/Cas9 expression vector;
4) and (3) transforming the constructed CRISPR/Cas9 expression vector into an agrobacterium competent cell EHA105 for genetic transformation.
Further, the target joint primer of the No. 2 exon is shown in SEQ ID NO. 1-2.
Further, the target joint primer of the No. 7 exon is shown in SEQ ID NO. 3-4.
Further, the dimer preparation system is 20 μ L: contains 18 mu L of Buffer Aneal, and 1 mu L of each upstream primer and downstream primer; the reaction procedure is as follows: after heating at 95 ℃ for 3min, the temperature was slowly decreased to 20 ℃ at 0.2 ℃/sec.
Further, the construction of the expression vector specifically comprises the following steps: the 10 μ L system contains 2 μ L of CRISPR/Cas9 vector, 1 μ L of target site dimer, 1 μ L of Enzyme Mix, and H2O6. mu.L was reacted at 20 ℃ for 60 min.
In another aspect of the invention, the method for editing the rice aroma gene Badh2 by using the CRISPR/Cas9 is applied to rice Badh2 gene mutation breeding.
The invention has the beneficial effects that:
the invention utilizes CRISPR/Cas9 gene editing technology, obtains different types of transgenic mutant strains through the directional editing of Badh2 gene, can separate homozygous strains without transgenic markers from transgenic offspring through PCR analysis and sequencing identification, and can stably inherit the mutant characteristics to the offspring, and the detection result of 2-AP content shows that the fragrance components of the transgenic strains are obviously higher than the wild type control, and the fragrance character is obviously improved.
Drawings
FIG. 1 is a schematic diagram showing the editing structure of exon2 gene of Badh2 gene;
FIG. 2 is a schematic diagram showing the editing structure of exon7 gene of Badh2 gene;
FIG. 3 is a type analysis of mutation of exon2 of the Badh2 gene;
FIG. 4 is a 7 th exon mutation type analysis of Badh2 gene;
FIG. 5 is T1PCR detection of hygromycin Hpt gene and nuclease Cas9 gene in the generation gene editing strain; a: the Hpt gene PCR detection result; b: cas9 gene detection results;
FIG. 6 is the 2-AP content in the gene-editing line.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following embodiment of the invention takes late japonica rice varieties Jia 58 and Xiushui 134 mainly popularized in Zhejiang province as materials, and inserts a plant expression vector T-DNA fragment into a rice genome by utilizing an agrobacterium-mediated genetic transformation method. The transgenic material is planted in a transgenic test area of a Yang du test base of agricultural academy of sciences of Zhejiang province, and conventional planting and field management are adopted.
Example 1 Gene editing vector construction
A DNA sequence of Nipponbare is taken as a template sequence, a No. 2 exon and a No. 7 exon of a rice Badh2 gene (Os08g32870) are taken as target sites, and conserved specific sequences are designed respectively through Blast sequence comparison, wherein Oligo-up1 and Oligo-lw1 are used as No. 2 exon design target point connectors, and Oligo-up2 and Oligo-lw2 are used as No. 7 exon design target point connectors. After the Oligo dimer was prepared, it was constructed into CRISPR/Cas9 expression vector (see fig. 1 and 2). The constructed plasmid vector utilizes primers SG-SR3 and PUV4-R to carry out PCR amplification, PCR products are sequenced to confirm that the sgRNA sequence is constructed in the expression vector, and the sgRNA sequence is transformed into agrobacterium-infected EHA105 to carry out genetic transformation.
Oligo dimer was prepared (20. mu.L system) containing 18. mu.L of BufferAneal, 1. mu.L of each of the upstream and downstream primers. After heating at 95 ℃ for 3min, the temperature was slowly decreased to 20 ℃ at 0.2 ℃/sec.
Constructing Oligo dimer into CRISPR/Cas9 vector, wherein the 10 mu L system comprises 2 mu L of CRISPR/Cas9 vector, 1 mu L of Oligo dimer, 1 mu L of Enzyme Mix, and adding H2O6 μ L was reacted at 20 ℃ for 60 min.
Wherein:
Oligo-up1：TGTGTGGCGATTGCGCGGAGGTACT；
Oligo-lw1：AAACAGTACCTCCGCGCAATCGCCA；
Oligo-up2：TGTGTGATGGCTTCAGCTGCTCCTA；
Oligo-lw2：AAACTAGGAGCAGCTGAAGCCATCA。
example 2 target analysis of Gene editing lines
The verified plasmid vector is transformed into an agrobacterium tumefaciens competent cell EHA105, rice Jia 58 and Xiushui 134 callus are infected and transformed, and a transgenic positive regeneration plant is obtained through hygromycin screening.
The rice leaf genome DNA is extracted by a CTAB method, and positive transgenic plants are identified by PCR by using Hpt gene primers (Hpt-F and Hpt-R) and Cas9 gene primers (Cas9-F and Cas 9-R). PCR reaction (20. mu.L): 2.0 XPCR Buffer 10. mu.L, upstream and downstream primers 0.5. mu.L each, DNA 2. mu.L, plus H2O7. mu.L. PCR reaction procedure: extension at 95 ℃ for 3min, 95 ℃ for 30sec, 60 ℃ for 30sec, 72 ℃ for 30sec, after 35 cycles, 72 ℃ for 5 min.
The PCR reaction product was electrophoresed on a 1.5% agarose gel and photographed under an ultraviolet lamp. According to the characteristics of the target site of the positive transgenic plant, primers of exon2 and exon7 are used for amplifying the adjacent sequence of the target site by using a high-fidelity KOD Fx enzyme. PCR reaction (20. mu.L): 2.0 XKod Fxbuffer 10. mu.L, dNTP (2mM) 2. mu.L, primers 1.0. mu.L each, Kod Fx 0.5. mu.L, plus H2O4.5. mu.L. PCR reaction procedure: extension at 95 ℃ for 3min, 95 ℃ for 30sec, 60 ℃ for 30sec, 68 ℃ for 30sec, 35 cycles later, 68 ℃ for 5 min. Subjecting the PCR reaction product to electrophoresis in 1.5% agarose gel, cutting and recovering the gel, sequencing, and analyzing the transgenic T according to the sequencing result0Plant generation target pointThe mutation type (primer sequence is shown in Table 1).
Various primers and sequences used in Table 1
The agrobacterium-mediated rice transgenic method is utilized to transform calli of Jia 58 and Xishui 134, and 26 independent transformation strains and 18 independent transformation strains are obtained through hygromycin selection. In order to identify the mutation condition of the transgenic strain in the 2 nd exon and the 7 th exon of the Badh2 gene respectively, the target adjacent sequences of the Badh2 gene are subjected to PCR amplification and sequencing by using a high fidelity enzyme through an exon2-F primer pair and an exon2-R primer pair and an exon7-F primer pair and an exon7-R primer pair respectively. The result shows that the 26 Jia 58 transgenic lines have 10 strains at the exon2, wherein 2 strains without mutation and 8 transgenic mutant lines have 5 different mutation modes, and different single bases are inserted into different mutation sites; the transgenic lines of exon7 had 16 strains, 9 of which were not mutated and 7 of which were mutated in 5 ways, all of which were fragments or base deletions (see FIGS. 3 and 4). Thus, 15 strains of the Jia 58 gene editing line were obtained altogether. The transgenic lines of 18 Xiuhui 134 are 8 lines at the 2 nd exon, wherein 3 lines have no mutation, and 5 lines have single base insertion; there were 10 strains at exon7, of which 4 were not mutated and 6 were all fragment-deleted (see FIGS. 3 and 4). Therefore, the Xiushui 134 gene editing line 11 strain was obtained altogether.
Example 3 marker-free transgenic line identification
To obtain a gene-editing line without transgenic components, two T's of Xiushui 134 gene editing were selected0The generation lines Bh57 and Bh53 were subjected to further analysis, wherein Bh57 caused a frame shift mutation for single base insertion and translation of Badh2 protein was terminated prematurely; bh53 is a fragment deletion that encodes an amino acid that is 6 amino acids shorter than the wild type. Planting 24 strains in each strain, extracting single strain genome DNA, respectively utilizing hygromycin selective marker Hpt gene primer pair and nuclease Cas9 gene primer pair, and analyzing the DNA in T through PCR detection1In the lines of the generations.
The results show that the molecular weight distribution in Bh524T of 71In the generation individual strains, 5 strains without Hpt and Cas9 genes are detected in total (see figure 5), and the sequencing result shows that the mutation characteristics of the strains are similar to T0Generation is consistent; at the same time, 24T at Bh531In the generation individual strains, 11 strains without Hpt and Cas9 genes are detected in total (see figure 5), and the sequencing result shows that the mutation characteristics of the strains are similar to T0The generations are consistent. Thus, can be selected from T1Gene editing strains without transgenic components are separated from the generation strains, and the mutation characteristics of the strains at target sites and T of the strains0The generation is consistent, and the mutation of gene editing can be stably inherited to the next generation. From two Ts according to field performance12 individuals are selected from the generation strain respectively and propagated, and are named as Bh1, Bh2, Bh3 and Bh4 respectively, and the content of the aroma substance 2-AP in the brown rice is detected.
Example 4 determination of Rice aroma 2-AP content
And respectively taking 30g of mature seeds of the gene editing plant and the control, shelling and grinding the brown rice into rice flour, and then determining the content of the aromatic substance 2-acetyl-1-pyrroline (2-AP). Taking 2,4, 6-trimethylpyridine as an internal standard (the concentration is 229.25ng/ml), weighing 400mg of rice flour sample each time, repeating the steps for three times, placing the rice flour sample in a10 ml narrow-mouth glass bottle, adding 0.8ml of ethanol leaching reagent containing the internal standard, leaching the rice flour sample in an oven at 80 ℃ for 3 hours, taking out the rice flour sample, standing the rice flour sample to room temperature, filtering the rice flour sample by using a 0.22 mu m disposable needle filter membrane, taking 150 mu l of the rice flour sample in a lining tube, placing the rice flour sample in a 2ml sample bottle, measuring the rice flour sample by using a GC-MS gas phase mass spectrometer (Shimadzu corporation), and analyzing detection data by Excel.
To determine the content of the fragrant substance 2-AP in the Gene-editing lines, two Ts from example 3 were used according to field Performance1And 2 individuals are selected from the generation strain system to breed, are respectively named as Bh1, Bh2, Bh3 and Bh4, detect the content of aroma substances 2-AP in the brown rice, are milled into brown rice flour after shelling, and are determined by a GC-MS method, wherein Bh1 and Bh2 are single base insertions and cause frame shift mutation, and Bh3 and Bh4 are 18bp fragment deletions to obtain deletion mutation.
The 2-AP content detection result shows that the 2-AP content of all gene editing strains is obviously higher than that of a wild type control variety (0.046 mu g/g), wherein the 2-AP content of Bh2 and the 2-AP content of Bh3 are respectively 0.347 mu g/g and 0.332 mu g/g and are extremely obviously higher than that of the wild type control; whereas the 2-AP of Bh1 and Bh4 was 0.309 and 0.295. mu.g/g, respectively, which was significantly higher than the wild-type control (see FIG. 6). Therefore, by mutating the rice Badh2 gene by gene editing technology, a flavor material with improved flavor properties can be obtained.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Zhejiang province academy of agricultural sciences
<120> method for editing rice aroma gene Badh2 by CRISPR/Cas9
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgtgtggcga ttgcgcggag gtact 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaacagtacc tccgcgcaat cgcca 25
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgtgtgatgg cttcagctgc tccta 25
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aaactaggag cagctgaagc catca 25
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggccatttgt ctgcagaat 19
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcccagtcac gacgttgtaa 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
catccagaaa gcccaggtgt 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gttcctggtc cacgtacata 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gggtgtcacg ttgcaagacc 20
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgcctccgc tcgaagtagc 20
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
caccctctgc ttctgcctct 20
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cagccatgct tccaacttat tc 22
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tggtcttcct tcaggtgtgc 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tccagtgaaa caggctgtca 20
Claims (7)
1. A method for editing rice aroma gene Badh2 by CRISPR/Cas9, which comprises the following steps:
1) respectively designing target point joint primers by taking the No. 2 exon and the No. 7 exon of the rice Badh2 gene as target sites;
2) preparing target site dimers by using the obtained target site linker primers respectively;
3) constructing target site dimers into a CRISPR/Cas9 expression vector;
4) the constructed CRISPR/Cas9 expression vector is transformed into agrobacterium competent cell EHA105, and then rice callus is used for genetic transformation.
2. The method for editing rice aroma gene Badh2 with CRISPR/Cas9 as claimed in claim 1, wherein the target joint primer of exon2 is shown as SEQ ID No. 1-2.
3. The method for editing rice aroma gene Badh2 with CRISPR/Cas9 as claimed in claim 1, wherein the target joint primer of exon7 is shown as SEQ ID No. 3-4.
4. The method for editing rice aroma gene Badh2 by CRISPR/Cas9 according to claim 1, wherein the dimer preparation system is 20 μ L: contains 18. mu.L of Buffer Aneal, and 1. mu.L of each of the upstream and downstream primers.
5. The method for editing rice aroma gene Badh2 by CRISPR/Cas9 according to claim 1, wherein the dimer preparation reaction program is as follows: after heating at 95 ℃ for 3min, the temperature was slowly decreased to 20 ℃ at 0.2 ℃/sec.
6. The method for editing rice aroma gene Badh2 by CRISPR/Cas9 according to claim 1, wherein the expression vector is specifically constructed as follows: the 10 μ L system contains 2 μ L of CRISPR/Cas9 vector, 1 μ L of target site dimer, 1 μ L of Enzyme Mix, and H2O6. mu.L was reacted at 20 ℃ for 60 min.
7. The use of the method for editing rice aroma gene Badh2 according to CRISPR/Cas9 of claim 1 in rice Badh2 gene mutation breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910900591.0A CN110592135A (en) | 2019-09-23 | 2019-09-23 | Method for editing rice aroma gene Badh2 by CRISPR/Cas9 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910900591.0A CN110592135A (en) | 2019-09-23 | 2019-09-23 | Method for editing rice aroma gene Badh2 by CRISPR/Cas9 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110592135A true CN110592135A (en) | 2019-12-20 |
Family
ID=68862526
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910900591.0A Pending CN110592135A (en) | 2019-09-23 | 2019-09-23 | Method for editing rice aroma gene Badh2 by CRISPR/Cas9 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110592135A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111411123A (en) * | 2020-04-08 | 2020-07-14 | 上海市农业生物基因中心 | Method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using CRISPR/Cas9 system and expression vector |
| CN113215187A (en) * | 2020-01-21 | 2021-08-06 | 江苏省农业科学院 | Method for rapidly obtaining fragrant rice material by using CRISPR/Cas9 technology |
| CN113215156A (en) * | 2021-05-11 | 2021-08-06 | 北京市农林科学院 | Method for efficiently creating fragrant corn by using CRISPR/Cas9 technology |
| CN113481214A (en) * | 2021-08-27 | 2021-10-08 | 深圳市兆农农业科技有限公司 | Method for creating long-grain fragrance type rice germplasm resources |
| CN113604501A (en) * | 2021-08-24 | 2021-11-05 | 北大荒垦丰种业股份有限公司 | Gene editing method of flavor control gene of indica rice improved line and application thereof |
| 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 |
| CN117417957A (en) * | 2023-10-23 | 2024-01-19 | 黄冈市农业科学院 | Method for increasing rice fragrance |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505979A (en) * | 2015-11-28 | 2016-04-20 | 湖北大学 | Method for acquiring aromatic rice strain by targeting Badh2 gene via CRISPR/Cas9 gene editing technology |
| CN105543228A (en) * | 2016-01-25 | 2016-05-04 | 宁夏农林科学院 | Method for transforming rice into fragrant rice rapidly |
| CN106676130A (en) * | 2016-12-30 | 2017-05-17 | 华智水稻生物技术有限公司 | Paddy rice BADH2 gene site-directed mutagenesis method through using CRISPR-CAS9 technology |
| CN108913714A (en) * | 2018-07-05 | 2018-11-30 | 江西省超级水稻研究发展中心 | A method of BADH2 gene, which is knocked out, using CRISPR/Cas9 system formulates fragrant rice |
-
2019
- 2019-09-23 CN CN201910900591.0A patent/CN110592135A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505979A (en) * | 2015-11-28 | 2016-04-20 | 湖北大学 | Method for acquiring aromatic rice strain by targeting Badh2 gene via CRISPR/Cas9 gene editing technology |
| CN105543228A (en) * | 2016-01-25 | 2016-05-04 | 宁夏农林科学院 | Method for transforming rice into fragrant rice rapidly |
| CN106676130A (en) * | 2016-12-30 | 2017-05-17 | 华智水稻生物技术有限公司 | Paddy rice BADH2 gene site-directed mutagenesis method through using CRISPR-CAS9 technology |
| CN108913714A (en) * | 2018-07-05 | 2018-11-30 | 江西省超级水稻研究发展中心 | A method of BADH2 gene, which is knocked out, using CRISPR/Cas9 system formulates fragrant rice |
Non-Patent Citations (2)
| Title |
|---|
| 孙慧宇 等: "利用CRISPR/Cas9技术编辑Badh2基因改良粳稻香味", 《华北农学报》 * |
| 邵高能 等: "利用CRISPR/CAS9技术编辑水稻香味基因Badh2", 《中国水稻科学》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215187A (en) * | 2020-01-21 | 2021-08-06 | 江苏省农业科学院 | Method for rapidly obtaining fragrant rice material by using CRISPR/Cas9 technology |
| CN111411123A (en) * | 2020-04-08 | 2020-07-14 | 上海市农业生物基因中心 | Method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using CRISPR/Cas9 system and expression vector |
| CN111411123B (en) * | 2020-04-08 | 2023-04-11 | 上海市农业生物基因中心 | Method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using CRISPR/Cas9 system and expression vector |
| 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 |
| CN113215156A (en) * | 2021-05-11 | 2021-08-06 | 北京市农林科学院 | Method for efficiently creating fragrant corn by using CRISPR/Cas9 technology |
| CN113604501A (en) * | 2021-08-24 | 2021-11-05 | 北大荒垦丰种业股份有限公司 | Gene editing method of flavor control gene of indica rice improved line and application thereof |
| CN113481214A (en) * | 2021-08-27 | 2021-10-08 | 深圳市兆农农业科技有限公司 | Method for creating long-grain fragrance type rice germplasm resources |
| CN117417957A (en) * | 2023-10-23 | 2024-01-19 | 黄冈市农业科学院 | Method for increasing rice fragrance |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110592135A (en) | Method for editing rice aroma gene Badh2 by CRISPR/Cas9 | |
| Zhang et al. | Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat | |
| CN109097387B (en) | Method for creating purple tomato mutant by using CRISPR/Cas9 gene editing system and application | |
| Sheng et al. | Improvement of the rice “easy-to-shatter” trait via CRISPR/Cas9-mediated mutagenesis of the qSH1 gene | |
| US20210189419A1 (en) | Loss-of-function gene of dominant gene eifiso4e-s resistant to tvbmv and uses thereof | |
| EP4342290A2 (en) | Tobacco plants having increased nitrogen efficiency and methods of using such plants | |
| Kumar et al. | Agrobacterium-and a single Cas9-sgRNA transcript system-mediated high efficiency gene editing in perennial ryegrass | |
| CN109971763A (en) | Florescence control gene C MP1 and relevant carrier and its application | |
| CN109371055B (en) | Method for breeding broad-spectrum potato virus Y resistant tobacco plant | |
| CN115851824B (en) | Method for reducing height of semen ginkgo waxy plants, improving yield and shortening growth period, SD1 gene core promoter and application | |
| CN113817033B (en) | Application of ZmELF3.1 protein and its functional deletion mutant in regulating and controlling crop aerial root number or layer number | |
| JP2021501602A5 (en) | ||
| JP7308870B2 (en) | Method for producing dry tobacco material | |
| CN111411123B (en) | Method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using CRISPR/Cas9 system and expression vector | |
| WO2021058485A1 (en) | Generation of haploids based on mutation of sad2 | |
| EP3975694A1 (en) | Compositions and methods for producing tobacco plants and products having reduced or eliminated suckers | |
| CA3131193A1 (en) | Methods and compositions for generating dominant short stature alleles using genome editing | |
| CN113544277A (en) | Compositions and methods for driving T1 event diversity | |
| Yang et al. | A method for generating genome edited plant lines from CRISPR-transformed Shanxin poplar plants | |
| CN114058639B (en) | Method for improving amylose content of rice by mutating OsWaxy gene by single base gene editing technology | |
| WO2023165627A1 (en) | Application of polynucleotide, protein and biological material in regulation and control of plant tuber development, and related product and cultivation method therefor | |
| CN115029359B (en) | Improvement method considering both crop yield and quality | |
| EP3975701A1 (en) | Methods and compositions for generating dominant short stature alleles using genome editing | |
| CN115960950A (en) | Tomato SlBBX31 gene, inDel marker and application | |
| CN117143911A (en) | Method for increasing phosphorus absorption rate of soybean |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191220 |