CN113493787A - Method for improving grain traits by blocking or weakening rice OsMIR7695 expression - Google Patents
Method for improving grain traits by blocking or weakening rice OsMIR7695 expression Download PDFInfo
- Publication number
- CN113493787A CN113493787A CN202010269792.8A CN202010269792A CN113493787A CN 113493787 A CN113493787 A CN 113493787A CN 202010269792 A CN202010269792 A CN 202010269792A CN 113493787 A CN113493787 A CN 113493787A
- Authority
- CN
- China
- Prior art keywords
- osmir7695
- rice
- crispr
- expression
- coding 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.)
- Granted
Links
Images
Classifications
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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]
-
- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Adjustment And Processing Of Grains (AREA)
Abstract
The invention belongs to the technical field of plant biology, and particularly relates to a method for improving rice grain traits by blocking or weakening expression of OsMIR7695 in rice. The technical problem to be solved by the invention is to improve the rice grain character. The technical scheme for solving the technical problem is to provide a method for improving the rice grain character by blocking or weakening the expression of OsMIR7695 in rice. Furthermore, the method can block or weaken the expression of OsMIR7695 in rice by knocking out the rice OsMIR7695 coding gene or interfering the action of the OsMIR7695 coding gene expression product. The rice mutant with the increased thousand kernel weight, length or width of the kernel can be obtained by using the method, which shows that the kernel property is greatly improved. The method has simple steps, is easy to operate and has good prospect.
Description
Technical Field
The invention belongs to the technical field of plant biology, and particularly relates to a method for improving rice grain traits by blocking or weakening expression of OsMIR7695 in rice.
Background
Micrornas (mirnas) are a class of non-coding single-stranded RNA molecules that are widely found in eukaryotes and viruses, encoded by endogenous genes, and about 20-24 nucleotides in length. Most of miRNA genes exist in the form of single copy, multiple copies or gene cluster in the genome, are transcribed into primary miRNA (primary miRNA, pri-miRNA) with the length of about 300-1000 bases by RNA polymerase II, are processed into precursor miRNA (pre-miRNA) with the length of about 70-90 bases and containing stem-loop structures, and are cut by Dicer enzyme to become mature miRNA with the length of about 20-24 bases. Mature miRNA is assembled into an RNA-induced silencing complex (RISC), target gene mRNA is identified and integrated in a base complementary pairing mode, and the RISC is guided to degrade the target mRNA (complete complementation) or suppress the translation of the target mRNA (incomplete complementation) according to different complementary degrees, so that the mature miRNA participates in the post-transcriptional gene expression regulation of eukaryotes.
The length of the plant miRNA is about 21nt, most of the plant miRNA and the target mRNA are in complete complementary pairing effect, and the binding region can be located in any region of the target mRNA. In the growth and development process of plants, miRNA plays an important role and participates in the processes of morphogenesis of each organ, phytohormone signal transduction, various biotic and abiotic stress responses and the like. Therefore, the method utilizes abundant miRNA gene resources to improve the genetic traits of plants, and becomes a new development direction for the production and living services of people. However, as more and more plant mirnas are identified and obtained, the biological functions of most mirnas are not clear. For example, the rice OsMIR7695 gene is a rice miRNA discovered based on high-throughput sequencing, the length of the annotated precursor sequence is 487bp, the accession number of the miRBase is MI0025202, and the annotated precursor sequence is located in the interval of 11666716 and 11667202bp of the rice chromosome 1, but no report about the function of the rice OsMIR7695 is found at present.
Recently, a target gene analog (TM) or short tandem target analog (STTM) strategy is used for miRNA molecular function research, and a miRNA derepression influence degree on a target gene is evaluated by obtaining a material interfering (or blocking) a target miRNA, so as to analyze miRNA molecular functions. In addition, RNA function interference research based on CRISPR-Cas13 is reported at present.
CRISPR-Cas9 is a gene editing system, and the creation of a mutant for knocking out plant miRNA encoding genes based on a CRISPR-Cas9 method is reported before. And the CRISPR-Cas12a (CRISPR-Cpf1) system is a new gene editing system discovered in recent years. Different from CRISPR-Cas9, due to the characteristics or advantages that the CRISPR-Cas9 has a PAM recognition site of a T/A enrichment region, guide RNA is a single crRNA molecule with a simple structure, a sticky end is generated, a deleted fragment is large (mostly 6bp-13bp), and the like, the method is expected to have specific advantages for directionally editing miRNA coding genes. However, at present, the work of specifically editing and creating mutant for plant miRNA coding genes based on CRISPR-Cas12a, effectively analyzing the biological functions corresponding to miRNA, and further excavating plant miRNA sites with breeding value is not reported.
Disclosure of Invention
The technical problem to be solved by the invention is to improve the rice grain character.
The technical scheme for solving the technical problem is to provide a method for improving the rice grain character. The method improves the rice grain character by blocking or weakening the expression of OsMIR7695 in rice.
Wherein, the blocking or weakening of the expression of OsMIR7695 in the rice can be performed by knocking out the rice OsMIR7695 coding gene or interfering the effect of the OsMIR7695 coding gene expression product.
The method for interfering the action of the OsMIR7695 expression product comprises at least one of an RNA interference method, an antisense RNA method, a target gene analogue (TM) method short tandem target analogue (STTM) method or a CRSPR-Cas13 method.
The method for knocking out the OsMIR7695 coding gene in the rice genome comprises at least one of a genome editing method, a homologous recombination method and a random insertion mutation method.
The genome editing method described in the above method includes at least one of Meganuclease (Meganuclease), ZFN, TALEN, and CRISPR-Cas.
When the CRISPR-Cas method is used for knocking out the OsMIR7695 coding gene in the rice genome in the method, the method comprises the following steps:
a. designing a guide RNA aiming at the OsMIR7695 coding gene of the rice;
b. constructing a Cas gene editing expression vector capable of expressing the guide crRNA;
c. b, transforming rice by using the expression vector obtained in the step a, and obtaining a transformed plant by using a CRISPR-Cas gene editing system;
d. collecting seeds of the transformed plants, and screening out rice OsMIR7695 coding gene directional editing mutant seeds to obtain the rice mutant with improved grain properties.
Wherein, the CRISPR-Cas method for knocking out the OsMIR7695 coding gene in the rice genome in the method comprises at least one of CRISPR-Cas9, CRISPR-Cas12a or CRISPR-Cas12b and the like.
Further, the nucleotide sequence of the guide RNA for the rice OsMIR7695 coding gene described in the above method is shown in Seq ID No.2 (5'-ATTGTGGTCTTGCCTATGTGGCA-3'). The guide RNA is used in the CRISPR-Cas12a gene editing system.
Wherein the method for improving the rice grain traits is at least one of thousand kernel weight, grain length and grain width.
On the basis, the invention provides the application of the reagent for blocking or weakening the expression of OsMIR7695 in rice in improving the rice grain traits.
Wherein the reagent for blocking or weakening the expression of OsMIR7695 in rice comprises at least one of a reagent for knocking out a rice OsMIR7695 coding gene or a reagent for interfering with an OsMIR7695 coding gene expression product.
The reagent for interfering the action of the OsMIR7695 expression product in the application comprises a reagent for weakening the action of the OsMIR7695 expression product by at least one of an RNA interference method, an antisense RNA method, a CRISPR-Cas13 method, a target gene analogue (TM) method or a short tandem target analogue (STTM) or a CRISPR-Cas13 method.
Wherein, the reagent used for weakening the effect of the OsMIR7695 expression product by the RNA interference method in the application comprises siRNA (Small interfering RNA) aiming at the OsMIR7695 expression product, antisense RNA, target gene analogues, short tandem target analogues and guide RNA used for the CRISPR-Cas13 method.
Wherein the reagent for reducing the OsMIR7695 expression of the rice comprises a reagent for knocking out an OsMIR7695 coding gene in a rice genome.
Further, the agent for knocking out an OsMIR 7695-encoding gene in a rice genome described in the above-mentioned uses includes at least one of a Meganuclease for Meganuclease (Meganuclease) method, a ZFN protein for ZFN method, a TALEN protein for TALEN method, a guide RNA for CRISPR-Cas method, a recombinant DNA fragment for homologous recombination method, or a T-DNA (Transfer DNA ) or transposon for OsMIR 7695-encoding gene.
Further, the nucleotide sequence of the guide for the CRISPR-Cas12a method in the application is shown as Seq ID No. 2.
Meanwhile, the invention also provides a guide RNA aiming at the OsMIR7695 coding gene of rice. Further, the coding nucleotide sequence of the guide RNA was 5'-ATTGTGGTCTTGCCTATGTGGCA-3' (Seq ID No. 2).
Meanwhile, the present invention provides a vector loaded with the above crRNA.
Further, the vector is a Cas editing expression vector for a CRISPR-Cas gene editing technology. Preferably, the CRISPR-Cas gene editing technology is CRISPR-Cas12a technology.
The invention has the beneficial effects that: the invention provides a method for improving rice grain traits, which improves the rice grain traits by blocking or weakening the expression of OsMIR7695 in rice. In the method, the rice OsMIR7695 coding gene is knocked out, or the effect of OsMIR7695 coding gene expression products is influenced, and the method is two main modes for blocking or weakening the expression of OsMIR7695 in rice. In the embodiment of the invention, the rice mutant with the increased thousand grain weight, grain length or grain width of grains is obtained after the OsMIR7695 coding gene of the rice is knocked out, which shows that the grain character is greatly improved, and other agronomic characters are not obviously changed. The method has simple steps, is easy to operate, and has good prospects in the aspects of rice quality improvement and genome function research and application.
Drawings
FIG. 1, OsMIR7695 locus stem-loop structure and CRISPR-Cas12a directional mutation vector schematic diagram. A. The stem-loop structure of the OsMIR7695 locus (MI0025202) is schematically shown, the underlined base region is mature OsMIR7695-5p (MIMAT0029961), and the underlined italic base region is mature OsMIR7695-3p (MIMAT 0029962). B. A T-DNA region structure schematic diagram of a rice directional knockout OsMIR7695 directional mutation vector (pTX377_ OsMIR7695) is thickened to form a PAM locus sequence, and an italic form is an OsMIR7695-crRNA sequence.
FIG. 2, molecular characterization of a part of rice T0 transformed plants with OsMIR7695 editing vector (pTX377_ OsMIR 7695). A. And (3) a positive PCR detection result of the rice OsMIR7695 editing vector transformation. B. And identifying the result of the SSCP of the OsMIR7695 editing site of the rice genome, wherein the italic numbered plant is a candidate editing mutant plant. C. According to the sequencing result of the PCR amplification product Sanger of the rice candidate editing mutant OsMIR7695 locus, the bold is a recognition sequence PAM locus edited by LbCas12a, the italic is a crRNA sequence, and the underlined is a mature body sequence of rice OsMIR7695-5p (MIMAT 0029961).
FIG. 3 shows that OsMIR7695 edits a mutant T3 generation plant seed phenotype. A. Comparison results of grain length of rice wild type plants (OsMIR7695_ WT) and rice OsMIR7695 locus editing mutant plants (OsMIR 7695-M1, M2 and M3) T3 generation grains. A. Comparison result Bar of grain width of rice wild type plants (OsMIR7695_ WT) and rice OsMIR7695 locus editing mutant plants (OsMIR 7695-M1, M2 and M3) T3 generation grains is 10 mm.
FIG. 4, identification of key agronomic traits of seeds of rice OsMIR7695 editing mutant T3 generation plants. A. Thousand grains weight, n is 3; B. the length of the kernel is 10; C. the width of the kernel is 10; **: very significant difference, p < 0.01; *: significant difference, 0.01< p < 0.05.
Detailed Description
At present, most of the recorded plant miRNA molecules in plant miRNA public database miRBase (http:// www.mirbase.org/index. shtml) are obtained based on small RNA high-throughput sequencing, and no specific biological function experiment result exists. One piece of rice miRNA molecular information discovered based on high-throughput sequencing is OsMIR7695, the length of the annotated precursor sequence is 487bp, the accession number of miRBase is MI0025202, and the annotated precursor sequence is located in the interval of 11666716-11667202bp of rice 1. Further data retrieval shows that the rice OsMIR7695 biological function is not reported in public except for sequencing data.
On the basis of carrying out a large amount of work of directionally editing mutant creation aiming at unknown biological function miRNA encoding genes of rice in the earlier stage of the invention, the invention knocks out OsMIR7695 encoding genes in the rice by a genome editing technology, and results unexpectedly discover that plants with obviously improved grain agronomic characters are obtained, and the improved characters are embodied in the aspects of grain weight increase, grain length increase, grain width increase and the like. Meanwhile, no obvious change of other agronomic characters is observed, which indicates that the rice yield can be improved.
Based on the experiments, the OsMIR7695 coding gene of the rice has close correlation with the agronomic characters of rice grains, and the expression of the OsMIR7695 in the rice is blocked or weakened, so that the agronomic characters of the grains can be effectively improved.
Therefore, the invention establishes and discloses a novel method for improving the rice grain character. The method improves the agronomic traits of grains by blocking or weakening the expression of OsMIR7695 coding genes in rice.
It is known that the genes encoding mirnas will first give primary transcripts (pri-mirnas) during expression, and then be processed into miRNA precursors (pre-mirnas), which are then further processed into mature mirnas. The mature miRNA molecules can exert the biological functions thereof. Obviously, any step of the whole process of OsMIR7695 coding gene expression is influenced, the effect of OsMIR7695 expression products can be reduced, and the agronomic traits of rice grains can be improved. Knocking out the rice OsMIR7695 coding gene or interfering the effect of the OsMIR7695 coding gene expression product is two main modes for blocking or weakening the expression of the OsMIR7695 coding gene in rice in the field.
As is known to those skilled in the art, there are several existing methods for interfering with the action of OsMIR7695 expression products, that is, with the biological function of OsMIR 7695.
For example, antisense RNA method can be used to design antisense RNA for OsMIR7695, so as to achieve the purpose of reducing the biological function of OsMIR7695 and improving the rice grain character.
In recent years, target gene analog (TM) and short tandem target analog (STTM) approaches have also been developed in the art to interfere with (or block) the effects of target mirnas. Obviously, after the invention is read, a person skilled in the art can design a target gene analogue or a tandem target analogue to reduce the biological function of OsMIR7695 and improve the rice grain character.
In recent years, the CRISPR-Cas13 method is applied as a newly developed editing tool for RNA, and a person skilled in the art can understand that the CRISPR-Cas13 method can be used for blocking or weakening the effect of an OsMIR7695 coding gene expression product.
While the other direction starts with the influence on the transcription of OsMIR 7695. At present, the most common method is to knock out the OsMIR7695 coding gene from the genome, and the adopted technical means are a genome editing method, a homologous recombination method or a random insertion mutation method and the like.
When the homologous recombination method is used, a specific recombinant DNA fragment can be designed and replaced into a genome through homologous recombination, so that the rice does not express the OsMIR7695 coding gene. In the random insertion mutation method, a T-DNA (Transfer DNA) insertion mutation method, a transposon insertion mutation method, or the like can be used, and similar effects can be achieved.
In recent years, genome editing techniques have been attracting much attention in the art. Meanwhile, currently, genome Meganuclease (Meganuclease) methods, ZFN (zinc finger nuclease) methods, TALEN (Transcription activator-like effectors) methods, and CRISPR-Cas methods are commonly used as genome editing techniques for gene knockout. The methods can be used for the creation of rice directional editing mutants with OsMIR7695 coding genes knocked out, and mutant plants with improved grain properties are obtained.
Meanwhile, the skilled person can also think that any rice variety can block or weaken the expression of OsMIR7695 in the rice and improve the agronomic characters of the seeds by using the method.
Meanwhile, the invention also provides application of the reagent for blocking or weakening the expression of OsMIR7695 in rice in improving the rice grain traits.
These agents include, on the one hand, various molecules that interfere with OsMIR7695 expression products and make them difficult to function normally, including, but not limited to, RNAi molecules, antisense RNA molecules, target gene analog (TM) molecules, short tandem target analog (STTM) molecules designed for OsMIR7695 expression products, guide RNAs for CRISPR-Cas13 method.
On the other hand, the gene coding OsMIR7695 in the rice genome can be knocked out by the gene coding OsMIR 7695. These agents include, but are not limited to, meganucleases for Meganuclease (Meganuclease) method, ZFN proteins for ZFN method, TALEN proteins for TALEN method, guide RNA molecules for CRISPR-Cas method, recombinant DNA fragments for homologous recombination method, or T-DNA (Transfer DNA) molecules or transposon molecules for random insertion mutation method against OsMIR7695 encoding gene.
The CRISPR-Cas method for knocking out the coding gene currently has a plurality of specific technical systems such as CRISPR-Cas9, CRISPR-Cas12a and CRISPR-Cas12b, and when the method is implemented by the technical personnel in the field, the targeted editing work of the OsMIR7695 coding gene can be easily carried out according to the requirements of each specific technology.
In one embodiment of the invention, a specially designed guide RNA for the gene encoding OsMIR7695 in rice is used. The nucleotide sequence is 5'-ATTGTGGTCTTGCCTATGTGGCA-3' (Seq ID No.2), and the guide RNA is used for the CRISPR-Cas12a gene editing system to edit the rice OsMIR7695 coding gene. Of course, those skilled in the art know that the crRNA molecule specifically functions as a guide RNA in the CRISPR-Cas12a gene editing system.
On the basis, the Cas12a oriented editing expression vector (pTX377_ OsMIR7695) capable of expressing the guide crRNA is also obtained in the embodiment of the invention. Of course, there are many reported alternatives to such Cas12 a-directed editing of the backbone vector of the expression vector. For this crRNA, pTX377, pYPQ230 (TangX, Lowder LG, Zhang T, Malzahn A, ZHENG X, Voytas DF, ZHONG Z, Chen Y, Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y2017. ACRISPR-Cpf1 system for effective gene editing and transcriptional expression in Plants Nature Plants,3: 17018; TangX, Ren Q, Yang L, Bao Y, ZHONG Z, He Y, Liu S, Qi C, Liu B, Wang Y, Sreenovic S, Zhang Y, ZHENG X, Zhang T, Zhang Y2019. Sitting Y, Stir Z, Stir Y, Stir T, Lowder LG, Zhang T, Skys DF, Zhang Z, Zhang Y, Zhang X, Zhang S, Zhang Z, S, Zhang Z, K5, Zhang S, S. In one embodiment of the invention, the pTX377 vector is used as a framework vector, so that a good effect is achieved, and the rice OsMIR7695 coding gene directional editing mutant material with obviously improved grain properties is obtained.
Specifically, the method for preparing the rice OsMIR7695 coding gene targeted editing mutant material knocked out by using the Cas12a targeted editing guide crRNA (OsMIR7695_ crRNA1) to improve the kernel character comprises the following main steps:
a. preparing a Cas12a gene editing expression vector with a guide RNA sequence such as Seq ID No. 2;
b. b, transforming rice by using the expression vector obtained in the step a, and obtaining a transformed plant by using a CRISPR-Cas12a gene editing system;
c. and (3) collecting seeds of the transformed plants, and screening seeds of the rice OsMIR7695 coding gene directional editing mutant to obtain the rice OsMIR7695 coding gene directional editing mutant with the rice grain properties remarkably improved.
Furthermore, the method of the invention can be carried out according to the following more specific steps:
(1) selection of crRNA target sites
The rice OsMIR7695 coding gene is located on chromosome 1, the precursor DNA coding sequence with the annotation 487bp length is shown as Seq ID No.1, and the stem-loop structure (stem-loop) schematic diagram in the maturation process is shown as figure 1A. The crRNA sequence is shown as Seq ID No.2, the length is 23bp, the target site is 127bp-149bp in OsMIR7695 precursor sequence shown as Seq ID No.1, and the PAM site is 123bp-126bp (5 '-TTTG-3') in OsMIR7695 precursor sequence. Two single-stranded nucleotide sequences OsMIR7695-crRNA1-F annealing to form cohesive ends are designed and synthesized according to the sequence of the crRNA, such as the sequence ID No.3 and OsMIR7695-crRNA1-R, and the sequence is shown as the sequence ID No. 4.
(2) Cas12a directed editing expression vector construction
OsMIR7695-crRNA1-F and OsMIR7695-crRNA1-R are respectively diluted by 10 times, 10 mu L of each are mixed together and denatured at 98 ℃ for 5min, natural cooling is carried out, and the annealed product is diluted by 20 times. The annealed product was ligated with BsaI digested backbone vector pTX 377. Through transformation of Escherichia coli, single colony PCR and sequencing identification, the directional editing expression vector pTX377_ OsMIR7695 for the rice OsMIR7695 coding gene is obtained, and the structural schematic diagram of the T-DNA region is shown in figure 1B. Specific methods of construction are described in the literature references (Tang X, Lowder LG, Zhang T, Malzahn A, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y.2017.ACRISPR-Cpf1 system for effective gene evaluation and translational repression in Plants Nature Plants,3: 17018; Tang X, Ren Q, Yang L, Bao Y, Zhang Z, He Y, Liu S, Qi C, Liu B, Wang Y, Sretogenic S, Zhang Y, Zhang X, Zhang T, Zhang Y, Zhang Y.2019.Single transform 2.0system for crisper 19. Cas 19. Biotechnology Cas 19. 11. Biocode 19. 11. Cas 19. 11. Biotechnology).
(3) Genetic transformation and molecular identification of rice
The rice OsMIR7695 coding gene directional editing expression vector pTX377_ OsMIR7695 obtains a transformed plant through agrobacterium-mediated rice genetic transformation, screening and regeneration, and extracts single plant DNA for positive identification. PCR amplification was performed with the designed specific primers OsMIR7695-SSCP-F (Seq ID No.5) and OsMIR7695-SSCP-R (Seq ID No.6), and through SSCP and Sanger sequencing verification (FIG. 2), OsMIR7695 targeted knockout mutants were obtained. Rice genetic transformation and molecular characterization specific Process references (Tang X, Lowder LG, Zhang T, Malzahn A, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y.2017.ACRISPR-Cpf1 system for efficacy gene mapping and transcription expression in Plants. Nature Plants,3: 17018).
The present invention will be described in further detail with reference to examples.
Example 1 construction of OsMIR7695 knockout vector CRISPR-Cas12a for rice
(1) crRNA design
The crRNA is designed according to the recognition and cleavage rules of the target site by CRISPR-Cas12 a. Single-stranded nucleotide sequences OsMIR7695-crRNA1-F (5'-agatATTGTGGTCTTGCCTATGTGGCA-3' SEQ ID No.3) and OsMIR7695-crRNA1-R (5'-ggccTGCCACATAGGCAAGACCACAAT-3', SEQ ID No.4) were designed based on the rice OsMIR7695 precursor genome sequence (SEQ ID No. 1).
(2) Annealing of single-stranded nucleotide sequences
Diluting 10 times of single-stranded nucleotide sequences of OsMIR7695-crRNA1-F and OsMIR7695-crRNA1-R at the upstream and downstream of the target site respectively, taking 10 mu L of each single-stranded nucleotide sequence, denaturing at 98 ℃ for 5min, naturally cooling, and diluting 20 times of annealed products for later use.
(3) Enzyme digestion, glue recovery, connection
The backbone vector used in the experiment was pTX377 and was constructed in the laboratory from the vector pYPQ230 shown in the literature (Tang et al, A CRISPR-Cpf1 system for effective gene injection and translational expression in plants. nat. 2017,3: 17103). pTX377 was digested with BsaI, and the establishment of the digestion system and the digestion conditions were carried out according to the restriction enzyme instruction of Thermo Scientific Co. The specific enzyme digestion system is as follows: 10 XFast digest buffer5 μ L, plasmid DNA or PCR product 10 μ L (1-1.5 μ g), restriction enzyme 1 μ L, ddH2O make up to 50 μ L.
Reacting in a constant temperature incubator at 37 ℃ for 2h, adding 10 mu L of 6 × loading buff after the reaction is finished, carrying out 1% agarose gel electrophoresis, cutting the gel and recovering. The Gel recovery method was carried out according to the AXYGEN AxyPrepTM DNA Gel Extraction Kit method.
The restriction enzyme recovery product of pTX377 is connected with the annealing dilution product of OsMIR7695-crRNA1-F, OsMIR7695-crRNA1-R, and the connection system and conditions refer to the specification of T4 DNA ligase of New England Biolabs company, wherein the specific connection system is as follows: 2. mu.L of 10 XT 4 DNA ligase reaction buffer, 1. mu.L of T4 DNA ligase, 5. mu.L of pTX377 cleavage product, 5. mu.L of annealing product, and 20. mu.L of ddH 2O.
(4) Transformation of E.coli
Coli DH 5. alpha. was thawed slowly on ice, 1. mu.g of plasmid was added and left on ice for 20 min. Heat shock is carried out for 1min at 42 ℃, and the mixture is placed on ice for 1-2 min. Adding 350 μ L liquid LB, mixing well, shake culturing at 37 deg.C for 45 min. Centrifuge at 12000rpm for 1min, remove 300. mu.L of supernatant, and resuspend the remaining 100. mu.L of the bacterial suspension. And (3) coating the whole resuspended bacterial liquid on an LB plate containing corresponding antibiotics (50mg/L Kan), and performing inverted culture at 37 ℃ for 18-22 h.
(5) Colony PCR
Single colonies on LB plates were picked with sterilized toothpicks and dissolved in 50. mu.L ddH2In O water, PCR amplification was performed using the bacterial solution as a template. A25 uL system was used, as follows: 10 XPCR Buffer 2.5. mu.L, dNTP 0.5. mu.L, OsMIR7695-crRNA 1-F0.5. mu.L, ZY010-R1 (5'-AAGACCGGCAACAGGATTC-3') 0.5. mu.L, Taq DNAenzyme 0.2. mu.L, Template 1. mu.L, ddH2O19.8. mu.L. The PCR procedure was: 94 ℃, 5min → (94 ℃, 30s → 56 ℃, 30s → 72 ℃, 10-60s)32 cycles → 72 ℃, 5min → 10 ℃, 5min (Taq DNAenzyme, dNTP, etc. from Tiangen Bio Inc.). After the PCR was completed, 5. mu.L of 6 Xbromophenol blue was added,detection was by agarose gel electrophoresis.
(6) Plasmid extraction and sequencing verification
The correct single clone was confirmed by colony PCR, and the Plasmid in the bacterial solution was extracted by shaking LB containing 50mg/Lkan, and the Plasmid DNA was extracted according to the AXYGEN AxyPrepTM Plasmid Miniprep Kit. The extracted plasmid was sent to Scout Biotechnology Limited for sequencing verification. And the plasmid was named pTX377_ OsMIR7695, and its T-DNA structural diagram is shown in FIG. 1B.
Example 2 Agrobacterium-mediated genetic transformation of Rice
A method disclosed in the Agrobacterium-mediated transformation of rice reference (Toki et al, Early infection of cutellum tissue with Agrobacterium high-speed transformation of rice, 2006,47(6): 969-976.). The genetic transformation steps of rice are as follows:
shelling and sterilizing mature seeds of rice (Nipponbare); inoculating the disinfected seeds on an N-6-D solid culture medium containing 0.4% gellan gum, and continuously culturing for 1-5 days at 32 ℃ by illumination; transferring the plasmid pMIR390-1 into rice by agrobacterium-mediated transformation, and culturing the transformed rice seeds in an induction selective culture medium at 32 ℃ for 2 weeks; transferring the callus generated by proliferation into a RE-III culture medium; transfer of young plants produced from callus into HF medium induced root production. And when the obtained resistant regenerated seedlings grow to about 15cm, cleaning the root culture medium with clear water, transplanting the seedlings into nutrient soil, and culturing in a greenhouse.
Example 3 molecular characterization of OsMIR7695 mutant in Rice
(1) Extraction of rice seedling genome DNA
The DNA extraction of the rice seedlings adopts a CTAB method, and the specific operation steps are as follows:
preheating CTAB extracting solution in a water bath kettle at 65 ℃. Taking a single leaf, putting the single leaf into a 2mL centrifuge tube with steel balls, putting into liquid nitrogen for quick freezing, and then shaking into powder. Adding 500 mu L of preheated CTAB extracting solution, and carrying out water bath at 65 ℃ for 30-50 min, wherein the mixture is fully and uniformly mixed. Add 500. mu.L of chloroform: isoamyl alcohol (24: 1), fully reversing and mixing uniformly, centrifuging at 4 ℃ and 10000rpmFor 10 min. Taking supernatant, adding isopropanol with the same volume for precipitation, and precipitating for 30 min-2 h at-20 ℃. The precipitate was collected by centrifugation at 12000rpm for 10min at room temperature. The supernatant was removed, rinsed with 75% ethanol and centrifuged at 12000rpm for 2 min. The supernatant was removed and the DNA was air-dried. Adding 30-50 mu L ddH2O dissolving DNA, and storing in a refrigerator at-20 ℃ for later use.
(2) Positive detection of rice seedling transgene
The fragment size of the amplified fragment was 1068bp by PCR amplification using Intron-F1 (5'-TTCTGATCCTCTCCGTTCCT-3', SEQ ID No.7) and ZY010-R1 (5'-AAGACCGGCAACAGGATTC-3', SEQ ID No.8), the results are shown in FIG. 2A. The PCR amplification system and the reaction procedure are the same as the colony PCR.
(3) Detection Using SSCP mutants
Firstly, PCR is carried out to amplify a target fragment, then a PCR product is denatured, and preliminary screening of mutants is carried out through polyacrylamide gel electrophoresis. The specific method is shown in the article (Zheng et al, Effective screen of CRISPR/Cas9-induced microorganisms in a single-strand transformation polymorphism. plant Cell Rep,2016, (7):1545-54.) the specific operation steps are as follows:
the positive plants obtained by detection are subjected to PCR amplification by using primers OsMIR7695-SSCP-F (5'-ATCGGGAGGTTTCACTTAAG-3', SEQ ID No.5) and OsMIR7695-SSCP-R (5'-GGTGGTGAGAGGCGGAACGA-3', SEQ ID No.6), and the length of the amplified fragment is 223 bp. The PCR amplification system and the reaction procedure are the same as before. Adding 5 μ LPCR product into 5 μ L SSCP denaturant, mixing, and denaturing at 95 deg.C for 5 min; and (5) after the denaturation is finished, quickly putting the mixture into an ice box, and cooling for 10 min. Preparing 15% PAGE (29: 1) gel by the following method: 21mL of Acr/Bis (29: 1) gelatin solution, 150. mu.L of 10% Aps and 10. mu.L of TEMED, and rapidly stirring and mixing uniformly for injecting gelatin. Acrylamide Acr, methylene bisacrylamide Bis, ammonium persulfate Aps, and tetramethylethylenediamine TEMED were obtained from AMRESCO. After the gel is solidified, performing electrophoresis for about 20min at the constant current of 45mA at the temperature of 4 ℃. Then, the power was turned off, and 5. mu.L of each denatured sample was sequentially applied. And performing constant current electrophoresis at 4 ℃ for 4-5 h at 45 mA. Unloading the glue, and washing for 2-3 times by using water; dyeing: adding AgNO3 dye solution, and dyeing for 10 min; color development: adding NaOH color developing agent, and placing in a shaking table for color development for about 5min until clear strips are seen; after the completion of the dyeing, the reaction was immediately stopped by rinsing with water. And (4) observation: the glue is laid on the lamp box, observed and photographed.
The SSCP results (fig. 2B) show: the bands of the single strains OsMIR7695-1-1, OsMIR7695-1-2, OsMIR7695-1-3, OsMIR7695-1-5, OsMIR7695-1-6 and OsMIR7695-1-7 are inconsistent with the Wild Type (WT), and the mutants are preliminarily shown. The CRISPR-Cas12 system is shown to be capable of carrying out directional editing on OsMIR7695, so that the mutant is obtained.
(4) Sequencing verification of knockout mutants
Single strains OsMIR7695-1-1, OsMIR7695-1-2 and OsMIR7695-1-3 which are obtained by screening SSCP and are different from wild type are subjected to PCR amplification by using primers OsMIR7695-SSCP-F (SEQ ID No.5) and OsMIR7695-SSCP-R (SEQ ID No.6), and the PCR amplification system and the reaction program are the same as those in the previous step. The deliveries were sequenced by the department of biology. The sequencing result (FIG. 2C) shows that the single-plant OsMIR7695-1-1 is a (-13bp/-13bp) homozygous mutant, OsMIR7695-1-2 is a (-16bp/-8bp) biallelic mutation, and OsMIR7695-1-3 is a (-13bp/wt) heterozygous mutant.
(5) Detection of agronomic traits of knockout mutants
The T2 generation plants of OsMIR7695-1-6 are further screened to obtain homozygous mutants (number OsMIR7695-1-6), named OsMIR7695-M, and the T3 generation OsMIR7695-M1, OsMIR7695-M2 and OsMIR7695-M3 seeds are subjected to agronomic trait analysis respectively to find that the length and width of the seeds are obviously increased compared with the control (figure 3), and further analyzed to find that the thousand seed weight is increased from 25.56g to 27.16g, 26.87g and 27.3g, and the weight is increased respectively by 6.3%, 5.1% and 6.8% (figure 4A), the grain length is obviously increased, and is increased respectively from 7.46mm to 7.59mm, 7.57mm and 7.61mm (figure 4B), and the grain width is increased respectively from 3.28mm to 3.31mm, 3.30mm and 3.36mm (figure 4C). Other agronomic characters are not obviously changed.
Experimental results show that the rice OsMIR7695 coding gene targeted editing expression vector pTX377_ OsMIR7695 constructed based on the wizard crRNA (OsMIR7695_ crRNA1, the sequence of which is shown in Seq ID No.2) discovered by the invention and a corresponding experimental method can effectively obtain OsMIR7695 coding gene targeted editing mutant rice material without transgenic components, increase of thousand grain weight, grain length and grain width of grains is realized, and rice yield is further improved.
<110> university of electronic technology
<120> a method for improving grain traits by blocking or weakening OsMIR7695 expression of rice
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 487
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gagtaaaatg cactagcggt ccttaaactt atcgggaggt ttcacttaag tccacgaact 60
tgcaaagcgc acatcgaggt ctctaaactt ggtttattgt atcatcccgg tccaaagcca 120
cgtttgattg tggtcttgcc tatgtggcac gccacgtgaa tgatgacatg gaattttttt 180
tatttttttc tcccttcttc cttctttttt caccactgcg acaacccctc ccctcgttcc 240
gcctctcacc accgagaaaa aagaaggaag aaagagaaaa aaagaaggaa gaagggagaa 300
aaaaataaaa aaaattccat gtcattatcc acgtgatgtg ccacgtaggc aagaccacag 360
tcaaatgcgg ctttgaaccg gaatgatata ataaaccaag tttagggatc tcgatgtgca 420
ctttgcaagt tcgtggatct aagtgaaacc tcctgacaag tttaagaacc gctggtgtat 480
tttactc 487
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
attgtggtct tgcctatgtg gca 23
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agatattgtg gtcttgccta tgtggca 27
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggcctgccac ataggcaaga ccacaat 27
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atcgggaggt ttcacttaag 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggtggtgaga ggcggaacga 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ttctgatcct ctccgttcct 20
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aagaccggca acaggattc 19
Claims (14)
1. A method for improving rice grain traits is characterized by comprising the following steps: the expression of OsMIR7695 in rice is blocked or weakened, and the rice grain character is improved.
2. The method of claim 1, wherein: the blocking or weakening of the expression of OsMIR7695 in rice is performed by knocking out the rice OsMIR7695 coding gene or interfering the effect of the OsMIR7695 coding gene expression product.
3. The method of claim 1, wherein: the method for interfering the action of the OsMIR7695 coding gene expression product comprises at least one of an RNA interference method, an antisense RNA method, a target gene analogue method, a short tandem target analogue method or a CRISPR-Cas13 method.
4. The method of claim 1, wherein: the method for knocking out the OsMIR7695 coding gene in the rice genome is carried out by at least one of a genome editing method, a homologous recombination method or a random insertion mutation method; preferably, the genome editing method comprises at least one of Meganuclease (Meganuclease) method, ZFN method, TALEN method or CRISPR-Cas method.
5. The method of claim 4, wherein: when the CRISPR-Cas method is used for knocking out the OsMIR7695 coding gene in the rice genome, the method comprises the following steps:
a. designing a guide RNA aiming at the OsMIR7695 coding gene of the rice;
b. constructing a Cas editing expression vector capable of expressing the guide RNA;
c. b, transforming rice by using the expression vector obtained in the step a, and obtaining a transformed plant by using a CRISPR-Cas gene editing system;
d. collecting seeds of the transformed plants, and screening out rice OsMIR7695 coding gene directional editing mutant seeds to obtain rice mutants with improved grain properties;
preferably, the CRISPR-Cas method comprises at least one of CRISPR-Cas9, CRISPR-Cas12a or CRISPR-Cas12 b.
6. The method of claim 5, wherein: the nucleotide sequence of the guide RNA aiming at the rice OsMIR7695 coding gene in the step a is shown as Seq ID No. 2.
7. The method of claim 1, wherein: the rice grain trait is at least one of thousand kernel weight, grain length and grain width.
8. The application of the reagent for blocking or weakening the expression of OsMIR7695 in rice in improving the rice grain traits.
9. Use according to claim 8, characterized in that: the reagent for blocking or weakening the expression of the OsMIR7695 in the rice comprises at least one of a reagent for knocking out the rice OsMIR7695 coding gene or a reagent for interfering with the expression product of the OsMIR7695 coding gene.
10. Use according to claim 9, characterized in that: the reagent for interfering the OsMIR7695 coding gene expression product comprises a reagent used in at least one of an RNA interference method, an antisense RNA method, a CRISPR-Cas13 method, a target gene analogue method or a short tandem target analogue method; preferably, the agent that interferes with the action of an OsMIR7695 expression product comprises at least one of a siRNA against an OsMIR7695 expression product, an antisense RNA against an OsMIR7695 expression product, a target gene analog, a short tandem target analog, a guide RNA for CRISPR-Cas13 method to edit an OsMIR7695 expression product.
11. Use according to claim 9, characterized in that: the reagent for knocking out the OsMIR7695 coding gene in the rice genome comprises at least one of giant nuclease aiming at the OsMIR7695 coding gene and used for a giant nuclease method, ZFN protein used for a ZFN method, TALEN protein used for a TALEN method, guide RNA used for editing the OsMIR7695 coding gene by a CRISPR-Cas method, a recombinant DNA fragment used for a homologous recombination method, and T-DNA or transposon used for a random insertion mutation method.
12. Use according to claim 11, characterized in that: the nucleotide sequence of the crRNA used for CRISPR-Cas12a is shown as Seq ID No. 2.
13. crRNA aiming at the OsMIR7695 coding gene of the rice; preferably, the coding nucleotide sequence of the crRNA is shown as Seq ID No. 2.
14. A vector loaded with the guide RNA of claim 15 or 16; preferably, the vector is a Cas editing expression vector for CRISPR-Cas gene editing technology; more preferably, the CRISPR-Cas gene editing technology is CRISPR-Cas12a technology.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010269792.8A CN113493787B (en) | 2020-04-08 | 2020-04-08 | Method for blocking or weakening expression of rice OsMIR7695 to improve seed shape |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010269792.8A CN113493787B (en) | 2020-04-08 | 2020-04-08 | Method for blocking or weakening expression of rice OsMIR7695 to improve seed shape |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113493787A true CN113493787A (en) | 2021-10-12 |
| CN113493787B CN113493787B (en) | 2023-07-28 |
Family
ID=77995635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010269792.8A Active CN113493787B (en) | 2020-04-08 | 2020-04-08 | Method for blocking or weakening expression of rice OsMIR7695 to improve seed shape |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113493787B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014100252A1 (en) * | 2012-12-18 | 2014-06-26 | University Of Washington Through Its Center For Commercialization | Methods and compositions to modulate rna processing |
| CN106367435A (en) * | 2016-09-07 | 2017-02-01 | 电子科技大学 | Method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice |
| CN111926009A (en) * | 2020-06-30 | 2020-11-13 | 成都极谷基因科技有限公司 | Method for improving rice grain traits by blocking or weakening rice OsMIR394 gene expression |
| CN112175990A (en) * | 2020-09-23 | 2021-01-05 | 电子科技大学 | Method for blocking or weakening expression of rice circRNA coding site to improve rice seedling growth traits |
| CN113493786A (en) * | 2020-04-07 | 2021-10-12 | 电子科技大学 | Method for improving rice grain traits by blocking or weakening expression of OsMIR3979 in rice |
-
2020
- 2020-04-08 CN CN202010269792.8A patent/CN113493787B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014100252A1 (en) * | 2012-12-18 | 2014-06-26 | University Of Washington Through Its Center For Commercialization | Methods and compositions to modulate rna processing |
| CN106367435A (en) * | 2016-09-07 | 2017-02-01 | 电子科技大学 | Method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice |
| CN113493786A (en) * | 2020-04-07 | 2021-10-12 | 电子科技大学 | Method for improving rice grain traits by blocking or weakening expression of OsMIR3979 in rice |
| CN111926009A (en) * | 2020-06-30 | 2020-11-13 | 成都极谷基因科技有限公司 | Method for improving rice grain traits by blocking or weakening rice OsMIR394 gene expression |
| CN112175990A (en) * | 2020-09-23 | 2021-01-05 | 电子科技大学 | Method for blocking or weakening expression of rice circRNA coding site to improve rice seedling growth traits |
Non-Patent Citations (10)
| Title |
|---|
| CAMPO S等: "Identification of a novel microRNA(miRNA) from rice that targets an alternatively spliced transcript of the Nramp6(Natural resistance-associated macrophage protein 6) gene involved in pathogen resistance" * |
| FERRAN SS等: "Osa-miR7695 enhances transcriptional priming in defense responses against the rice blast fungus" * |
| LING-LING ZHENG等: "Application of microRNA gene resources in the improvement of agronomic traits in rice" * |
| NGUYEN BAO QUOC等: "Expression of osa-miR7695 against the blast fungus Magnaporthe oryzae in Vietnamese rice cultivars" * |
| SANCHEZ-SANUY等: "The microRNA miR7695 is involved in the rice immune response to pathogen infection" * |
| 于雅洁等: "miRNA对水稻胁迫应答及产量调控影响的研究进展" * |
| 刘潮;韩利红;褚洪龙;王海波;高永;唐利洲;: "植物与病原菌互作的分子机制研究进展" * |
| 刘潮;韩利红;褚洪龙;王海波;高永;唐利洲;: "植物抗病研究进展与展望" * |
| 周建平;吉琳艺;何瑶;郑雪莲;罗樊;蔡光泽;张勇;: "利用CRISPR-Cas9系统高效创制水稻品系"17318"的OsGW2、OsGN1a基因定向突变体" * |
| 肖之源;王青霞;王贺;李金璐;赵胜利;樊晶;李燕;王文明;: "水稻miR444b.2调控稻瘟病抗性及分蘖" * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113493787B (en) | 2023-07-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hu et al. | Engineering non-transgenic gynoecious cucumber using an improved transformation protocol and optimized CRISPR/Cas9 system | |
| CN108642055B (en) | sgRNA capable of effectively editing pig miR-17-92 gene cluster | |
| CN106367435A (en) | Method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice | |
| Wang et al. | Construction of a genomewide RNA i mutant library in rice | |
| CN106282231B (en) | Construction method and application of mucopolysaccharide storage disease type II animal model | |
| CN110684777B (en) | Application of isolated nucleotide sequence in construction of zebra fish with reduced intramuscular stings | |
| CN108034671B (en) | Plasmid vector and method for establishing plant population by using same | |
| CN115820685A (en) | Citrus CsGSTF1 gene and application thereof | |
| CN113493786B (en) | Method for blocking or weakening expression of OsMIR3979 in rice to improve rice seed shape | |
| CN112175990B (en) | Method for blocking or weakening expression of rice circRNA coding site to improve rice seedling growth traits | |
| CN114807138B (en) | Plant annular RNA over-expression vector, construction method and application thereof | |
| CN113493787B (en) | Method for blocking or weakening expression of rice OsMIR7695 to improve seed shape | |
| CN111909956B (en) | Method for blocking or weakening OsNAC092 gene expression of rice to improve drought resistance of rice | |
| CN115820686A (en) | Citrus CsGSTU18 gene and application thereof | |
| CN110684797B (en) | VIGS vector based on TCV with simultaneous silencing of 2 endogenous genes | |
| CN112852805B (en) | Preparation method of rice miRNA homozygous lethal mutant | |
| CN111893119A (en) | Method for obtaining SCD1 gene editing goat embryo by CRISPR/Cas9 system and microinjection | |
| CN110129359B (en) | Method for detecting gene editing event and determining gene editing efficiency and application thereof | |
| CN112322622B (en) | Application of lncRNA17978 in improvement of disease resistance of rice | |
| CN114934073B (en) | Construction method and application of hoxa a gene knockout zebra fish mutant | |
| CN106701820A (en) | Method for improving and utilizing key genes of wild rice | |
| CN117802148A (en) | Method for regulating rice plant type | |
| CN117987460A (en) | LbCas12a plant genome editing system for coexpression TypeV-A anti-CRISPR protein | |
| CN117568388A (en) | CRISPR vector of BnanINJA gene of brassica napus, construction method, detection/identification method and application thereof | |
| CN116536309A (en) | Modified pilot editing system and application thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |