64 results about "Deletion mutation" patented technology

The present application relates to a kind of deletion mutant nucleic acid and its application in anti-herbicide.The deletion mutation occurs in the promoter of rice OsHPPD Gene, the length of deletion sequence is at least 10 bp, and the A in the start codon ATG of gene is 0, at least the bases located in the upstream of start codon ATG from 2041 to 2032 are deleted. OsHPPD The present application finds that by deleting part of the sequence in the promoter of rice OsHPPD Gene, rice can obtain resistance to HPPD inhibitor herbicides, which is of great application value for ensuring agricultural production safety and improving the efficiency of herbicide use.

The invention relates to the technical field of gene editing, and particularly provides a method for creating herbicide-resistant rice through gene editing of an OsEPSPS gene promoter region, and the method is characterized in that WT / 29bp single allelic deletion mutation is introduced into the OsEPSPS gene promoter region, i.e., A in an OsEPSPS gene initiation codon ATG is 0 site, A in the OsEPSPS gene initiation codon ATG is 0 site, A in the OsEPSPS gene initiation codon ATG is 0 site, and A in the OsEPSPS gene initiation codon ATG is 0 site; 29bp is deleted from the 112nd site to the 1150th site at the upstream of the initiation codon ATG on one allele, and the other allele is kept unchanged. By introducing the single allelic deletion mutation, the rice can obtain resistance to EPSPS inhibitor herbicides, the resistance is identified in T0-generation plants carrying the mutation and can be stably inherited to offspring plants, and the application value is high for guaranteeing agricultural production safety and improving the use efficiency of the herbicides.

The invention provides a primer probe set for digital PCR detection of non-small cell lung cancer EGFR mutation and application thereof, and relates to the technical field of digital PCR detection, the primer probe set comprises a primer pair and a probe for specific detection of EGFR gene exon 19 deletion mutation (19del) and exon 21 L858R point mutation, and the 19del mutation probe comprises a plurality of sequences to cover common subtypes. The primer probe group can be combined with a microfluidic digital PCR platform for use, a sample is divided into tens of thousands of microdroplets for amplification, and FAM, HEX, ROX and CY5 fluorescence channel signals are detected to realize absolute quantification. The system provided by the invention has high sensitivity and excellent linearity and precision, is particularly suitable for detecting extremely-low-frequency EGFR mutation in body fluid such as plasma, and has important application value in individualized diagnosis and treatment, curative effect monitoring and drug resistance evaluation of non-small cell lung cancer.

The application provides a mussel mucin synthesis method and relates to the technical field of genetic engineering.The application improves the yield of mussel mucin by carrying out deletion mutation on the N-terminal sequence of the secondary structure of the mussel mucin amino acid sequence such as SEQ ID NO.1.The yield of mussel mucin in the prior art is about 2.57g / L, while the yield of mussel mucin in the application is about 5g / L, which is greatly improved compared with the yield of mussel mucin in the prior art.On this basis, the mussel mucin is modified by dopa through co-expression of tyrosinase, and the expression amount of the tyrosinase is controlled through different intensities of RBS, so as to regulate the modification degree of dopa, improve the yield of mussel mucin, and realize controllable modification of dopa in the mussel mucin, finally obtaining mussel mucin with high yield and different modification degrees of dopa.

The invention belongs to the technical field of biological detection, and provides a kit for detecting EGFR (epidermal growth factor receptor) 19del subtypes. According to the invention, by designing four universal upstream primer target segments and four probes combined with the upstream primers, different subtypes of EGFR19del can be accurately detected by a Tm value; by changing the fluorophore combination of the probe, single-tube multi-channel accurate typing can be realized. The primer tag segment and detection probe combination developed by the invention is not only suitable for deletion mutation, but also suitable for detection of fusion mutation and pathogenic microorganisms, and has wide applicability.

A primer pair, method, and use for rapidly identifying newborn nude mice. On the basis that a deletion mutation occurs in the base G at position 337 of a coding region of the Foxn1 gene in nude mice, a primer pair introducing an enzyme digestion site is designed according to sequence characteristics. Using the genome of a mouse to be tested as a template, the Foxn1 gene is amplified according to a PCR-RFLP method. The PCR product is then digested with a SmaI restriction endonuclease, and the digested product is genotyped by agarose gel electrophoresis. In addition, a reaction system that can be combined with lateral flow technology is screened, thereby achieving instant detection of the newborn nude mice. The method has the characteristics of simple operation, short time consumption, low cost, instant detection, etc.

This invention discloses transcription factors As NF-YB3 Its application in regulating drought resistance in oats falls under the field of biotechnology. (Transcription factors) As NF-YB3 The amino acid sequence is shown in SEQ ID NO.1. As NF-YB3 The CDS sequence of the gene is shown in SEQ ID NO.2, and the gDNA sequence is shown in SEQ ID NO.3. Overexpression As NF-YB3 The gene significantly enhances the drought resistance of oats, while also increasing the activity of CAT and SOD enzymes in oats, slowing down the water loss rate of detached leaves, and improving seedling survival rate under drought stress. AsNF-YB3 forms a heterotrimeric complex with AsNF-YA1 and AsNF-YC5, and its 4bp insertion / deletion mutation in the promoter region regulates oat drought resistance by affecting AsARF1 binding and gene expression. This provides a new gene resource and technical pathway for agricultural stress-resistance breeding.

This invention relates to the field of gene editing technology, specifically providing a method for creating herbicide-resistant rice by gene editing the promoter region of the OsEPSPS gene. The method involves introducing a WT / -29bp monoallelic deletion mutation into the OsEPSPS gene promoter region. Specifically, with position A of the OsEPSPS gene start codon ATG set to 0, a 29bp deletion is made in one allele located at positions 1122 to 1150 upstream of the start codon ATG, while the other allele remains unchanged. This invention demonstrates that by introducing this monoallelic deletion mutation, rice can acquire resistance to EPSPS inhibitor herbicides. This resistance was identified in the T0 generation plants carrying this mutation and can be stably inherited by their offspring, possessing significant application value for ensuring agricultural production safety and improving herbicide application efficiency.

The invention belongs to the technical field of gene editing and disease model construction, and particularly relates to a construction method and application of a Wilson disease animal model. Aiming at a large fragment deletion mutation type which exists clinically in Wilson disease but lacks a corresponding animal model, a CRISPR / Cas9 gene editing technology is utilized, a pair of sgRNAs is specifically designed, an eighth exon region of a mouse Atp7b gene is precisely cut and deleted, and a WD mouse model with deletion mutation (c.2333340delGACGGTGG) of eight basic groups of the eighth exon of the Atp7b gene is successfully constructed. The method can be used for research on pathogenesis of WD, screening of novel diagnostic markers and evaluation of curative effect of therapeutic drugs, is particularly suitable for preclinical evaluation of gene therapy strategies and development of adjuvant therapeutic drugs of targeted NLRP3 inflammasomes, and has great scientific research value and clinical transformation prospect.

The invention discloses a primer group, a kit and a detection system for thalassemia gene detection. In order to more comprehensively and accurately detect point mutation, small insertion, small deletion, gene fusion mutation, large fragment deletion and gene structure variation on a thalassemia core gene and a modified gene in single detection, the invention provides a thalassemia gene detection primer group and a kit. Based on third-generation sequencing platforms such as a single-base nanopore sequencing platform and the like, all point mutation, small insertion, small deletion and gene fusion mutation in the amplification range of the thalassemia core gene and the modified gene can be detected by utilizing the primer group or the kit; and 60 alpha large fragment deletion mutations, 60 beta large fragment deletion mutations and 7 gene structure variations. The method is beneficial to rapid and comprehensive screening of thalassemia, and is high in detection sensitivity and good in accuracy.

The invention relates to the technical field of gene editing, and particularly provides a method for creating fluazifop-p-butyl herbicide resistant rice through gene editing of an OsACCase gene promoter region. The method comprises the following steps: constructing a crRNA plasmid library covering an OsACCase gene promoter region on the basis of a CRISPR / LbCas12a system, performing agrobacterium-mediated transformation on rice callus, and performing resistance screening, fluazifop-p-butyl herbicide screening and molecular identification to obtain a fluazifop-p-butyl resistant rice strain. The strain is subjected to-4bp / -5bp double allelic deletion mutation in an upstream 1865-site region of an initiation codon ATG of the OsACCase gene, and a homozygous mutant strain and a double allelic deletion mutant strain can normally grow after being sprayed with 6 times of field recommended dose of fluazifop-p-butyl. The herbicide resistance is realized by editing the gene promoter region, the growth defect of coding region mutation is avoided, the breeding technology is enriched, and weed prevention and control are assisted.

The invention relates to a T7RNA polymerase mutant for synthesizing single-stranded DNA as well as a screening method and application of the T7RNA polymerase mutant, and belongs to the technical field of enzyme engineering. The invention provides a method for directed evolution and screening of a T7RNA polymerase mutant, which comprises the following steps: knocking out exonuclease and mismatch repair protein from a chassis strain genome, knocking in single-stranded annealing protein, resistance gene containing deletion mutation and a single-stranded DNA synthesis template, and constructing to obtain a genetically engineered bacterium for screening; a T7RNA polymerase mutant library is constructed, recombinant plasmids with the mutant library are transferred into engineering bacteria, T7RNA polymerase mutants synthesize single-stranded DNA to repair deletion mutation of resistance genes, the transformed engineering bacteria can grow on a resistance plate, the T7RNA polymerase mutants obtained through multiple rounds of screening can efficiently synthesize the single-stranded DNA through in-vitro verification, and the T7RNA polymerase mutant can be used for preparing the single-stranded DNA. A brand new tool is provided for synthesis of single-stranded DNA, and the method has the potential of being applied to the fields of gene editing, evolutionary engineering and the like.

The invention relates to the technical field of gene editing, and particularly provides rice OsEPSPS gene promoter region deletion mutation and application thereof in creation of herbicide-resistant rice, the deletion mutation is that a sequence with the length of 22bp or 28bp is deleted in the OsEPSPS gene promoter region, namely, A in an OsEPSPS gene initiation codon ATG is 0 site, A in the OsEPSPS gene initiation codon ATG is 0 site, A in the OsEPSPS gene initiation codon ATG is 0 site, and A in the OsEPSPS gene initiation codon ATG is 0 site; and deleting the basic groups at sites 2534 to 2555 upstream of the initiation codon ATG or the basic groups at sites 2527 to 2554 upstream of the initiation codon ATG. According to the application disclosed by the invention, the rice can obtain resistance to EPSPS inhibitor herbicides by deleting part of sequences in the promoter of the rice OsEPSPS gene, so that the application has great application values in guaranteeing agricultural production safety and improving the use efficiency of the herbicides.

A method of collecting data for diagnosing cancer, determining an onset risk, determining a malignancy grade, and / or predicting a prognosis of cancer according to the present disclosure includes the steps of bringing a sample derived from a subject into contact with tumor-associated antigens to cause an antigen-antibody reaction; measuring an amount of anti-p53 antibody, in the sample, that specifically binds to any of the tumor-associated antigens; and comparing the measured amount of the anti-p53 antibody with a predetermined reference level of the anti-p53 antibody against the tumor-associated antigens. The tumor-associated antigens include one or more p53 isoform variants consisting of amino acid sequences having a mutation in an amino acid sequence of any one of SEQ ID NOs: 1 to 3, and the mutation includes any one of an N-terminal deletion mutation, a C-terminal deletion mutation, and both the N-terminal deletion and the C-terminal deletion mutation.

The application belongs to the technical field of animal breeding, and particularly relates to application of a detection reagent of a molecular marker related to a long hair trait of a cashmere goat. The molecular marker is located at 155,344,701-155,346,600 on a goat chromosome 1, and a nucleotide sequence thereof is shown as SEQ ID NO. 1. It is found by the application that the length of wool of an individual with a normal type of the molecular marker region is greater than the length of wool of an individual with a deletion type of the molecular marker region, and thus a cashmere goat breed with long wool can be selected by detecting a deletion mutation of the molecular marker on the goat chromosome 1.

The invention relates to a rice OsACC gene promoter mutant for resisting ACCase inhibitor herbicides and application of the rice OsACC gene promoter mutant. The rice OsACC gene mutant is a deletion mutant or a deletion and combination insertion mutant; the deletion mutation occurs in the promoter of the rice OsACC gene, and the sequence of the nucleic acid before deletion mutation is as shown in SEQ ID No. 1; the length of the sequence of the deleted nucleic acid is 29 bp to 97 bp, A in the initiation codon ATG of the OsACC gene is 0 site, the deleted nucleic acid occurs in the 305-209 sites of the upstream of the initiation codon ATG, and at least the 271-243 sites of the upstream of the initiation codon ATG are deleted; the inserted nucleic acid occurs within sites 271 to 243 upstream of the initiation codon ATG. The mutant can enable rice to obtain resistance to at least one of herbicides such as haloxyfop-R-methyl, fluazifop-p-butyl and clethodim.

The invention discloses a monoclonal antibody capable of simultaneously identifying mycobacterium tuberculosis secretory protein 64 of a wild type (W-MPT64) and a 63bp deletion mutant type (D-MPT64). The amino acid sequences of variable regions of a heavy chain and a light chain of the antibody are shown as SEQ ID NO.1 and SEQ ID NO.5. The lowest detection limits of W-MPT64 and D-MPT64 by a mycobacterium tuberculosis MPT64 double-antibody sandwich enzyme-linked immunosorbent assay and a fluorescence immunochromatography assay established by using the monoclonal antibody both reach 1.5625 pg / mL, which are significantly higher than the lowest detection limit in the prior art, and the monoclonal antibody can be used for qualitative and quantitative detection of mycobacterium tuberculosis secretory protein 64.

This invention discloses a *Lactobacillus paracasei* S-NB gene deletion mutant strain, its construction method, and its application. The S-NB gene deletion mutant strain uses *Lactobacillus paracasei* S-NB as the starting strain, and the mutant strain contains *Lactobacillus paracasei* S-NB gene deletion mutants. cps The upstream segment of the gene was knocked out by the plasmid. cps Upstream homologous arm gene substitution in *Lactobacillus paracasei* S-NB cps The downstream gene fragment carried by the knockout plasmid cps Gene substitution in the downstream homologous arm of the gene; among which, cps The gene base sequence of the upstream homologous arm is shown in SEQ ID NO:1. cps The downstream homologous arm gene base sequence is shown in SEQ ID NO:2. Compared with traditional methods, the success rate of knockout vector construction in this invention reaches over 95%, and the operation is simple, time-saving, and the operation cycle is only 6-7 days; the knockout vector constructed using this invention can knock out Lactobacillus paracasei S-NB. cps Genes, the result cps Gene deletion mutants are unaffected in growth, but their capsule layer becomes thinner and their ability to produce biofilm decreases.

The present invention provides a novel therapeutic means for cancer cells resistant to epidermal growth factor receptor (EGFR)-targeted anticancer agents. Almost no cell death was observed when cancer cells having an exon 19 deletion mutation in the EGFR gene (EGFR Del19), cancer cells having a substitution mutation in which the 790th amino acid threonine in the EGFR protein is substituted with methionine (EGFR T790M), and cancer cells having a substitution mutation in which the 858th amino acid leucine is substituted with arginine (EGFR L858R) were treated solely with aripiprazole or solely with an EGFR-targeted anticancer agent. However, cell viability decreased and cleavage of the apoptosis marker PARP was observed when the cancer cells were treated with aripiprazole in combination with an EGFR-targeted anticancer agent. Therefore, aripiprazole is provided as an enhancer of the anticancer effect of EGFR-targeted anticancer agents.

The invention relates to a herbicide-resistant rice OsACC gene promoter mutant and application thereof. The rice OsACC gene mutant is a deletion mutant; wherein the deletion mutation occurs in the promoter of the rice OsACC gene, and the sequence of the nucleic acid before the deletion mutation is shown as SEQ ID No.1; the length of the sequence of the deleted nucleic acid is 14 bp to 30 bp, A in the initiation codon ATG of the OsACC gene is 0 site, the deleted nucleic acid occurs in 964 to 935 sites on the upstream of the initiation codon ATG, and at least bases on 958 to 945 sites on the upstream of the initiation codon ATG are deleted. The mutant can enable rice to obtain resistance to herbicide fluazifop-p-butyl and / or clethodim.

To provide a technique for inducing early flowering of a gramineous plant.SOLUTION: The method for inducing early flowering comprises culturing a gramineous plant (e.g. rice variety'Kyo no Yume') having a functionally deficient mutation of a gene encoding gibberellin biosynthetic enzyme protein GA3ox2 under gibberellin treatment and a method for inducing early flowering of the gramineous plant comprising gibberellin is provided.SELECTED DRAWING: Figure 3

The invention relates to application of PDCD5 in preparation of a diagnostic agent or a therapeutic agent for asthenospermia. The invention also discloses a method for constructing an asthenospermia mouse model or inhibiting fibroblast cilia generation by knocking out or knocking down the PDCD5. The application shows that PDCD5 participates in sperm generation, and asthenozoospermia can be caused if the PDCD5 has function deletion mutation in testis. Therefore, the gene locus can be used as a target spot for asthenospermia diagnosis and medication guidance. According to the method disclosed by the invention, the dysfunction of the site is supplemented by the PDCD5 RNA wrapped by the testis injection liposome, so that the asthenospermia caused by the mutation is rescued, the sperm quantity is increased, the sperm malformation rate is reduced, and a new path is provided for the treatment of the asthenospermia.

The application belongs to the technical field of aquatic organism breeding, and discloses a method for creating grass carp resistant to hemorrhagic disease by knocking out dhcr7 a gene and application thereof. dhcr7 The method utilizes CRISPR-Cas9 technology to generate an insertion / deletion mutation in a grass carp dhcr7 gene, so that the gene cannot encode a protein or the encoded protein does not have a function. dhcr7 The application first knocks out the innate immune negative regulation gene dhcr7 in grass carp, obtains a F0 generation population with a high mutation rate, and finds that knocking out significantly improves the resistance of grass carp to GCRV-II virus, thereby providing a method and breeding material for breeding grass carp resistant to hemorrhagic disease and having important industrial application value.

The application relates to the technical field of gene editing, and provides a method for creating herbicide-resistant rice through gene editing of an intron region of an OsEPSPS gene, specifically, a WT / -22bp single allelic deletion mutation or a WT / -32+6bp single allelic deletion insertion mutation is introduced into the intron region of the OsEPSPS gene, that is, taking A in an initiation codon ATG of the OsEPSPS gene as the 0 position, 22bp is deleted from 771 to 792 downstream of the 0 position on one allele or 32bp is deleted from 775 to 806 downstream of the 0 position and 6bp is inserted into the interval, and the other allele remains unchanged. It is found by the application that the rice can obtain resistance to EPSPS inhibitor herbicides by introducing the above-mentioned mutation, the resistance is identified in T0 generation plants carrying the mutation, and can be stably inherited to the offspring plants, and the application value for improving the use efficiency of herbicides is extremely high.

The invention relates to the technical field of gene engineering technology, in particular to a preparation method of an efficient and stable gene knockout cell line. According to the AKAP11 gene knockout cell line provided by the invention, the 6th exon of the AKAP11 gene in the cell line is subjected to 3395bp frameshift deletion mutation, so that a coding frame is shifted, and protein translation is terminated in advance; after continuous subculture, the homozygous knockout phenotype is still maintained through PCR sequencing verification. A CRISPR / Cas9 system is combined with puromycin screening, the positive cloning rate reaches 30% or above, the construction period is shortened to 45 days, the constructed cell line can be directly used for AKAP11 related cell signal path research, disease model construction and targeted drug screening, and a reliable tool is provided for basic research and clinical transformation.

The invention relates to the technical field of biological medicine, provides novel application of STC2, and particularly provides application of STC2 or combined immunotherapy in treatment of ARID1A deficiency lung cancer. According to the invention, by combining the siRNA technology with an exosome vesicle delivery system, an action mechanism that ARID1A affects a tumor cell ferroptosis process by regulating the transcriptional activity of an STC2 promoter region is disclosed for the first time. Experimental results show that an STC2-targeted inhibitor (such as siRNA) can specifically silence the mRNA expression of the STC2-targeted inhibitor, and the resistance effect on AA-resistant induced ferroptosis is reduced, so that the ARID1A is proved to possibly regulate cells to tolerate AA and IFN combined induced ferroptosis through STC2, further the drug resistance of tumor cells to a PD-1 inhibitor caused by deletion of ARID1A is reversed, and the anti-tumor curative effect is remarkably enhanced. The discovery provides a new thought and an experimental basis for an immunotherapy strategy of the ARID1A deletion mutation patient.

The invention provides application of mitochondrial vesicles in reduction of mitochondrial DNA deletion mutation. The mitochondrial vesicles are prepared by wrapping mitochondria with erythrocyte membranes, the mitochondrial vesicles can be tested and verified to effectively reduce the mitochondrial DNA deletion mutation level in cells, metabolism and functions of mutant cells are improved, verification is further carried out in muscle cells of CPEO patients, and the clinical application prospect is wide. By implanting the mitochondrial vesicles, the mitochondrial DNA deletion mutation level in the CPEO patient muscle cells can be effectively reduced, which indicates that the mitochondrial vesicles have the potential of treating CPEO, and a new thought is provided for treating diseases related to mitochondrial DNA deletion mutation.

The invention belongs to the field of gene engineering, and relates to a wheat TaPRR95 gene and application thereof in regulating early aging of wheat flag leaves. The gene mutant disclosed by the invention is formed by carrying out base deletion mutation on a coding region of a wheat wild type fielderTaPRR95 gene, and the gene numbers of a genome A, a genome B and a genome D of the TaPRR95 of heterohexaploid wheat are TraesCS5A02G320300, TraesCS5B02G320500 and TraesCS5D02G326200 in sequence. According to the present invention, the CRISPR / Cas9 gene editing technology is adopted to create the mutant, the mutant can cause early aging of wheat flag leaves, the photosynthesis and grouting characteristics of wheat are improved by regulating the TaPRR95 gene expression level, and the new approach is provided for wheat yield regulation. The invention fills the blank of wheat TaPRR95 gene function research, and provides key gene resources and breeding technical means.

The invention relates to the technical field of gene editing, and provides a method for creating herbicide-resistant rice through gene editing of an intron region of an OsEPSPS gene, and particularly relates to a method for introducing single allelic deletion mutation of WT / 22bp or single allelic deletion insertion mutation of WT / 32 + 6bp into the intron region of the OsEPSPS gene, i.e., A in an initiation codon ATG of the OsEPSPS gene is 0 site, A in the initiation codon ATG of the OsEPSPS gene is 0 site, A in the initiation codon ATG of the OsEPSPS gene is 0 site, and A in the initiation codon ATG of the OsEPSPS gene is 0 site; on one allele, 22bp is deleted from the 771 site to the 792 site at the downstream of the allele or 32bp is deleted from the 775 site to the 806 site at the downstream of the allele, and 6bp is inserted in the interval, and the other allele is kept unchanged. It is found that by introducing the mutation, rice can obtain resistance to EPSPS inhibitor herbicides, the resistance is identified in T0-generation plants carrying the mutation and can be stably inherited to offspring plants of the T0-generation plants, and the application value for improving the use efficiency of the herbicides is extremely high.

The invention discloses a preparation method of an Iars1 gene knockout zebrafish model, which comprises the following steps: mixing sgRNA fragments and Cas9 protein, injecting a mixed solution into zebrafish fertilized eggs in 1-2 cell stages through micro-injection, and obtaining Iars1 gene edited individuals after the fertilized eggs are hatched and cultured; after genotype identification, family screening and continuous breeding, the stably inherited Iars1 gene knockout zebrafish mutant strain is obtained. According to the invention, a zebrafish Iars1 gene is subjected to targeted editing by adopting a CRISPR / Cas9 technology, a zebrafish model carrying deletion mutation is obtained, and a stably inherited mutant is obtained through molecular identification. The model constructed by the invention can be used for researching early development abnormality related to Iars1 function deficiency, and an experimental model can be provided for evaluating early nutrition utilization abnormality, protein translation related abnormality and candidate interveners. The construction method is reliable, the model phenotype is clear, and the method has good research and application prospects.