44 results about "Insertional mutation" patented technology

The invention discloses enterobacter mori capable of resisting fish aeromonas and application of the enterobacter mori, the bacterial strain is classified and named as enterobacter mori SCH0241, the enterobacter mori SCH0241 is preserved in the China Center for Type Culture Collection on October 17, 2024, and the preservation number is CCTCC NO: M 20242236. The enterobacter mori SCH0241 disclosed by the invention is derived from the intestinal tract of high-stress-resistance healthy hybrid crucian carassius auratus No. 2, has good contact dependence antibacterial activity on aeromonas veronii, and can improve the capability of resisting aeromonas veronii infection of zebra fish. Through construction of an insertion mutation library and phenotype screening, it is found that a gene cluster of SCH0241, which exerts contact inhibition toxicity, is T6SS-2. The enterobacter mori SCH0241 disclosed by the invention provides a new powerful tool for biological prevention and treatment of fish diseases caused by aeromonas veronii.

The invention belongs to the technical field of microbial engineering, and discloses a method for producing eicosapentaenoic acid (EPA) with high yield by schizochytrium limacinum based on random mutation and low-temperature screening and application, and the method comprises the following steps: driving overexpression of a G418 resistance gene through a P2520 promoter, constructing a random insertion mutant library, and screening to obtain a schizochytrium limacinum mutant strain with a randomly mutated genome; coating in a culture medium containing G418 resistance for culturing, and screening out strains which are resistant to low temperature and good in growth state at 15 DEG C; and inoculating into a fermentation culture medium, adding MgSO4 with the final mass concentration of 0.1% at the initial stage of fermentation, supplementing MnCl2 with the final mass concentration of 0.05% when fermentation is performed for 72 hours, and culturing to obtain a mutant strain. According to the invention, the G418 resistance gene carried by the plasmid is randomly inserted into the schizochytrium limacinum genome, and low-temperature induced screening is combined, so that the target limitation of traditional homologous recombination is broken through, and a stable strain with multiple mutation superposition can be obtained through enrichment.

The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel elements that further improve expression. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery.

Disclosed is a detection method for a dominant clone caused by an exogenous DNA insertion mutation, comprising: carrying out pretreatment, including plus-minus strand correction and redundancy integration, on virus IS information data; step 2, when the pretreatment is completed, identifying ISs caused by non-specific amplification so as to remove a false positive IS detection result caused by PCR non-specific amplification; step 3, merging the ISs at neighboring positions on the basis of a site clustering algorithm, and clustering the ISs; and step 4, analyzing a clone plane on the basis of the clustering result, and describing a dominant clone. According to the present invention, a PCR primer similarity ranking algorithm is utilized to identify and eliminate non-specific amplification potentially caused by PCR primer similarity, significantly reducing false positives of IS identification in practical applications, improving the accuracy.

The invention discloses a bacteriophage genome editing vector based on a CRISPR-Cas9 system as well as an editing method and application of the bacteriophage genome editing vector. According to the invention, a plasmid containing a cas9 gene and an sgRNA element is successfully constructed, a specific primer is designed for an editing site, and a pTarget plasmid is obtained; a donor DNA sequence is constructed on a vector, and a pEdit plasmid is obtained. After the bacteriophage is infected with host bacteria containing pEdit plasmids, the mutant bacteriophage is reversely screened through pTarget plasmids, and efficient and rapid gene editing, including gene deletion mutation, mononucleotide replacement mutation and insertion mutation, of the bacteriophage genome is achieved. The method is simple to operate and high in gene editing efficiency, promotes the structural analysis and functional research development of the staphylococcus aureus bacteriophage genome, provides a new strategy for the transformation of engineering bacteriophages, and has wide application prospects and market values.

The present invention contemplates-allele-specific gene editing based on targeting a heterozygous single nucleotide polymorphism (SNP) in a protein coding sequence associated with a genetic disease. The data shown herein demonstrates that the outcome of such gene editing creates a nonesense mutation that results in a marked and selective reduction of mutant protein without affecting wild type protein expression. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion / deletion mutations and viral vector insertions. Thus, as disclosed herein, allele-specific targeting of InDel and insertion mutations to heterozygous coding SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.

The present disclosure provides methods of treating cancer in a patient determined to have an EGFR and / or HER2 exon 20 mutation, such as an insertion mutation, by administering a third-generation tyrosine kinase inhibitor, such as poziotinib or afatinib.

The application discloses a detection method based on dominant clones caused by exogenous DNA insertion mutation, and the method comprises the following steps: 1, preprocessing of virus IS information data by positive and negative strand correction and redundant integration; 2, after the preprocessing is completed, IS caused by non-specific amplification is identified to remove false positive IS detection results caused by PCR non-specific amplification; 3, according to a site clustering algorithm, IS in adjacent positions is combined and clustered; 4, according to the clustering results, a clone plane is analyzed, and dominant clones are described; and the application utilizes a PCR primer similarity ranking algorithm to identify and remove non-specific amplification caused by possible PCR primer similarity, so that the false positive of IS identification is significantly reduced in actual application, and the accuracy is improved.

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 discloses medicago truncatula plant type development control protein as well as a gene coded by the medicago truncatula plant type development control protein and application. The method comprises the following steps: firstly, finding a Tnt1 insertion mutant mini-1 with a changed plant type from a medicago truncatula Tnt1 insertion mutant library, cloning a control gene MINI for plant type development by combining whole genome re-sequencing with flanking sequence analysis, and then inversely screening a natural mutant mini-2 of the MINI gene in the mutant library in order to prove that the MINI is the control gene for plant type development. Meanwhile, a MINI gene knockout strain mini-3 is also constructed, and analysis finds that phenotypes of mini-2 and mini-3 are completely the same as those of a mini-1 mutant. The results prove that the MINI gene provided by the invention can regulate and control the plant type. The method lays a foundation for plant type development research of leguminous plants such as alfalfa and other dicotyledonous plants.

The invention discloses an epegRNA (Ribonucleic Acid) primer pair for detecting an Indel marker related to porcine diarrhea virus resistance and application of the epegRNA primer pair. According to the epegRNA sequence provided by the invention, accurate AACA insertion mutation can be realized on a sequence of which the pig genome position is ChrX: 56448702 through pilot editing; the A / AAACA mutation can be used as a molecular marker to be applied to breeding for resisting porcine epidemic diarrhea.

The present application belongs to the technical field of fungal genetic breeding, and particularly relates to a molecular marker associated with the number of Lentinula edodes and application. The molecular marker is located at the 3865993th locus of chromosome 7 of the genome of Lentinula edodes, and is a 15bp insertion mutation. By using the molecular marker and detection method provided by the present application, the number of fruiting bodies (number of mushrooms) of Lentinula edodes at the mushroom stage can be efficiently determined at the mycelium stage, so as to accelerate the breeding process and improve the breeding efficiency. The molecular marker is a universal molecular marker, is less limited by genetic materials, and is widely applied.

The invention relates to the technical field of gene editing. Specifically, the invention provides a method for creating herbicide-resistant rice through gene editing of a 3 'UTR region of an OsEPSPS gene, and the method is characterized in that-38 / -13 + 12bp double allelic deletion insertion mutation is introduced into the 3' UTR region of the OsEPSPS gene, i.e., A in an initiation codon ATG of the OsEPSPS gene is taken as a 0 site, two alleles are respectively positioned at the 3363 to 3400 site deletion 38bp of the downstream of the OsEPSPS gene and at the 3377 to 3389 site deletion 13bp of the downstream of the OsEPSPS gene, and 12bp is inserted in the section. It is found that by introducing the double allelic deletion insertion 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 guaranteeing agricultural production safety and improving the use efficiency of the herbicides is extremely high.

The invention discloses an arabidopsis thaliana VHA-B1 gene, a coding protein thereof, a mutant, and preparation, identification and application thereof, and the preparation of the mutant comprises the following steps: respectively targeting a ninth exon and / or a first exon of a VHA-B1 gene (such as SEQ ID NO.1) through a T-DNA insertion and / or gene editing technology to obtain an insertion mutant and / or a gene editing mutant, and carrying out amplification on the insertion mutant and / or the gene editing mutant to obtain the VHA-B1 gene. And identifying the insertion mutant and / or the gene editing mutant through PCR (Polymerase Chain Reaction) to obtain the homozygous mutant with VHA-B1 gene deletion. The tolerance of the obtained purified mutant to cucumber mosaic virus (CMV) is weakened, which shows that the Arabidopsis thaliana VHA-B1 gene can improve the CMV tolerance of the plant, and an application basis is provided for culturing antiviral crop varieties by using the VHA-B1 gene.

The present invention discloses a design method for primers and probes for amplifying low-concentration mutant target sequences. First, the mutation position (mutation point, i.e., position 0) of the target sequence to be amplified is determined; then, a 15-25bp nucleic acid fragment including the 0 base is selected in the negative direction of the mutation point as a forward primer, and a 12-25bp nucleic acid sequence is selected from the ‑1 base or the 0 base in the positive direction of the mutation point as the probe sequence of the amplification system; finally, a reverse primer is designed according to a conventional method at a suitable position downstream of the 3' direction of the probe sequence. The primers and probes designed according to the method of the present invention can effectively (high specificity and high efficiency) amplify the target fragment under the background of a higher content of wild-type template, especially for point mutations, deletion mutations, and insertion mutations in gene mutations, etc., combined with fluorescent real-time PCR technology, can effectively solve the difficulty of insufficient sensitivity in current clinical tumor detection and drug sensitivity detection.

The present application discloses a strain of Enterobacter mori against Aeromonas veronii and its application, which is classified and named as Enterobacter mori (SCH0241). Enterobacter mori SCH0241, preserved in China Center for Type Culture Collection on October 17, 2024, with a preservation number of CCTCC NO: M 20242236. The Enterobacter mori SCH0241 of the present application is derived from the intestine of a highly stress-resistant healthy hybrid crucian carp “Hefang crucian carp No. 2”, has good contact-dependent bacteriostatic activity against Aeromonas veronii, and can improve the ability of zebrafish to resist Aeromonas veronii infection. Through the construction of an insertion mutation library and phenotype screening, it is found that the gene cluster of T6SS-2 of SCH0241 plays a contact inhibition toxicity. The Enterobacter mori SCH0241 of the present application provides a new powerful tool for the biological control of fish diseases caused by Aeromonas veronii.

To provide methods of treating cancer in a patient determined to have an EGFR and / or HER2 exon 20 mutation, such as an insertion mutation.SOLUTION: The present invention provides a method for treating cancer in a subject determined to have one or more EGFR exon 20 mutations, the method comprising a step of administering to the subject a third-generation tyrosine kinase inhibitor such as poziotinib or afatinib in an effective amount.SELECTED DRAWING: None

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 present disclosure provides compositions and methods of use of nucleic acids and complexes thereof targeted to nucleic acids. Genomic engineering can refer to the alteration of a genome by deletion, insertion, mutation, or replacement of a particular nucleic acid sequence. The alteration can be gene or position specific. Genomic engineering can utilize nucleases to cleave nucleic acids, thereby generating a site for alteration. Engineering of non-genomic nucleic acids is also contemplated.

The present invention discloses a primer composition and reagent for detecting human SMN1 and SMN2 gene mutations based on NGS method. The primer composition can specifically amplify all exons and part of introns or UTR adjacent regions of SMN1 and SMN2 genes in a single tube by multiple PCR method, and the constructed library is then used for NGS sequencing, so as to realize the report of the overall copy number of SMN1 and SMN2 genes of patients and the copy number variation of each of the 7th and 8th exons, and also can report the pathogenic point mutations and short deletion / insertion mutations in other regions of the two genes. The present invention can meet the needs of SMA pathogenic gene screening, genetic risk analysis, disease prognosis assessment, treatment guidance, etc. The overall process is simple and efficient, low cost, and has good prospects.

The invention belongs to the technical field of bioengineering, and particularly relates to a genetic manipulation method for a large-scale DNA fragment of a bacterial genome. The technical problem to be solved by the invention is to provide a new choice for genetic manipulation of large-scale DNA fragments. According to the technical scheme, the vector suitable for genetic manipulation of the large-scale DNA fragment of the bacterial genome takes a suicide plasmid vector as a skeleton vector and carries the following structures: a conditional replication starting point, a homologous recombination element, a positive selection marker, a negative selection marker and a transferable element; the homologous recombination element is a deletion homologous recombination element or an insertion homologous recombination element. The invention also provides a bacterial genome large-scale DNA fragment genetic manipulation method based on the vector. According to the technology, large-scale DNA fragments can be accurately inserted into bacterial genomes, and traceless insertion mutation without any antibiotic selection marker can be realized.

The invention discloses an InDel marker located on a chromosome 9 of a pig and capable of regulating and controlling the papillary number heterosis of the pig and application of the InDel marker. The nucleotide sequence of the InDel marker is shown as SEQ ID NO: 1 or SEQ ID NO: 2; the site is located between the 79th site and the 80th site from the 5'end of the nucleotide sequence shown in SEQ ID NO: 1, and corresponds to insertion mutation of C between the 11197897 bp and the 11197898 bp on the No.9 chromosome of the international pig reference genome version 11.1. The InDel marker provided by the invention can be applied to crossbreeding of pigs so as to improve the nipple number heterosis of offspring pigs and realize genetic improvement of the pigs, and by breeding the pigs with the InDel marker genotype of T / T, the reproductive capacity of sows can be remarkably improved, and the number of weaned piglets can be increased.

The invention belongs to the technical field of biology, and discloses a plasmid for genetic manipulation of bacillus licheniformis and application of the plasmid. The temperature-sensitive plasmid pTSMK created by the invention can be transferred into bacillus licheniformis in a conjugational transfer manner, and has both high efficiency and universality. The plasmid is used for carrying target DNA (deoxyribonucleic acid), so that the genetic manipulation of the bacillus licheniformis can be realized, such as DNA knockout, DNA knockin and transposon random insertion mutation.

The invention relates to the technical field of plant genetic engineering application, and particularly provides application of a mutant transcription factor AHL14 gene in regulating and controlling the oil content of plant seeds and a method for improving the oil content of the plant seeds. According to the invention, the homozygous mutants ahl14-1 and ahl14-2 are obtained by identifying the DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) levels of T-DNA insertion mutants of two AHL14 transcription factors. Phenotypic analysis shows that compared with wild Col-0, the total oil production of ahl14-1 and ahl14-2 mutant plant seeds is remarkably improved, and meanwhile, the seed size and thousand seed weight of the mutant plants are not reduced compared with those of the wild Col-0. The AHL14 gene disclosed by the invention provides a new excellent gene resource for molecular design breeding of oil crops, and has good reference value and application prospect in the aspect of high-oil breeding of the oil crops.

The invention discloses a circRNA-BMP2 delivery system for plant vesicles as well as a preparation method and application of the circRNA-BMP2 delivery system, and belongs to the technical field of biomedicine. According to the invention, the gastrodia elata vesicles are taken as a delivery carrier of circRNA-BMP2 and are fixed on the surface of the micro-arc oxidation implant, so that double-effect synergistic improvement of diabetes mellitus osseointegration is realized. Due to the beneficial components and the unique hollow biological membrane structure, the gastrodia elata vesicles are considered as potential candidate drugs for treating diseases, the plant-derived phospholipid structure of the gastrodia elata vesicles endows circRNA with a long-acting protection effect, and the risk of DNA insertion mutation is avoided. Meanwhile, the large-scale production cost of the gastrodia elata vesicles is reduced, and the gastrodia elata vesicles are free of animal The system breaks through the bottlenecks of short osteogenic factor release period, poor targeting property and unregulated inflammation microenvironment in the prior art, solves the problem that the inflammation microenvironment damages bone metabolism balance in a diabetic environment, and realizes long-acting slow release and stable expression of drugs.

The present invention discloses a phage genome editing vector based on the CRISPR-Cas9 system and its editing method and application. red9 A plasmid containing the gene and sgRNA elements is constructed, and specific primers are designed for the editing site to obtain the pTarget plasmid; the donor DNA sequence is constructed into a vector to obtain the pEdit plasmid. After phage is infected with a host bacteriophage containing the pEdit plasmid, the pTarget plasmid is used to reverse screen for mutant phage, achieving efficient and rapid gene editing of the phage genome, including gene deletion mutations, single nucleotide substitution mutations, and insertion mutations. This method is simple to operate and has high gene editing efficiency. It has promoted the development of structural analysis and functional research of Staphylococcus aureus phage genomes and provided a new strategy for the modification of engineered phages, with broad application prospects and market value.

The present invention relates to a new crystal form of Zipalertinib, a preparation method therefor, a pharmaceutical composition containing the crystal form, and the use of the crystal form in the preparation of an EGFR inhibitor drug and a drug for treating non-small cell lung cancer with an EGFR exon 20 insertion mutation.

The invention discloses arabidopsis thaliana myosin binding protein MyoBs and application thereof, and belongs to the technical field of biology. Mutant homozygous identification and screening are carried out on MyoB I (A) subfamily four members, pollen, pod length and seed phenotype of the homozygous inserted mutant are observed, it is found that flower morphology and pollen grain activity of a single mutant do not have abnormal phenotype, but the pod length of the single mutant is shorter than that of a wild type, and the single mutant has the advantages of being high in yield and high in yield. Therefore, the yield is also obviously lower than that of the wild type. According to the invention, phenotypes of short and small fruit pods and wizened seeds appear in a plant in an arabidopsis thaliana MyoBs protein single mutant, so that the importance of the MyoBs protein in a plant growth and development process is clarified, and a theoretical basis is provided for subsequent plant breeding.

The invention belongs to the technical field of fungus genetic breeding, and particularly relates to a molecular marker associated with the number of shiitake mushrooms and application. The molecular marker is located at the 3865993th site of a chromosome 7 of a mushroom genome and is a section of insertion mutation of 15bp. By utilizing the molecular marker and the detection method provided by the invention, the quantity (mushroom number) of mushroom sporocarp in the fruiting stage can be efficiently judged in the mushroom hypha stage, so that the breeding process is accelerated, and the breeding efficiency is improved. The molecular marker is a universal molecular marker, is slightly limited by genetic materials and is wide in application.

The invention relates to the technical field of plant genetic engineering application, and particularly provides application of a mutant transcription factor AHL29 gene in regulating and controlling the oil content of plant seeds and a method for improving the oil content of the plant seeds. According to the invention, the homozygous mutants ahl29-2 and ahl29-3 are obtained by identifying the DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) levels of T-DNA insertion mutants of two AHL29 transcription factors. Phenotypic analysis shows that compared with wild Col-0, the total oil production of ahl29-2 and ahl29-3 mutant plant seeds is remarkably improved, and meanwhile, the seed size and thousand seed weight of the mutant plants are not reduced compared with those of the wild Col-0. The AHL29 gene disclosed by the invention provides a new excellent gene resource for molecular design breeding of oil crops, and has good reference value and application prospect in the aspect of high-oil breeding of the oil crops.