31 results about "Exogenous DNA" patented technology

The invention belongs to the technical field of agricultural biology, and particularly relates to a plant constitutive promoter OsSULTR2. 2pro and an application thereof. The promoter is characterized in that the promoter is OsSULTR2; the nucleotide sequence of the 2pro is as shown in SEQ ID NO. 1. The promoter provided by the invention is OsSULTR2; 2pro is a rice endogenous constitutive promoter, can drive a target gene to realize efficient and stable expression in plant callus, roots, stems, leaves, young ears, seeds and other tissues in a vegetative growth period, and can replace an existing non-plant-source promoter. The method has important application value in the field of plant genetic engineering, and can effectively reduce potential safety risks of transgenic plants caused by introduction of exogenous DNA.

The invention provides a method for cultivating anti-aging glutinous highland barley, highland barley starch as well as a preparation method and application of the highland barley starch, and particularly relates to a method for improving the starch quality character of an existing high-quality glutinous highland barley variety, in particular to a method for cultivating anti-aging glutinous highland barley by utilizing a gene editing technology without exogenous DNA (Deoxyribose Nucleic Acid) integration. A method for fixed-point knockout of an SSIIa gene to cultivate an anti-aging glutinous highland barley new strain under the background of a "Zhengnongglutinous 232" variety comprises the following steps: providing an acceptor material derived from a highland barley variety "Zhengnongglutinous 232" with a glutinous character, and deleting a Wx gene function of the "Zhengnongglutinous 232"; the method comprises the following steps: assembling guide RNA (Ribonucleic Acid) designed aiming at an SSIIa gene and Cas9 protein in vitro to form a ribonucleoprotein RNP complex; introducing the RNP complex into cells of an acceptor material so as to enable Cas9 protein to generate double-chain breakage at a target site of the SSIIa gene; carrying out culture and plant regeneration on the introduced cells to obtain a T0-generation plant of which the SSIIa gene is edited; and screening plants with the function loss of the SSIIa gene, wherein the plants keep the glutinous character of adult agricultural glutinous 232.

The invention relates to a method for splitting, assembling and site-specific integration of large-fragment exogenous DNA (deoxyribonucleic acid) in Trichoderma reesei. Comprising the following steps: (1) constructing an arg:: Ptcu-Cas9-TtrpC-hph strain containing a Cas9 protein; (2) constructing a pUC19 <-> integration site: a pyr4 plasmid; (3) obtaining a plurality of target gene segments containing integration site homologous arms and having overlapping regions at the tail ends of adjacent DNA segments; (4) obtaining a gRNA expression cassette of a targeted genome integration site; (5) arg conversion: the arg is converted into a Ptcu-Cas9-TtrpC-hph strain; and (6) screening and verifying. According to the method, the CRISPR / Cas9 system is combined with the TAR in the trichoderma reesei for the first time, the exogenous large-fragment DNA is split into a plurality of small fragments, then the plurality of small-fragment exogenous DNA are assembled in the trichoderma reesei and are subjected to fixed-point integration, one-step editing of the large-fragment exogenous DNA is realized, and compared with a traditional genetics method, the gene editing efficiency in the trichoderma reesei is greatly improved.

This invention discloses a gene editing method and application for *Trichoderma reesei* based on a ribonucleoprotein complex. It aims to overcome the problems of low editing efficiency, easy generation of heterozygotes, and risk of exogenous DNA integration in *Trichoderma reesei* using plasmid-based CRISPR / Cas9 systems. This invention combines a dual-site cleavage strategy guided by double sgRNAs with direct RNP delivery. The method involves: designing two guide RNAs spaced 50-700 bp apart for the target gene, pre-assembling them with Cas9 protein in vitro to form an active RNP complex; delivering this complex along with exogenous donor DNA to *Trichoderma reesei* protoplasts. The exogenous donor DNA contains a left homologous arm, an editing sequence, and a right homologous arm, wherein the left and right homologous arms are homologous to the genomic sequences outside the dual cleavage sites on the target gene determined by the double sgRNAs, respectively; upon entering the cell, the RNP complex introduces dual-site cleavage at the target site, achieving precise editing under the guidance of the exogenous donor DNA.

The invention discloses a construction method and application of a kluyveromyces marxianus mutant, and belongs to the field of synthetic biology and molecular biology. According to the method, single-stranded DNA binding protein and cytosine deaminase pmCDA1 are fused, a random base editing system is constructed, and whole-genome efficient C-T / G-A mutation is realized in kluyveromyces marxianus by taking MCM2-7 as a verification target. The system does not need a specific target or an exogenous DNA template, the mutation efficiency is high, the distribution is wide, the mutation rate is improved by 303.1 times compared with that of a control group through optimal combination, and the application potential of kluyveromyces marxianus in industrial production is expanded. The method is combined with an efficient screening system, mutant strains with the intracellular protein expression quantity remarkably improved are screened out, the constructed mutation system is not only suitable for kluyveromyces marxianus cell factory transformation, but also can be popularized to genome evolution of other yeasts or eukaryotic hosts, and a new strategy is provided for industrial microorganism breeding and protein engineering.

This invention belongs to the field of agricultural biotechnology, specifically relating to a plant constitutive promoter OsSULTR2;2pro and its applications. The nucleotide sequence of the promoter OsSULTR2;2pro is shown in SEQ ID NO.1. The promoter OsSULTR2;2pro of this invention is an endogenous constitutive promoter for rice, capable of driving efficient and stable expression of target genes in plant callus tissue, roots, stems, leaves during vegetative growth, as well as young panicles and seeds, and can replace existing non-plant-derived promoters. It has significant application value in the field of plant genetic engineering and can effectively reduce the potential safety risks of transgenic plants caused by the introduction of exogenous DNA.

The invention belongs to the technical field of agricultural biology, and particularly relates to a rice endogenous space-time specific promoter OsSULTR1; the invention discloses 3pro and application thereof. The promoter OsSULTR1 is characterized in that the promoter OsSULTR1 The nucleotide sequence of 3pro is as shown in SEQ ID NO. 1. The invention provides an OsSULTR1; 3pro is a space-time specific expression regulation element from rice, can efficiently and stably drive a target gene to be expressed in a plant callus, and can realize targeted efficient and stable expression in root and stem tissues in a plant vegetative growth period. The promoter can effectively replace a non-plant-source promoter commonly used in existing plant genetic engineering, has an important industrial application value in the field of plant genetic engineering, and not only can improve the targeting and stability of target gene expression in transgenic plants, but also can improve the yield of the transgenic plants. And the potential biological safety risk of the transgenic plant caused after the exogenous DNA is introduced into the plant genome can be obviously reduced.

The invention discloses a method for creating high-anthocyanin tomatoes through a gene knock-in technology. A homologous recombination mediated gene knock-in technology is utilized, the Aft gene of the tomato is knocked into the downstream of a promoter of a fruit ripening specific gene E8 at a fixed point, the E8 promoter is successfully utilized to activate Aft gene expression, and the high-anthocyanin tomato with purple black tomato fruit is obtained; and further performing selfing on the high-anthocyanin tomatoes, and screening out non-transgenic high-anthocyanin tomatoes which are subjected to Aft gene homozygous insertion and do not carry exogenous DNA fragments from selfing filial generations of the high-anthocyanin tomatoes. Therefore, the gene editing method disclosed by the invention can be used for quickly converting the common tomato without accumulation of anthocyanin into a high-nutrition tomato material rich in anthocyanin, and has great breeding application prospect and economic value.

The present application belongs to the field of microbial metabolic engineering and industrial microorganism technology, and particularly relates to a method for stably integrating exogenous DNA into Pseudozyma roseolata, an iterative gene editing method and application thereof. The preservation number of the strain MYL-2 is CCTCC M 20252968, and the strain can efficiently accumulate oil under nitrogen-limited culture conditions, the main fatty acid components in the cells are C16-C18 fatty acids, and the strain has application potential as a microbial oil production strain. The present application provides a method for stably integrating exogenous DNA into Pseudozyma roseolata, and constructs a recombinant expression vector pNFH-loxp-RAD51 and a Cre recombination vector containing a loxP site, and for the first time, precise removal and repeated use of a screening marker mediated by a Cre-LoxP system are realized in the strain, thereby laying a foundation for multi-round gene editing and efficient engineering modification of the strain.

The present application relates to the field of agricultural biotechnology, and particularly relates to a CEPro722 promoter and application thereof.The CEPro722 promoter comprises a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence as shown in SEQ ID NO.2.The present application is screened from a GRMZM2G069722 gene, and the CEPro722 promoter can drive gene expression in plants such as rice, wheat or corn; and particularly, the promoter can drive high and stable expression in various tissues such as plant callus, leaf in the vegetative growth period, young ear and seed, and can replace a non-plant source promoter.The CEPro722 promoter provided in the present application has important significance in the field of plant genetic engineering, and can effectively reduce potential safety risks of transgenic plants caused by exogenous DNA.

The present invention is directed to a genome editing kit for targeted integration of an exogenous DNA in a target site of a large gene by non-homologous end joining (NHEJ) or by microhomology-mediated end joining (MMEJ), wherein said kit comprises donor nucleic acids for replacing the 5' and 3' portions of the target gene relative to the target site.

The invention discloses a method for targeted blocking of antibiotic resistance gene horizontal transfer based on replication-defective engineering bacteriophage and application, and belongs to the technical field of environmental microbiology and gene engineering. In order to solve the problems of ARGs release caused by a splitting strategy and biosafety risk of the engineering bacteriophage, the replication-defective engineering bacteriophage obtained by packaging a CRISPR-Cas9 system with a targeted cleavage resistance gene through a packaging system related to a bacteriophage display technology can reduce the risk of drug-resistant gene diffusion in an environment and a medical scene. The method has the main advantages that: 1) dual specificity: the method has the recognition capability of phage on specific host phenotype and the specific recognition of CRISPR-Cas9 on ARGs sequence, and dual precise targeting is realized; 2) the biosafety is high: after the element is delivered, the element cannot be copied in a host, the exogenous DNA is easily degraded by the host, and the residue and unexpected evolution of the engineering bacteriophage in the environment are avoided;

The application belongs to the field of agricultural biotechnology, and particularly relates to a plant constitutive promoter OsSULTR1;2pro and application thereof. The nucleotide sequence of the promoter OsSULTR1;2pro is shown as SEQ ID NO. 1. The promoter OsSULTR1;2pro is an endogenous constitutive promoter of rice, and can drive efficient and stable expression of a target gene in plant callus, roots, stems, leaves and grains in the vegetative growth period, and can replace existing non-plant origin promoters. The application has important application value in the field of plant genetic engineering, and can effectively reduce potential safety risks of transgenic plants caused by introduction of exogenous DNA.

The invention discloses a herbicide-resistant alfalfa transformation event TWMS2-17 and a detection method thereof, an exogenous DNA fragment is inserted into an alfalfa genome, and herbicide-resistant plants TWMS2-17 are obtained through herbicide spraying treatment and single-copy plant screening. Through analysis and verification of a flanking sequence of a T-DNA insertion site, the position of an exogenous gene inserted into a genome is a No.3 chromosome, a 270bp genome sequence is replaced, and after asexual cuttage planting, excellent glyphosate and glufosinate resistance is shown, and normal growth is realized. The DNA molecule of the transformation event and the detection method have remarkable application value on breeding of herbicide-resistant alfalfa.

The invention relates to an in vitro method for modifying double stranded DNA (dsDNA) in a eukaryotic cell. The method comprises: a) introducing a base editor system into the cell, said system comprising i) a base editor, comprising an RNA guided DNA nickase linked to a single-stranded DNA nucleobase modifying enzyme, or a nucleic acid encoding said base editor, and ii) at least two guide RNA (sgRNA) molecules, comprising a first sgRNA that hybridizes to a first target sequence in the dsDNA, and a second sgRNA that hybridizes to a second target sequence in an opposing strand of the dsDNA, b) generating at least two single-strand breaks (SSBs), in opposing strands, of the dsDNA, wherein a first base editor complex, comprising the first sgRNA, introduces a nick cleavage of one strand of the dsDNA in the first target sequence, and a second base editor complex, comprising the second sgRNA, introduces a nick cleavage of the opposite strand of the dsDNA in the second target sequence, thereby producing a 5' or 3' overhang, c) inserting an exogenous DNA repair template sequence in proximity to the two single strand breaks (SSBs). The invention further relates to a genetically modified eukaryotic cell obtained by the method according to the present invention. The invention also relates to a kit for use in the in vitro method.

The invention provides a method for identifying and eliminating pollutants during quantification of microbe cfDNA in peripheral blood, which comprises the following steps: S1, screening common bacteria of peripheral blood and esophageal-gastric junction tumor, and extracting bacterial nucleic acid; s2, designing a corresponding primer probe combination according to the specific nucleic acid sequence of the bacteria, and carrying out multiple amplification on the bacterial nucleic acid by utilizing fluorescent quantitative PCR (Polymerase Chain Reaction); s3, carrying out statistical classification on the amplified specific nucleic acid sequence in the step S2 by utilizing R statistical software decontam to obtain a statistical classification program; the method comprises the following steps: adding artificially synthesized exogenous DNA during preparation of a sample of microorganism cfDNA in peripheral blood, and setting an identification sample confusion method; and S4, identifying and eliminating pollutants through a statistical classification program, and quantifying the microbe cfDNA in the peripheral blood. The method disclosed by the invention can be used for accurately, quickly and conveniently detecting the bacterial nucleic acid in the peripheral blood, identifying and eliminating polluted samples at the same time, and avoiding false positive during detection.

This invention relates to a novel engineered restriction-modification system for salt-tolerant Jeju bacteria to improve the efficiency of exogenous DNA transformation, belonging to the fields of bioengineering, metabolic engineering, and synthetic biology. This invention involves in-depth analysis of the whole-genome methylation map of Jeju bacteria to identify its unique methylation motif SAGCTS, and further identifies the corresponding restriction endonuclease Sen. Based on this, the restriction-modification system of Jeju bacteria is edited and modified, thereby improving the efficiency of exogenous DNA transformation.

The invention belongs to the technical field of agricultural biology, and particularly relates to a rice endogenous space-time specific promoter OsMOT1; the invention relates to 1 pro and application thereof. The promoter OsMOT1 and the promoter OsMOT1 are selected from the And the nucleotide sequence of 1pro is as shown in SEQ ID NO. 1. According to the invention, OsMOT1; 1pro is a space-time specific expression regulation element from rice, can efficiently and stably drive a target gene to be expressed in a plant callus, and can realize targeted efficient and stable expression in root and stem tissues in a plant vegetative growth period. The promoter can effectively replace a non-plant-source promoter commonly used in existing plant genetic engineering, has an important industrial application value in the field of plant genetic engineering, and not only can improve the targeting and stability of target gene expression in transgenic plants, but also can improve the yield of the transgenic plants. And the potential biological safety risk of the transgenic plant caused after the exogenous DNA is introduced into the plant genome can be obviously reduced.

The present invention relates to a gene editing system comprising: a) a donor nucleic acid comprising: —a degradation signal sequence, —an enzymatic cleavage site, —a ribosomal skipping sequence, —an exogenous DNA sequence, wherein said donor nucleic acid is flanked at 5′ and 3′ by inverted targeting sequences; b) an oligonucleotide complementary to the targeting sequence and c) a nuclease that recognizes the targeting sequence.

The present invention relates inter alia to rodents and cells that are engineered to contain the exogenous DNA of cats, their use in medicine and the study of disease, methods for production of rodents and cells, and antibodies and antibody chains produced by such rodents and derivatives thereof.

Transduction aids such as polymers, enzyme inhibitors, cytokines, exogenous DNA components, or any combination thereof. Transduction compositions that include one or more transduction aids. Transduction methods that include the use of a transduction composition that include one or more transduction aids.

The invention discloses an application of cinacalcet hydrochloride in preparation of drugs for resisting drug-resistant bacteria. The invention finds that the cinacalcet hydrochloride (CC) has dual effects, on one hand, the sensitivity of a strain carrying a tmexCD-toprJ gene cluster to tigecycline can be recovered, and on the other hand, the conjugational metastasis of antibiotic resistance genes can be inhibited by reducing over 99% of plasmid propagation. The synergistic effect of the CC and the tigecycline is that the minimum inhibitory concentration of the tigecycline is reduced by at least 8 times by dissipating proton dynamic potential and inducing active oxygen to damage an excretion function. Besides, the CC also reduces propagation of various drug-resistant plasmids by synergistically inhibiting expression of an Mpf and Dtr conjugation system and inhibiting SOS response, so that plasmid transfer and stable colonization of exogenous DNA (Deoxyribose Nucleic Acid) are blocked. The CC overcomes the tigecycline drug resistance mediated by tmexCD-topJ by destroying energy metabolism and oxidative stress, and meanwhile, limits horizontal transfer of other drug-resistant genes.

The present application relates to the technical field of acellular matrix repair materials, and discloses a preparation method of human acellular alveolar bone matrix microparticles, comprising the following steps: step S1: obtaining fresh and clean human alveolar bone from a clinic; and step S2: processing the obtained human alveolar bone and performing two cleaning operations on the human alveolar bone. According to the preparation method, after the human alveolar bone obtained from the clinic is cleaned, the cells, blood stains and exogenous DNA in the alveolar bone are successfully removed by using an ultrasonic cleaner and a common deionized water treatment liquid on the market without relying on a nuclease, the immunogenicity of the material is effectively reduced, the biocompatibility and safety are significantly improved, the natural structure and collagen integrity of the alveolar bone are retained, the preparation process is simplified, and the use of chemical reagents is reduced, so that the acellular alveolar bone matrix microparticles which are safe, non-toxic and suitable for scientific research and clinical application are prepared.

The present invention relates to a gene editing system comprising: a) a donor nucleic acid comprising a degradation signal sequence, an enzyme cleavage site, a ribosome skip sequence, and an exogenous DNA sequence, wherein the donor nucleic acid is adjacent to the inverse targeting sequence at 5' and 3'; b) an oligonucleotide complementary to the targeting sequence; and c) a nuclease that recognizes the targeting sequence.

This invention belongs to the field of gene editing technology, specifically relating to a scarless gene editing system for *Sphingomonas* and its application. The scarless gene editing system comprises a first inducing plasmid, a donor plasmid, and a second inducing plasmid. The first inducing plasmid includes the following elements: the EXO gene, the Gam gene, the Beta gene, a tetracycline resistance gene, and replicon 3233. The donor plasmid includes an exogenous DNA fragment for homologous recombination capable of amplifying upstream and downstream homologous arms of the target gene, an FRT fragment, and a chloramphenicol resistance gene. The second inducing plasmid includes the following elements: the FLP gene, a bleomycin resistance gene, and replicon 3233. Based on this scarless gene editing system, this invention knocks out the *Sphingomonas* 1535 gene, achieving an engineered strain capable of producing zeaxanthin, and further knocks out the 516 gene to increase zeaxanthin yield.

The present application relates to the field of microbial technology, and more particularly to a method for improving transformation efficiency of schizochytrium sp. The method comprises the following steps: before the electrotransformation, the schizochytrium sp. is subjected to enzyme treatment; the enzyme treatment comprises the following steps: before the electrotransformation, the schizochytrium sp. is subjected to enzyme treatment; the enzyme is protease or snailase; the enzyme activity of the protease is greater than or equal to 200 U / g, and the enzyme activity of the snailase is greater than or equal to 12.5 U / g. The present application studies an enzymatic hydrolysis method applied to the electrotransformation of schizochytrium sp., and the transformation efficiency of exogenous DNA of schizochytrium sp. can be effectively improved by subjecting the schizochytrium sp. to enzymatic hydrolysis treatment by alkaline protease or snailase, and the voltage size and the concentration requirement of exogenous DNA in the electrotransformation process are reduced, which has important application value in the field of microbial engineering related to schizochytrium sp.

The application provides a method for improving transformation efficiency of competent cells and application, and belongs to the technical field of molecular biology, and the method comprises the following steps: S1, mixing the competent cells with a plasmid solution, and then sequentially performing ice bath combination and heat shock treatment to obtain a heat shock system; S2, adding a SOC liquid medium containing a synergistic reagent to the heat shock system for recovery culture to obtain recovered bacterial liquid; S3, diluting the recovered bacterial liquid, and then culturing on LB solid culture medium containing a resistance component to obtain transformed bacterial colonies, and the transformation is completed; the method can be applied to library construction, rare plasmid transformation and large fragment plasmid transformation; the application does not need to adjust the formula in the preparation stage of the competent cells, and only needs to add the synergistic reagent in the recovery link, so that the recovery survival rate of the competent cells and the expression efficiency of the exogenous DNA are improved, and high transformation efficiency is realized.

This invention provides a method, tool, and application for improving the gene editing efficiency of *Eimeria tenella*. The *Eimeria tenella* strain with KU80 deletion is obtained by knocking out the KU80 gene in *Eimeria tenella* using a CRISPR-Cas9 gene editing system. Compared with existing technologies, the tool and method of this invention can significantly improve gene-targeted integration efficiency, significantly reduce random integration events of exogenous DNA, and improve the accuracy and stability of gene editing. It is a universal gene editing platform that can be widely applied to gene knockout, gene knock-in, and endogenous marker research. The strain using this tool can significantly shorten the screening cycle for positive recombinant strains, improve experimental reproducibility, and provide important technical support for functional genomics research on *Eimeria tenella*.

The invention discloses a preparation method and application of a yeast super competent cell based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9), and belongs to the technical field of genetic engineering. According to the invention, a CRISPR-Cas9 system and a LiAc / PEG / ssDNA transformation method are utilized to construct a fission yeast super competent cell capable of taking in a large amount of exogenous DNA, and a method capable of improving gene transformation efficiency. By utilizing the cell, the conversion rate and the integration rate of an exogenous gene can be remarkably improved, the number of transformants can be increased, the cell can also be used for conversion of large-fragment exogenous DNA, the research requirements of yeast mutant library construction and the like can be met, and an efficient experimental tool and a new strategy are provided for research in the field of genetic engineering.

The invention discloses an insect-resistant and herbicide-resistant transgenic maize event RF1117 and a detection method thereof. The transgenic maize event RF1117 is a DNA molecule obtained by inserting an exogenous DNA molecule into a No.3 chromosome of a maize genome. A transformed plant of the transgenic maize event RF1117 has better tolerance to herbicide glyphosate, has high resistance to lepidoptera pests such as spodoptera frugiperda, prodenia litura and ostrinia nubilalis, and can effectively control the occurrence and harm of the target pests. The DNA detection method provided by the invention can be used for accurately and stably identifying the existence of the RF1117 transgenic event by applying a nucleic acid amplification method, and is of great significance for realizing the traceability and whole-process supervision of the research, production processing and application of the RF1117.