528 results about "Mutant gene" patented technology

The invention belongs to the technical field of biology, and relates to an escherichia coli engineering strain for producing L-isoleucine through a methyl malic acid way as well as a construction method and application of the escherichia coli engineering strain. According to the invention, Escherichia coli BW25113 is used as a chassis strain, and a methyl malic acid synthase high-activity mutant gene cimA3.7, an isopropyl malic acid isomerase gene GsleuCD, a 3-isopropyl malic acid dehydrogenase gene AfleuB and a leucine dehydrogenase gene Lsleudh are over-expressed; a methyl malic acid absorption protein gene is over-expressed, and a branched chain amino acid transport system is modified to improve the exosome of the L-isoleucine; all methyl malic acid pathway related genes are integrated to a genome of a chassis strain, and a plasmid-free L-isoleucine production strain is constructed; the copy number of a key gene on a genome is optimized, and the yield of the engineering strain L-isoleucine is increased by introducing a non-oxidative glycolysis approach. The yield of the L-isoleucine after the engineering strain is fermented for 34 hours in a fermentation tank is 56.6 g / L.

The invention relates to a rice embryo specific expression transfluorescent protein gene and Malt gene editing integrated vector and a preparation method thereof, the vector comprises a gene editing fragment and a fluorescent protein gene fragment, the sequence of the gene editing fragment is shown as SEQ ID NO.1; the sequence of the fluorescent protein gene segment is as shown in SEQ ID NO. 2; when the vector is applied to a molecular detection technology, a rice induction line with a homozygous embryo-specific expression transfluorescent protein gene and a mutant gene can be screened out, and the time for creating the induction line with the embryo-specific expression transfluorescent protein gene is saved, so that the cost is saved.

The invention discloses a genetically engineered bacterium and an application of the genetically engineered bacterium in production of cis-3-hydroxy-L-proline. According to the genetically engineered bacterium, a proline dehydrogenase gene putA and a proline transporter gene putP in a starting bacterium are silenced, and meanwhile, a gamma-glutamyl kinase mutant gene proBD107N, a gamma-glutamyl phosphate reductase gene proA and a cis-3-hydroxy-L-proline hydroxylase gene P3H are subjected to overexpression. And by comparing a plurality of common escherichia coli, the cis-3-hydroxy-L-proline hydroxylase gene P3H and different gamma-glutamyl kinase mutant genes, the highest yield combination is preferably selected. The strain takes glucose as a raw material, so that the production cost is greatly saved, and the strain has a relatively good industrial utilization value.

The invention discloses a laccase mutant and application thereof in oxidative conversion of aflatoxin. According to the invention, laccase of coprinus cinerea is used as a starting enzyme, and through molecular docking, a structure-function relationship is analyzed, and mutation is designed. After induced expression of engineering bacteria containing mutant genes, mutant laccase Lcc5-T485E with improved detoxification efficiency is obtained through screening. When the enzyme with the same protein mass is used, the AFB1 conversion efficiency of the mutant Lcc5-T485E is obviously improved compared with that of Lcc5, and the mutant enzyme has potential application value in oxidative conversion of aflatoxin.

The invention discloses a laccase mutant and application of the laccase mutant in preparation of polyflavone by oxidative conversion of daidzein. According to the invention, laccase Lcc5 of coprinus cinerea is used as a starting enzyme, and through molecular docking, a position structure is analyzed, and mutation is designed. Synthesizing a mutant gene, and constructing a recombinant expression engineering bacterium of the mutant gene. After engineering bacteria containing mutant genes are subjected to induced expression, the Lcc5 mutant Lcc5-T485E is obtained through separation and purification, the specific enzyme activity and ethanol tolerance of the mutant are improved, and the conversion rate of daidzein is increased to about 1.26 times under the same enzyme activity; the antioxidant activity, the enzyme inhibition activity and the antibacterial activity of the obtained conversion product are also remarkably improved. The mutant enzyme has potential application value in oxidative conversion of daidzein and improvement of biological value of daidzein.

The invention belongs to the technical field of bioengineering, and particularly relates to an activity-improved tyrosinase mutant obtained by site-directed mutagenesis through an overlapping PCR technology, and preparation and application thereof. The tyrosinase mutant is obtained by mutation containing F337I / F352I on the basis of wild type tyrosinase as shown in SEQ ID NO.1, the enzyme activity of the tyrosinase mutant is obviously improved compared with that of the wild type tyrosinase, the F337I / F352I mutant gene is used for constructing a recombinant vector, and the recombinant vector is successfully expressed in bacillus subtilis WB600, bacillus amyloliquefaciens CGMCC No.11218 and bacillus licheniformis 2709, so that the tyrosinase mutant is obtained. The recombinant strain with improved enzyme production activity is further optimized through a fermentation process to obtain the novel tyrosinase.

The invention discloses a male sterility gene ZmUCE4 and application thereof in creating a male sterility line of corn, and belongs to the field of plant biotechnology breeding. According to the invention, site-directed mutagenesis of the ZmUCE4 gene in wild type corn is carried out through a CRISPR / Cas9 gene editing technology, pollen development abnormality and abortion can be caused, complete male sterility is caused, and it is proved that the ZmUCE4 gene has a regulation and control function on corn male reproductive development. Through offspring screening, a sterile line without transgenic components can be obtained, a stable maize male sterile line is created, and the method has important significance on maize male fertility control and hybrid seed production. The invention also designs a functional molecular marker aiming at the obtained uce4 male sterility mutant gene, and has important application value in maize male sterility line cultivation, sterile hybrid seed production and molecular marker-assisted selection.

The invention relates to the technical field of gene engineering, in particular to an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain and application. The amino acid sequence of the mutant is as shown in SEQ ID NO.4, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.3. A specific mutant gene sequence is designed, a recombinant plasmid pET28a (+)-L-Rhi is successfully constructed, the gene sequence is connected between enzyme cutting sites BamH I and Hind III of an escherichia coli expression vector pET-28a (+) to construct a mutant expression vector (the recombinant plasmid pET-28a (+)-L-Rhi), the mutant expression vector is transformed into an escherichia coli BL21 competent cell, a mutant strain is obtained, and the recombinant plasmid pET-28a (+)-L-Rhi is obtained. The obtained mutant strain can overexpress L-rhamnose isomerase, the conversion rate of D-allose is remarkably improved, the thermal stability of the enzyme is remarkably improved through the design of mutation sites, and the half-life period is effectively prolonged.

The invention relates to a mutant gene of a rice OsSPL10 gene and application of the mutant gene. The nucleotide sequence of the mutant gene is shown as SEQ IN NO.1, and the mutant gene can endow rice with resistance to glufosinate-ammonium herbicide; according to the invention, a rice variety 'Yangxian 9B' is treated by an EMS chemical mutagenesis technology, mutation of the OsSPL10 gene is successfully induced, and the mutation causes that the 225th amino acid in the amino acid sequence of the encoded protein is changed into tyrosine from cysteine; the application also develops a KASP molecular marker for the variation site, molecular marker detection and field glufosinate-ammonium resistance screening and verification prove that rice with the mutant gene can improve the glufosinate-ammonium resistance, and the gene has significant application potential in agricultural production, and can reduce the use of herbicides and improve the crop yield and quality.

The present invention provides a 5-hydroxytryptophan production strain and a construction method and application thereof. The strain has a clear genetic background and is effective in producing 5-hydroxytryptophan. E.coli HTP11 Subtilis subtilis spores pureF bsu K316Q Mutated gene, upregulated strain E.coli HTP11 of guaB , guaA , gmk , ndk The transcription level of the gene was increased by heterologous introduction of the gapX Gene, missing aromatic amino acid transporter serB , tnaB , aroP Gene, up-regulated inner membrane efflux transporter yhjV The transcription level of the gene was increased and electroporated into the pTm-serB plasmid, which could stably provide 5-hydroxytryptophan, the direct precursor of 5-hydroxytryptophan, and had a high efficiency of 5-hydroxytryptophan output.

The invention relates to the technical field of gene engineering, in particular to an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain and application. The amino acid sequence of the mutant is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. A specific mutant gene sequence is designed, a recombinant plasmid pET28a (+)-L-Rhi is successfully constructed, the gene sequence is connected between enzyme cutting sites BamH I and Hind III of an escherichia coli expression vector pET-28a (+) to construct a mutant expression vector (the recombinant plasmid pET-28a (+)-L-Rhi), the mutant expression vector is transformed into an escherichia coli BL21 competent cell, a mutant strain is obtained, and the recombinant plasmid pET-28a (+)-L-Rhi is obtained. The obtained mutant strain can overexpress L-rhamnose isomerase, the conversion rate of D-allose is remarkably improved, the thermal stability of the enzyme is remarkably improved through the design of mutation sites, and the half-life period is effectively prolonged.

The invention relates to the technical field of biological genes, in particular to a rice herbicide-resistant OsEPSPS mutant gene, a protein and application thereof. The rice herbicide-resistant OsEPSPS mutant gene provided by the invention is characterized in that the 836th site of the nucleotide sequence of a wild rice OsEPSPS gene is mutated, and T is mutated into C. A plant containing the rice herbicide-resistant OsEPSPS mutant gene or the rice herbicide-resistant OsEPSPS mutant protein has remarkable resistance to glyphosate herbicide and can normally grow in the herbicide almost without being influenced, and under the same condition, the growth of a wild plant is remarkably inhibited until the wild plant dies, so that the rice herbicide-resistant OsEPSPS mutant gene or the rice herbicide-resistant OsEPSPS mutant protein has a good application prospect. The mutant gene and the mutant protein have huge market economic prospects.

The invention discloses a bifunctional flavin adenine dinucleotide synthetase mutant and an application of the bifunctional flavin adenine dinucleotide synthetase mutant in FAD synthesis. The mutant disclosed by the invention is RFK / FADSC18R or RFK / FADSC18R-R46H, and the structural formula of the mutant is as shown in the specification. The amino acid sequence of the RFK / FADSC18R is as shown in SEQ ID NO. 15, and the amino acid sequence of the RFK / FADSC18R-R46H is as shown in SEQ ID NO. 16. A genetically engineered bacterium carrying the bifunctional flavin adenine dinucleotide synthetase mutant gene plasmid is used for producing flavin adenine dinucleotide by using glucose as a substrate through a simple fermentation process under a shake flask aerobic condition, and the FAD yields of strains XF02 and XF03 containing mutant genes are 79.31 mg / l and 167.56 mg / l in sequence; and compared with a strain XF01 containing a non-mutated gene, the mutant gene is improved by 58.62% and 235.12% respectively. A microbial'cell factory 'is constructed through a metabolic engineering strategy, and the modified microorganisms can generate required natural products in microbial cells by utilizing low-price raw materials. The progress not only reduces the production cost of the FAD, but also improves the environmental sustainability of the production process of the FAD.

The invention provides a strain of high-yield gamma-cyclodextrin glucosyltransferase and application of the strain. Specifically, the invention relates to a gamma-cyclodextrin production method, which comprises the following steps: (a) in the presence of a gamma-cyclodextrin glucosyltransferase mutant, carrying out enzymatic reaction on a substrate starch so as to form gamma-cyclodextrin. The invention also provides a strain for high yield of gamma-cyclodextrin glucosyltransferase, and a gamma-cyclodextrin glucosyltransferase mutant gene is integrated in a strain genome. The mutant enzyme and the strain provided by the invention can be used for generating high-yield gamma-cyclodextrin.

The application discloses a kind of fungal laccase mutant Lcc5-G and its expression strain and application.The application is based on the fungal laccase from Coprinopsis cinerea heterologous expression since, by site-directed mutagenesis, obtain mutant gene.After fungal induction expression containing mutant plasmid, obtain laccase mutant enzyme Lcc5-G with improved specific activity, stability and AFB1 removal efficiency.When ABTS is used as substrate, the specific activity of the mutant is improved by 3 times, and the stability of the mutant enzyme is improved to 5 times of the original enzyme at 45 DEG C and pH 7.The mutant has potential application value in oxidizing aflatoxin.

The invention discloses escherichia coli recombinant engineering bacteria for producing citicoline as well as a construction method and application of the escherichia coli recombinant engineering bacteria, and belongs to the technical field of biology. A genome cytidine deaminase coding gene cdd is knocked out on the basis of Escherichia coli K-12 MG1655, a temperature control expression vector PBV220 is used for expressing a SpnCCT-SpnCKI-pRpLpyrG mutant gene cluster, the expression of choline transporter protein BetT is enhanced, the gene locus of Escherichia coli ptsH-ptsI-crr is destroyed, meanwhile, a glucose permease gene glf and a glucokinase gene glk from Zymomonas mobilis are introduced, and the mutant gene cluster of the Escherichia coli K-12 MG1655 is obtained. The escherichia coli recombinant engineering bacterium ZMCB02NCL capable of efficiently biosynthesizing citicoline is obtained, and the preservation number of the escherichia coli recombinant engineering bacterium ZMCB02NCL is CCTCC NO: M 2025483.

Using chemical genetic screening, we discovered a small molecule Cellulosin (aka endosidin20 or ES20) that causes cell swollen and inhibits plant growth, but does not disrupt global vesicle trafficking. By doing mutant screening, we obtained multiple alleles of Arabidopsis thaliana that are resistant to Cellulosin inhibition in growth. Those mutated amino acid residues are conserved across plant species. Cellulosin targets a group of cellulose synthases (CesAs) of Arabidopsis thaliana by binding to a conserved domain essential for the catalytic activity of CesA. Cellulosin may target and inhibit all subtypes of CesAs in plants. The present invention relates to Cellulosin, a cellulose synthase inhibitor, its analogs or derivatives as a broad-spectrum herbicide. The mutated genes, their protein products and a cell or a plant having those mutated genes or expressing those protein products are within the scope of this disclosure.

The invention discloses a method and application for enhancing cytidine production, and belongs to the field of genetic engineering and microbial engineering. The present invention knocks out cytidine deaminase gene cdd, uridine kinase gene udk, pyrimidine-specific ribonucleoside hydrolase gene rihB, nucleotide 5'-monophosphate nucleosidase gene ppnN, pyruvate oxidase gene poxB and integrates and expresses 5'-CTP diphosphohydrolase, orotate phosphoribosyltransferase, uridylate kinase mutant gene PyrH (R92G / D93G), as well as phosphoribosyl pyrophosphate synthetase gene prs, glucose-6-phosphate dehydrogenase gene zwf and 6-phosphogluconate dehydrogenase gene gnd, and constructs a recombinant Escherichia coli with enhanced cytidine synthesis ability, and the cytidine yield reaches 61.9g / L in a 30L fermenter without adding antibiotics and inducers, which can be used to realize the industrial production of cytidine and realize green biomanufacturing.

The invention discloses a sorghum brown midrib mutant gene bmr34 related to high digestibility and application of the sorghum brown midrib mutant gene bmr34. The haplotype of the mutant gene bmr34 can specifically reduce the lignin content by 2.99% without affecting the biological yield. The invention provides the KASP marker which can accurately locate the bmr34 gene of sorghum and is closely linked with the bmr34 gene of sorghum, so that the breeding efficiency can be greatly improved, the breeding process is accelerated, and the KASP marker has important theoretical and practical significance for improving the quality of sorghum feed.

The invention discloses a rice seed dormancy gene OsPHS1 and application of a protein encoded by the gene OsPHS1, and belongs to the technical field of plant genetic engineering, and the gene and the protein encoded by the gene are used for regulating the dormancy of rice seeds. The nucleotide sequence of the gene is shown as Seq ID No: 1, the CDS sequence is shown as Seq ID No: 2, and the coded protein sequence is shown as Seq ID No: 3. The function of the rice seed dormancy gene OsPHS1 is verified by cloning and identifying the rice seed dormancy gene OsPHS1, and research finds that the dormancy of rice seeds can be remarkably reduced by knocking out or mutating the OsPHS1 gene, and the dormancy of the rice seeds can be improved to a certain extent by overexpressing the gene; the invention plays an important role in developing rice varieties with moderate dormancy.

Disclosed herein is a novel Cas9 protein. Further described herein are fusion proteins, compositions, and methods comprising the same. The novel Cas9 protein may be used, for example, in compositions and methods for modulating expression of a gene, for correcting a mutant gene, and for treating a disease.

The invention discloses a circulating tumor DNA (deoxyribonucleic acid) standard substance as well as a preparation method and a valuing method thereof. The preparation method comprises the following steps: (1) culturing cells; (2) cell nucleus extraction; (3) carrying out enzyme digestion on cell nucleuses; (4) purifying the fragmented DNA; (5) sampling a sample, and quantifying the sample by nanodrop measurement; (6) sampling the length of a verification fragment; and (7) mixing different ctDNAs according to the finally obtained theoretical mutation abundance of 1%, 5% and 10% to prepare the standard substance. The valuing method comprises the following steps: carrying out digital PCR amplification on a sample through a double-labeled probe to obtain the copy number concentration of a mutant gene and a wild gene or the copy number concentration of a target gene and a reference gene, and calculating the gene mutation frequency and the amplification multiple. According to the method disclosed by the invention, the fragmented DNA with the length similar to that of the ctDNA is obtained; 330bp and large fragments can be removed through two rounds of purification of a paramagnetic particle method, and a single-fragment standard substance is obtained.

The invention discloses a gene ZmKO1 for regulating flowering and pollination of corn male flower spikelet and application of the gene ZmKO1 in corn male sterility hybrid seed production, and belongs to the field of plant genetic engineering. The invention provides a gene ZmKO1 for regulating and controlling flowering and pollination of maize male flower spikelet, and the phenomenon of nuclear male sterility can be caused by mutation of the gene ZmKO1. It is also clear that on the basis of the ZmKO1 gene locus, a CRISPR-Cas9 technology or a mutant gene backcross transformation technology is utilized, and cultivation of the maize nuclear male sterile line can be achieved. The invention also finds that in the spinning period of the sterile line, the male flower fertility of the sterile line can be recovered by spraying exogenous gibberellin to tassels, normal selfing propagation can be realized, and the sterile line can be economically and efficiently maintained as a maintainer line. Therefore, the cell nucleus male sterile line created based on the ZmKO1 gene locus has the characteristic of dual purposes and can be used for maize male sterility hybrid seed production, and the invention also provides a technical system for realizing maize two-line method sterile hybrid seed production by utilizing ZmKO1 gene mutation.

The invention discloses a rice ACCase mutant protein and a preparation method thereof. The amino acid sequence of the ACCase mutant gene encoding protein has the following mutations: the 2052nd site corresponding to the amino acid sequence of wild rice ACCase is mutated, isoleucine (I) is changed into asparagine (N), or isoleucine (I) is changed into glutamine (Q), or isoleucine (I) is changed into cysteine (C), or isoleucine (I) is changed into cysteine (C), or isoleucine (I) is changed into cysteine (C), or isoleucine (I) is changed into cysteine (C), or isoleucine (I) is changed into cysteine (C). And the plant can have extremely strong resistance (tolerance) to acetyl-coenzyme A carboxylase inhibitor herbicides. After the rice containing the rice ACCase mutant protein disclosed by the invention is applied with an acetyl-coenzyme A carboxylase inhibitor herbicide with 8 times of recommended use concentration, plants still grow normally, and the tolerance of the herbicide is greatly enhanced, so that the rice ACCase mutant protein can be widely applied to breeding of herbicide-resistant plants.

The present invention relates to a MeCP2 gene-specific trans-splicing ribozyme, and a use thereof. The trans-splicing ribozyme according to the present invention replaces the RNA of a mutant MeCP2 gene, which causes Rett syndrome, with a wild type MeCP2 RNA, so as to reduce the expression of the mutant MeCP2 protein and increase the expression of the wild type MeCP2 protein, and enables regulated expression so that wild type MeCP2 expression can be prevented from exceeding physiological levels, and thus can be effectively used for preventing or treating Rett syndrome.

In traditional plant breeding approaches, chemical mutagenesis may be utilized to introduce nucleotide substitutions at random in the genome of a plant, i.e. without possibilities to control the sites of nucleotide changes. Because of genome complexities, the statistical probability is extremely little when it comes to finding a predetermined nucleotide substitution. The present invention, however, demonstrates how a novel, alternative use of digital polymerase chain reaction (dPCR), preferably droplet dPCR (ddPCR), is developed to exploit finding of specific nucleotide substitutions in mutated genes. The entire platform comprises a screening method with a library of mutagenized organisms, digital PCR-based systems and a set-up to propagate and analyze identified, mutated organisms.

The invention relates to a gene ZmILR1 for controlling the size of a corn kernel as well as a molecular identification method and application of the gene ZmILR1, and belongs to the technical field of plant genetic breeding and functional gene research. The ZmILR1 gene encodes an IAA-amino acid hydrolase, mutation of the IAA-amino acid hydrolase causes corn kernels to become smaller, and overexpression of the gene can create a corn material with increased kernel length, kernel width and hundred-kernel weight. A functional molecular marker A1A4 is developed according to the sequence difference of the mutant gene and a wild type gene at the DNA level, and can be used for rapid identification and screening of small-grain corn materials. The invention provides a theoretical basis and an application tool for analyzing a molecular mechanism of the corn grain size and developing molecular marker-assisted breeding and hybrid seed breeding, and provides important gene resources and technical support for high-yield and high-quality molecular breeding of corn.

The invention discloses a corn ALS mutant gene and application of the corn ALS mutant gene in herbicide resistance. The invention provides a method for improving the herbicide resistance of corn, which comprises the following steps: mutating 493th nucleotides of a ZmALS1 gene and a ZmALS2 gene in a corn genome from C to G so as to improve the herbicide resistance of the corn, the nucleotide sequence of the ZmALS1 gene is shown in SEQ ID No. 1; the nucleotide sequence of the ZmALS2 gene is shown in SEQ ID No. 2. Specific sites in corn ZmALS1 and ZmALS2 serve as sgRNA recognition sites on the basis of the principle of a base editing technology, a single base replacement editing vector capable of simultaneously targeting the ZmALS1 and ZmALS2 is designed, the gene editing vector is stably transformed through agrobacterium to obtain a transgenic plant, and the resistance to herbicides is remarkably improved.

The invention discloses a rice mutant gene and an identification method. Compared with an OsNRAMP5 gene sequence of an indica rice Shuhui 498 genome, the OsNRAMP5 mutant gene of the rice is subjected to base A deletion mutation at the downstream + 1083 site of an initiation codon ATG. The invention creatively proposes that groups are firstly grouped, grouping screening of OsNRAMP5 gene variants is carried out by utilizing high-throughput targeted sequencing of seed endosperm mixed sample DNA, and then only seeds of a variation group in which the screened variant mixed sample DNA with the highest mutation possibility is located are sown into seedlings. And subsequently screening and identifying single plants carrying corresponding variation in the variation group according to single plants. The technology can greatly save the cost of time, land, reagents and the like, and improves the screening efficiency and land utilization efficiency. The method disclosed by the invention is high in efficiency, good in accuracy, simple and convenient to operate, capable of better realizing high throughput, low cost and operation convenience, and suitable for popularization and application.

An Interferon Gamma Inducible Protein 16 (IFI16) mutant gene and its use as a marker for predicting, diagnosing, or prognosticating risk or severity of chronic liver disease is described. As a result of performing genomic analysis on NAFLD and NASH patient groups, it was confirmed that the frequency of IFI16 single-nucleotide variants (SNVs) including rs2276404, rs73021847, rs7532207, and rs6940 was increased, and the expression of the IFI16 mutant gene was increased depending on the disease stage of liver disease. The IFI16 SNV was highly expressed in infiltrating macrophages, playing a role in macrophage-induced inflammatory processes, and the IFI16 variant bound more strongly to dsDNA than wild-type IFI16, exacerbating the impaired mitochondrial DNA-sensing response signaling of the IFI16-PYCARD-CASP1 pathway. Thus, the IFI16 mutant gene may be used for predicting, diagnosing, or prognosticating risk or severity of chronic liver disease.