61 results about "Double mutant" patented technology

The invention belongs to the technical field of biology, and particularly provides UCRT X heat-resistant reverse transcriptase mutants with improved thermal stability, the UCRT heat-resistant reverse transcriptase mutants provided by the invention comprise four single-point mutants, six double mutants and four three mutants, and compared with wild-type UCRT X heat-resistant reverse transcriptase, the mutant has longer half-life period at 65 DEG C; the double mutant has a better effect, and the half-life period of the double mutant is about four times of that of wild type DNA polymerase. The invention also provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for synthesizing DNA (Deoxyribonucleic Acid) by reverse transcription at higher temperature, and has higher application potential.

The invention belongs to the technical field of biology, and particularly provides reverse transcriptase UCRT vZ mutants with improved thermal stability, the reverse transcriptase UCRT vZ mutants comprise four single-point mutants, six double mutants and four three mutants, and compared with wild type reverse transcriptase UCRT vZ, the half-life period of the mutants is longer at 65 DEG C; the double-mutant has a better effect, and the half-life period of the double-mutant is about four times that of the wild reverse transcriptase UCRT vZ. The invention also provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vZ mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for reverse transcription of RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribose Nucleic Acid) at a higher temperature, and has higher application potential.

The invention belongs to the technical field of biology, and particularly provides a reverse transcriptase UCRT v6 mutant with improved thermal stability, the reverse transcriptase UCRT v6 mutant comprises four single-point mutants, six double mutants and four three-point mutants, and compared with wild type reverse transcriptase UCRT v6, the half-life period of the mutant is longer at 65 DEG C; the three mutants are better in effect, and the half-life period of the three mutants is about 5 times that of the wild reverse transcriptase UCRT v6. The invention also provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT v6 mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for reverse transcription of RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribonucleic Acid) at higher temperature, and has higher application potential.

The invention belongs to the technical field of biology, and particularly provides UCRT VI heat-resistant reverse transcriptase mutants with improved thermal stability, the mutants comprise four single-point mutants, six double mutants and four three mutants, and compared with wild-type UCRT VI heat-resistant reverse transcriptase, the mutants have longer half-life period at 65 DEG C; the double mutant has a better effect, and the half-life period of the double mutant is about three times of that of wild type DNA polymerase. The invention also provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for synthesizing DNA (Deoxyribonucleic Acid) by reverse transcription at higher temperature, and has higher application potential.

The invention belongs to the technical field of biology, and particularly provides reverse transcriptase UCRT vL mutants with improved thermal stability, the reverse transcriptase UCRT vL mutants comprise four single-point mutants, six double mutants and four three mutants, and compared with wild type reverse transcriptase UCRT vL, the half-life period of the mutants is longer at 65 DEG C; the three mutants are better in effect, and the half-life period of the three mutants is about three times that of the wild type reverse transcriptase UCRT. The invention also provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vL mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for reverse transcription of RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribose Nucleic Acid) at higher temperature, and has higher application potential.

The invention belongs to the technical field of biology, and particularly provides UCRT heat-resistant reverse transcriptase mutants with improved thermal stability, the mutants comprise four single-point mutants, six double mutants and four three mutants, and compared with wild-type UCRT III heat-resistant reverse transcriptase, the mutants have longer half-life period at 65 DEG C; the double mutant has a better effect, and the half-life period of the double mutant is about four times of that of wild type DNA polymerase. The invention also provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for synthesizing DNA (Deoxyribonucleic Acid) by reverse transcription at higher temperature, and has higher application potential.

The present disclosure relates to T cells engineered to comprise a heterologous nucleic acid sequence encoding a double mutant transforming growth factor [beta] 1 (dmTGFB1) under the control of a promoter sequence. In certain embodiments, the cells further comprise modifications of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modifications, e.g., knock-down, of an endogenous nucleic acid sequence encoding IFNG or IL17A; and insertion of sequences encoding regulatory T cell promoting molecules, and compositions and uses thereof.

The invention belongs to the technical field of biology, and particularly relates to recombinant escherichia coli for producing curcumin glucoside and application of the recombinant escherichia coli. According to the invention, escherichia coli BL21 (DE3) is taken as a host cell, and a double mutant of a polyphenol TDP-rhamnosyltransferase ppuG gene derived from bacillus subtilis 168, a glucose-6-phosphate mutase Pgm gene derived from escherichia coli MG1655 and a UDP-glucose pyrophosphorylase GalU gene are transferred into the escherichia coli host cell, so that the recombinant escherichia coli is obtained. The double mutants of the polyphenol TDP-rhamnosyltransferase ppuG gene comprise two mutation sites, namely, G51A and Q345R. The invention further discloses a preparation method of the polyphenol TDP-rhamnosyltransferase ppuG gene. The recombinant escherichia coli constructed by the invention can efficiently produce curcumin diglucoside, and the yield can reach 7.10 g / L, so that not only are the water solubility and bioavailability of curcumin improved, but also powerful support can be provided for industrial production of curcumin glucoside.

The invention discloses a TAPC esterase mutant with improved thermal stability as well as a construction method and application of the TAPC esterase mutant. The TAPC esterase mutant is obtained by mutating on an amino acid sequence as shown in SEQ ID NO.2, and a mutation site is selected from one or a combination of more than one of S275Y, S326F, S326M, G266M, R321G, H385L, S84E and H297L. The TAPC esterase mutant provided by the invention comprises eight single-site mutants and two double mutants, and compared with wild type TAPC esterase, the mutant has a longer half-life period at 49 DEG C; the double mutant has a better effect, and the half-life period of the double mutant is about 13 times of that of wild TAPC esterase. The TAPC esterase mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when an ester-containing compound is catalytically hydrolyzed at a higher temperature, and has higher industrial application potential.

The application discloses a 17beta-hydroxysteroid dehydrogenase mutant, an engineering bacterium and application, and uses the 17beta-hydroxysteroid dehydrogenase mutant to biotransform methyl diketone as a substrate to prepare nandrolone. The 17beta-hydroxysteroid dehydrogenase is 17beta-hydroxysteroid dehydrogenase (17beta-HSDcl) from a fungus Cochliobolus lunatus, and the mutant is a single mutant in which histidine His at the 164th position is mutated into valine Val, a single mutant in which threonine Thr at the 151st position is mutated into alanine Ala, or a double mutant in which the 164th position and the 151st position are jointly mutated. The application uses pET28a as an expression plasmid, realizes heterologous expression of the 17beta-HSDcl mutant in E.coli BL21 (DE3), and through 24h whole cell transformation of the engineering bacterium, the nandrolone yield of the single mutant H164V, the single mutant T151A and the double mutant H164V / T151A is 1.27 times, 1.30 times and 1.34 times that of before mutation, respectively. The application provides a basis for industrialized production of nandrolone by microorganisms.

The application discloses application of a PRR5 gene in regulating early flowering of soybean under high temperature, and belongs to the field of genetic engineering. The application is directed to a soybean PRR5 gene, and a double mutant prr5a prr5b in which PRR5a shown in a nucleotide sequence as shown in SEQ ID NO. 1 and PRR5b shown in a nucleotide sequence as shown in SEQ ID NO. 2 are simultaneously knocked out is constructed by using CRISPR technology. Experimental results show that the double mutant has a flowering time under high temperature (30 DEG C) consistent with a flowering time under normal temperature (25 DEG C), and the response mechanism of the soybean in which the PRR5 gene is knocked out is relieved from early flowering under high temperature. The application provides a new idea, technical means and biological material for changing early flowering of the soybean under high temperature and enhancing production management of the soybean under high temperature. The application has a wide application prospect in research and high-yield breeding of the soybean in response to high temperature stress, and is helpful to promoting development of a soybean breeding industry.

The application belongs to the technical field of biology, and specifically provides a reverse transcriptase UCRT vZ mutant with improved thermal stability, the reverse transcriptase UCRT vZ mutant includes four single-point mutants, six double mutants and four triple mutants, compared with a wild-type reverse transcriptase UCRT vZ, the half-life of the mutants is longer at 65 DEG C, the effect of the double mutants is better, and the half-life is about 4 times that of the wild-type reverse transcriptase UCRT vZ. The application further provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vZ mutant obtained by the construction method has better thermal stability, and when used for RNA reverse transcription into cDNA at a higher temperature, the reverse transcriptase UCRT vZ mutant has higher thermal stability and great application potential.

The invention relates to a method for the preparation of industrially useful strains of yeast Saccharomyces cerevisiae and the strain prepared by the method. In particular the invention relates to the creation of double mutant strains of the yeast that shows high ethanol conversion efficiency and substantially reduced acetic acid and glycerol production during fermentative production of ethanol from a sugar-based feedstock.

The invention belongs to the field of biotechnology and plant breeding, and discloses a method for creating haploids by using rice endogenous genes. The invention relates to an application of an OsKPL1 gene or an OsKPL1 gene and an OsKPL2 gene in creating a rice haploid induction system or creating a rice haploid, and the gene number of the OsKPL1 gene is LOCOs04g51260, and the gene number of the OsKPL2 gene is LOCOs04g51290. Researches show that a single mutant of the oskpl1 has haploid inducibility of about 0.5%, double mutants of the oskpl1 and the oskpl2 have haploid inducibility of about 1.6%, and additional gene resources are provided for haploid breeding in rice.

The present invention belongs to the field of plant biotechnology. Specifically, the present invention provides rapeseed BnBPR1 Gene and its application in rapeseed clubroot resistance. The present invention uses gene editing technology to knock out two homologous copies in rapeseed. BnaA10.PBR1 and BnaC09.PBR1 , obtained BnBPR1 The homozygous double mutant of the gene mutation and the analysis of its disease resistance function revealed that the gene had a negative regulatory effect on rapeseed resistance to clubroot, which clearly indicated that it could be used to BnBPR1 Two homologous copies of a gene BnaA10.PBR1 and BnaC09.PBR1 Gene editing is performed to obtain a new material of homozygous double mutant transgenic rapeseed with enhanced resistance to clubroot. The discovery of the present invention has important significance and application value for the prevention and treatment of rapeseed clubroot, and can be used in the breeding of new rapeseed varieties resistant to clubroot.

The invention discloses application of double-mutant zebrafish in preparation of a mast cell function defect animal model. The double-mutant zebra fish disclosed by the invention is cpa1- / -cpa5- / -double-mutant zebra fish, and is obtained by further knocking out a cpa1 gene on the basis of a cpa5- / -zebra fish mutant. According to the method, potential compensation effects of two key carboxypeptidases are eliminated at the same time, and the storage and release capacity of mast cell protease is remarkably weakened from the genetics level, so that the zebrafish animal model with the mast cell function defect, which is clear in phenotype and stable in function, is obtained. The animal model can be used for mast cell function mechanism research, related disease mechanism exploration and drug screening and evaluation, and has a good application prospect.

The present invention discloses a single mutant strain, double mutant strain or complement strain of Aeromonas dakarensis phoP or phoBR, and a construction method and application thereof. The single mutant strain is a wild strain of Aeromonas dakarensis that lacks phoP or phoBR The double mutant is a strain that lacks both phoP as well as phoBR The complement strain is obtained by complementing the expression of a single mutant or a double mutant. phoP The results of immune protection experiments showed that compared with the PBS group, Δ pho The relative immune protection efficiency (RPS) of the P mutant strain increased by 40%, and the double mutant strain was significantly higher than that of the Δ pho P mutant and Δ pho The BR mutant strains were reduced by 10% and 17.5%, respectively, and the complement strains were Δ pho P mutant and Δ pho The BR mutant strains increased by 2.5% and 10% respectively. This provides a research basis and technical means for the development of subsequent attenuated vaccines.

This invention discloses the application of reducing the expression of negatively regulated genes in improving resistance to Phytophthora sojae, belonging to the field of plant genetic engineering. Research has found that the soybean genes GmTIFY5a or GmTIFY5b have a negative regulatory function on resistance to Phytophthora sojae. Knocking out GmTIFY5a or GmTIFY5b, or a combination of GmTIFY5a and GmTIFY5b, can enhance soybean resistance to Phytophthora sojae. This can be applied to improving soybean resistance to diseases caused by Phytophthora sojae or to breeding soybean varieties resistant to these diseases. In the soybean cultivar "Williams 82," a double mutant lacking both GmTIFY5a and GmTIFY5b, the accumulation of Phytophthora sojae was reduced by 50-60% compared to the wild type, the lesion infection area was reduced by 80-90%, and the survival rate was increased by 20-30%. Knocking out GmTIFY5a and GmTIFY5b significantly improved plant resistance to Phytophthora sojae. By genetically engineering the above genes to combat plant root rot caused by septicemia, we can not only provide important genetic resources for plant molecular breeding, but also provide an effective means to achieve high plant yields.

The invention discloses a sucrose isomerase mutant with improved thermal stability and a construction method thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the sucrose isomerase mutant, the thermal stability and the expression quantity are simultaneously improved, the mutant is used for preparing isomaltulose, the half-life period of the single mutant V447E at the temperature of 40 DEG C is prolonged by 1.38 times, the optimum temperature is increased to 35 DEG C from 30 DEG C, and the expression quantity is increased to 33.06 U * mL <-1 > from 18.89 U * mL <-1 >; other mutations are superposed on the basis of the mutant V447E, and the thermal stability of the constructed double mutant V447E / D232P is further improved. Compared with a wild type, the V447E / D232P has the advantages that the optimal temperature is increased by 5 DEG C, the half-life period at 40 DEG C is prolonged by 1.52 times, and the expression quantity is increased from 18.89 U.mL <-1 > to 30.73 U.mL <-1 >.

The invention relates to the technical field of biology, in particular to a Zymomonas mobilis transcriptional activation system and application thereof.The transcriptional activation system comprises crRNA of a target gene promoter regulatory region and a sequence of a coding effect protein complex, dCas12a losing nuclease activity and a transcriptional activation factor SoxS double mutant (R93A / S101A) are subjected to fusion expression, and the Zymomonas mobilis transcriptional activation system is obtained. Or through mediation of SpyTag / SpyCatcher, the two are coupled, so that an effect protein complex is constructed, and effective transcriptional activation can be carried out on different exogenous promoters and zymomonas mobilis endogenous promoters.

The application belongs to the technical field of biology, and specifically provides a reverse transcriptase UCRT vL mutant with improved thermal stability, which includes four single-point mutants, six double mutants and four triple mutants, and the half-life of the mutants is longer than that of the wild-type reverse transcriptase UCRT vL at 65 DEG C; the triple mutants have better effects, and the half-life is about 3 times that of the wild-type reverse transcriptase UCRT. The application also provides a genetically engineered bacterium containing the mutants. The reverse transcriptase UCRT vL mutant obtained by the construction method has better thermal stability, and when used for RNA reverse transcription into cDNA at a higher temperature, the reverse transcriptase UCRT vL mutant has higher thermal stability and great application potential.

The invention discloses an application of FLOE2 protein and FLOE3 protein in regulation and control of plant disease resistance. A floe2 single mutant and a floe3 single mutant are hybridized to obtain a floe23 double-mutant material in which both an FLOE2 gene and an FLOE3 gene are mutated, and the analysis on the disease resistance of the floe23 double-mutant material shows that compared with wild arabidopsis thaliana, when the floe23 double-mutant material is infected by pseudomonas syringae carrying Hopz1 effect protein, the disease resistance of the floe23 double-mutant material is remarkably improved, and the disease resistance of the floe23 double-mutant material is remarkably improved. The resistance to pseudomonas syringae is obviously reduced. It is shown that the FLOE2 protein and the FLOE3 protein or biological materials related to the FLOE2 protein and the FLOE3 protein can regulate and control the resistance of plants to the pseudomonas syringae induced by the Hopz1 effect protein and can be used for cultivating disease-resistant plant varieties in agricultural production. The invention is of great significance to the cultivation of disease-resistant plant varieties.

The invention discloses a sorbose dehydrogenase mutant capable of efficiently degrading vomitoxin, and belongs to the technical field of agricultural biology. The mutant is based on wild type SDH, through rational design and a directed evolution strategy, the mutant comprises three mutation sites F103A, T492E and N427A, the amino acid sequence and the nucleotide sequence of the mutant are SEQ ID NO.1 and SEQ ID NO.2 respectively, the mutant SDHF103A / T492E / N427A can efficiently degrade DON without depending on phenazine methyl sulfate (PMS), and the degradation activity of the mutant SDHF103A / T492E is improved by about two times compared with that of a double mutant SDHF103A / T492E under the condition that PMS does not exist. The method can be applied to preparation of the vomitoxin detoxification enzyme, and has a good application prospect in the aspect of biological detoxification of DON in feeds, grains and food.

The present invention discloses a TAPC esterase mutant with improved thermal stability, a construction method, and applications thereof. The TAPC esterase mutant is mutated in the amino acid sequence shown in SEQ ID NO. 2, and the mutation site is selected from one or a combination of more than one of S275Y, S326F, S326M, G266M, R321G, H385L, S84E, and H297L. The TAPC esterase mutants provided by the present invention include 8 single-point mutants and 2 double mutants. Compared with the wild-type TAPC esterase, the mutants have a longer half-life at 49°C; the double mutant is even more effective, with a half-life approximately 13 times that of the wild-type TAPC esterase. The TAPC esterase mutant obtained by the construction method provided by the present invention has improved thermal stability and exhibits high thermal stability when catalyzing the hydrolysis of ester-containing compounds at higher temperatures, thus having great potential for industrial application.

The present invention provides a nitrilase mutant and application thereof in the synthesis of 1-cyanocyclohexyl acetic acid, the nitrilase mutant is obtained by mutating one or two of the amino acids at position 180 and 205 of the amino acid sequence shown in SEQ ID No. 2. In the present invention, by semi-rational design and protein molecular modification, the specific enzyme activity of the nitrilase double mutant AcN-G180D / A205C was increased by up to 1.6 folds, and the conversion rate>99%. And the reaction time was shortened to a quarter of the original using the recombinant Escherichia coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at high temperature (50° C.). Therefore, the mutants obtained by the present invention have a good application prospect in efficiently catalyzing 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate, 1-cyanocyclohexyl acetic acid.

The invention discloses a method for removing exogenous genomes in cassava. The method comprises the following steps: (1) selecting a transgenic cassava strain of which a target gene is mutated; (2) selecting tissue culture seedlings of the transgenic cassava strain, inducing axillary buds of the tissue culture seedlings of the transgenic cassava strain to form somatic embryos, continuing to culture the somatic embryos into calluses, performing heat shock treatment on the calluses, inducing the transgenic cassava strain to edit T-DNA of a carrier exogenous gene, and inducing the transgenic cassava strain to edit the T-DNA of the carrier exogenous gene; the Free of all T-DNA of the exogenous gene of the transgenic cassava strain editing carrier is realized. According to the invention, the copy number of T-DNA in a double mutant in a genome is reduced through heat shock treatment, and Free of T-DNA is realized.

The invention discloses a leucine transporter mutant capable of reducing methionine transport activity and application of the leucine transporter mutant, and belongs to the technical field of bioengineering. Compared with a wild leucine transporter with an amino acid sequence as shown in SEQ ID NO.1, the mutant provided by the invention contains amino acid mutation at the 30th site, the 108th site or the 123th site, and the amino acid mutation is F30K, F30P, S108F, S108Y or L123R respectively. In order to improve the ACC transfer efficiency, the invention also provides double mutations of S108F / L160Y. Experiments show that compared with a wild type, the Escherichia coli strain of the leucine transporter mutant has the advantages that the sensitivity to methionine analogues is obviously improved, and the concentration of methionine in the strain can be obviously improved. Meanwhile, the double mutants can effectively improve the ACC tolerance of escherichia coli. Therefore, the method has a good application prospect in the field of ACC fermentation preparation.

The present invention relates to a double mutant heat labile enterotoxin gene comprising a spacer and, more specifically, to a use of a heat labile enterotoxin protein, encoded by the gene, for enhancing immunity. It was found that when the double mutant heat labile enterotoxin gene comprising a spacer according to the present invention is introduced to host cells, the translations of LTA and LTB constituting the labile enterotoxin gene do not affect each other, and thus LTB expression increases more than fivefold. In addition, it was experimentally confirmed that the expressed double mutant heat labile enterotoxin protein exhibits weak enterotoxic activity and simultaneously activates humoral and cellular immunity. Thus, the double mutant heat labile enterotoxin gene according to the present invention can be variously utilized in the fields of vaccines and vaccine adjuvants.

The invention belongs to the technical field of plant biotechnology and crop molecular breeding, and particularly relates to an application and a method of a replication gene pair for inducing development of maize spontaneous endosperm and embryos, and the application of the replication gene pair for inducing development of maize spontaneous endosperm and embryos to ZmFIE develops a new approach for apomixis of maize. It is proved that ZmFIE2 (and the synergistic effect of ZmFIE2 and ZmFIE1) is a key factor for maintaining fertilization dependence of corn seed development, corn can be induced to generate autonomously developed endosperm and embryoid structures under the condition that exogenous genes are not introduced through mutation of the gene, and a core element is provided for fixing heterosis. An efficient germplasm innovation material is provided, the zmfie1 and zmfie2 double mutant shows more severe phenotype than a single mutant, endosperm cell differentiation in mutant grains is blocked, intracellular replication is remarkably reduced, mitotic activity is enhanced, and the strong cell cycle change is a characteristic necessary for constructing an efficient haploid induction line.

This invention discloses a strain for the efficient synthesis of D-tagatose, its preparation method, and its applications. Through strategies such as the discovery of novel galactitol dehydrogenase and promoter engineering, this invention initially increased tagatose yield by 28.48%. Simultaneously, a highly efficient and sensitive tagatose biosensor was developed, which was used as a screening tool for xylose reductase. Ss XR molecularly modified the strain to obtain the F128M-Q219K double mutant, which achieved a tagatose yield of 5.39 g / L in the producing strain. Based on this, amino acid complementation optimization was performed on the high-yielding strain, and the final engineered strain achieved a tagatose yield of 44.61 g / L in a 5 L bioreactor. This study provides a new strategy and core technology support for the efficient biosynthesis of tagatose.