21 results about "Double mutant" patented technology

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 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 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.

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 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 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.

The application belongs to the technical field of biology, and specifically provides a UCRT heat-resistant reverse transcriptase mutant with improved thermal stability, the mutant including four single-point mutants, six double mutants and four triple mutants, compared with the wild-type UCRT III heat-resistant reverse transcriptase, the mutant has a longer half-life at 65 DEG C; the double mutant has a better effect, and the half-life thereof is about 4 times that of the wild-type DNA polymerase. The application further provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained through the construction method has better thermal stability, and when reverse transcription and DNA synthesis are performed at a higher temperature, the mutant has higher thermal stability, and has great application potential.

The application discloses a kind of xylanase activity enhanced mutant optimized by gating adjacent residues and application thereof.The application takes GH11 family xylanase XynLC9 derived from Bacillus subtilis as research object, carries out modeling to its structure using AlphaFold 3, carries out site-directed saturation mutation and combination mutation to candidate site by combining gating region adjacent residue analysis, and obtains xylanase mutant with enhanced activity.The mutant is mutated at the 123th and / or 124th position of the amino acid sequence shown in SEQ ID NO:1, preferably D123G and / or R124E, more preferably double mutant D123G / R124E.Compared with wild type, the relative enzyme activity of double mutant D123G / R124E is increased by 4.0 times, and it can be used for xylan degradation and xylo-oligosaccharide preparation.

The application discloses application of an OsCATA gene in controlling rice heading stage and belongs to the technical field of genetic engineering. The application finds for the first time that the OsCATA gene (cDNA sequence as shown in SEQ ID NO:1) derived from rice Nipponbare is involved in the regulation of the rice heading stage, and knocking out the gene can significantly delay the heading of rice under short-day and long-day conditions; under long-day, the OsCATA gene regulates the heading stage in a manner dependent on OsELF3-1, down-regulates Ghd7 to inhibit the expression of Ehd1, Hd3a and RFT1, and finally delays the heading. The application also provides a knocking-out vector and method of the OsCATA gene and obtained mutant plants, the oscata single mutant delays the heading by 12.4 days under the long-day in Hangzhou and by 7.2 days under the short-day in Hainan, and the oself3-1 oscata double mutant has a more significant delay effect. The application provides a new gene target for the regulation of the rice heading stage, perfects the photoperiodic flowering regulation network of rice, and the provided method and mutant can be used for the directional breeding of new rice varieties suitable for different photoperiod regions, and has important breeding application value.

The application discloses a sucrose isomerase mutant with improved thermal stability and a construction method thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The application constructs a sucrose isomerase mutant with improved thermal stability and expression amount, and uses the mutant for preparation of isomaltulose, wherein a single mutant V447E has a half-life at 40 DEG C prolonged by 1.38 times, an optimum temperature increased from 30 DEG C to 35 DEG C, and an expression amount increased from 18.89 U.mL ‑1 -1 to 33.06 U.mL ‑1 -1; a double mutant V447E / D232P with other mutations superimposed on the basis of the mutant V447E has further improved thermal stability; compared with a wild type, the double mutant V447E / D232P has an optimum temperature increased by 5 DEG C, a half-life at 40 DEG C prolonged by 1.52 times, and an expression amount increased from 18.89 U.mL ‑1 -1 to 30.73 U.mL ‑1 -1.

The invention discloses a genetically engineered bacterium for efficiently producing butanediamine and application of the genetically engineered bacterium, and belongs to the field of bioengineering. According to the present invention, the optimal mutant AdiAD606R / H730D subjected to double-interface synergetic modification can maintain high activity in the whole neutral-alkaline range with the pH value of 7.0-9.0; the optimal pH of the arginine decarboxylase mutant is increased, the enzyme activity stability in a neutral pH range is improved, the arginine decarboxylase mutant is more suitable for the condition requirements of industrial microbial fermentation, and a foundation is laid for efficient synthesis of butanediamine. According to the present invention, with the recombinant whole-cell catalyst of the optimal double mutant AdiA D606R / H730D, the butanediamine yield of 149.7 g / L can be achieved within 12 h;

The invention provides a directionally modified 7beta-hydroxysteroid dehydrogenase iteration mutant as well as a coding gene and application thereof. According to the invention, the expression vector or recombinant bacterium containing the 7beta-hydroxysteroid dehydrogenase iterative mutant gene is successfully constructed. According to the invention, the 7beta-hydroxysteroid dehydrogenase double mutants are directionally modified by an enzyme modification technology, and alanine at the 203th site of the 7beta-hydroxysteroid dehydrogenase double mutants is mutated into glutamine, so that the pH tolerance and catalytic activity of the enzyme are remarkably improved. The 7beta-hydroxysteroid dehydrogenase iteration mutant disclosed by the invention can be used for preparing UCDA (Urban Cyclin Dehydrogenase).

The invention relates to a cellobiose 2-epimerase mutant with high catalytic efficiency and application of the cellobiose 2-epimerase mutant. The mutant is obtained by changing the specific amino acid site of a wild type enzyme, the double mutant psyCE-FR shows the optimal performance, and the catalytic efficiency (Kcat / Km) of the double mutant is improved by about 40% compared with that of the wild type enzyme. The mutant has excellent catalytic efficiency and substrate affinity at low temperature (4-10 DEG C), and is suitable for low-temperature dairy product processing and functional sugar preparation. Besides, the mutant retains the characteristic that the psychrophilic enzyme is easy to thermally inactivate, can be instantly inactivated at 50 DEG C, is compatible with a pasteurization process in the dairy industry, reduces the downstream treatment cost, and improves the product purity and safety. The invention also provides a gene for coding the mutant, a recombinant expression vector, a recombinant engineering bacterium and a method for producing epilactose by using the mutant enzyme, and the mutant enzyme has wide application prospect and commercial value.

The invention discloses application of reduction of expression of a negative regulation gene in improvement of phytophthora resistance of soybean, and belongs to the field of plant genetic engineering. The research finds that the gene GmTIFY5a or GmTIFY5b has the function of negatively regulating and controlling the resistance of the soybean phytophthora root rot. The knockout of GmTIFY5a or GmTIFY5b or the combination of GmTIFY5a and GmTIFY5b can enhance the resistance of soybeans to phytophthora sojae, and can be applied to the improvement of the resistance of soybeans to diseases caused by phytophthora sojae or the cultivation of soybean varieties resistant to the diseases caused by phytophthora sojae. According to the present invention, the accumulation amount of the double mutant soybean phytophthora of the soybean cultivar Williams 82 without GmTIFY5a and GmTIFY5b is reduced by 50-60% compared to the wild type, the disease spot infection area is reduced by 80-90% compared to the wild type infection area, the survival rate is increased by 20-30% compared to the wild type, and the resistance of the plant to the phytophthora can be significantly improved by knocking out the GmTIFY5a and GmTIFY5b. Genetic engineering improvement of plant phytophthora root rot resistance is carried out through the gene, important gene resources are provided for plant molecular breeding, and an effective means is provided for high yield of plants.

The present application belongs to the field of bioengineering technology, and particularly relates to a xylanase mutant with improved heat resistance, an engineering bacterium and application thereof. The present application takes the wild-type xylanase shown in SEQ ID NO. 1 as a template, and obtains a series of xylanase mutants with improved heat resistance, such as G38P, D160R, E263R single mutant, G38P / D160R, G38P / E263R, D160R / E263R double mutant and G38P / D160R / E263R triple mutant, and a recombinant engineering bacterium containing the mutant gene, especially the triple mutant G38P / D160R / E263R, which shows the best heat resistance, and the enzyme activity residual rate is greater than 90% after 2 h of treatment at 90℃, far more than that of the wild type (less than 30%), and can completely adapt to the high-temperature industrial process of 80-100℃, effectively solving the problem of high-temperature inactivation of traditional xylanase, so that it can be widely applied in the fields of feed, cellulose bioconversion, food processing and the like.

The invention belongs to the field of bioengineering, and relates to an N-acetyl amino acid racemase mutant with improved racemization activity, the sequence of which is obtained by mutating the 293rd site and / or the 21st site of the amino acid sequence shown as SEQ ID NO.1 of the original N-acetyl amino acid racemase from the N terminal to the C terminal. The mutant of the N-acetyl amino acid racemase provided by the invention is obtained through site-directed mutagenesis and has relatively high enzyme activity to N-acetyl-L-tryptophan, the racemization activity of a single mutant I293L to the N-acetyl-L-tryptophan is 1.43 times that of a proenzyme, and the racemization activity of a double mutant T21A / I293L to the N-acetyl-L-tryptophan is 4.89 times that of the proenzyme; the N-acetyl amino acid racemase mutant has better efficiency when applied to preparation of D-tryptophan.

The invention discloses a recombinant escherichia coli engineering strain for producing hydroxytyrosol and application, and belongs to the technical field of genetic engineering and biological engineering. According to the invention, by knocking out tyrR, ptsG, crr, pheA, feaB and pykA genes of escherichia coli, the competition between feedback inhibition and carbon metabolism is eliminated; the mutant aroGfbr and the mutant tyrC are subjected to overexpression, feedback and inhibition, and tyrosine precursor supply is strengthened; the method comprises the following steps: carrying out rational design on a key rate-limiting enzyme 4-hydroxyphenylacetic acid 3-hydroxylase to obtain an S462A / M293Y double mutant, and carrying out overexpression on an nadC gene to optimize cofactor balance, so that the hydroxytyrosol yield of the constructed engineering strain in a shake flask reaches 4.90 g / L; in a 5L bioreactor, the yield is further increased to 13.25 g / L, and the method can be widely applied to industrial production of hydroxytyrosol-containing products in the fields of food, health care products and medicines.

The invention belongs to the technical field of biological catalysis, and particularly relates to an enzyme saturation mutation library, a construction method and application. The enzyme saturation mutation library comprises PGA enzyme mutants; compared with an amino acid sequence shown in SEQ ID No: 1, the amino acid sequence of the PGA enzyme mutant comprises one or two of the following mutations: (1) Q235H; (2) Y513C; (3) N411T; (4) L392I is carried out; (5) L4P; (6) T311C; (7) V403Y; (8) L269T; the PGA enzyme mutant does not comprise double mutants, namely, L392I / N411T and L392I / L4P. The invention further provides a biological synthesis method of the cefradine. The method comprises the step of incubating the biological catalyst containing the PGA enzyme mutant in a reaction system containing the substrates 7-ADCA and DHPGM for enough time so as to convert the substrates into the cefradine. The method is mild in condition, the product yield reaches 99.0% or above after the reaction is carried out for 50 h, the method is suitable for industrial production, and the problem that the catalytic activity of wild type PGA enzyme is low is solved.