54results about How to "Increase mutation frequency" patented technology

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

A minicell display method has been developed which has significant advantages for screening peptide libraries for candidates that can bind and effectively modulate a particular biological process. The method, based on the small, anucleate minicell, has increased versatility in generating unique sequences to screen as well as increasing the size of the peptides to be screened. In vivo mutagenesis, at the level of protein synthesis, as well as DNA replication, increases diversification of the library to be screened and therefore substantially increases the number of potential peptides that can modulate a particular biological response or mechanism.

This invention provides methods and reagents for identifying genes involved in cell cycle progression, growth promotion, modulation of apoptosis, cellular senescence and aging, and methods for identifying compounds that inhibit or potentiate cellular senescence.

The invention provides compositions and methods used to inhibit USP1 deubiquitinase activity and to identify new inhibitors of USP1 deubiquitinase. The inhibitors can be used to treat or prevent cancer, bone marrow failure, and damage to cells or DNA resulting from genotoxic agents such as antineoplasic agents, including chemotherapeutic agents and radiation. The inhibitors include siRNA directed at inhibiting the expression of USP1 or UAF1, a protein which forms a heterodimeric complex with USP1. The inhibitors can be used to enhance cell survival if administered either before or after radiation exposure. Methods are also provided to enhance chemotherapy or radiotherapy of cancer and to enhance DNA repair. Transgenic knockout animals and knockdown cells are provided, whose USP1 expression is impaired.

The invention provides compositions and methods used to inhibit USP1 deubiquitinase activity and to identify new inhibitors of USP1 deubiquitinase. The inhibitors can be used to treat or prevent cancer, bone marrow failure, and damage to cells or DNA resulting from genotoxic agents such as antineoplasic agents, including chemotherapeutic agents and radiation. The inhibitors include siRNA directed at inhibiting the expression of USP1 or UAF1, a protein which forms a heterodimeric complex with USP1. The inhibitors can be used to enhance cell survival if administered either before or after radiation exposure. Methods are also provided to enhance chemotherapy or radiotherapy of cancer and to enhance DNA repair. Transgenic knockout animals and knockdown cells are provided, whose USP1 expression is impaired.

The invention provides a polymorphic hansenula polymorpha mutant strain and application of the polymorphic hansenula polymorpha mutant strain in glutathione biosynthesis. Firstly, low-energy ions N+ serve as a mutagenesis source, mutagenesis treatment for Hansenula polymorpha DL-1 is achieved, a glutathione (GSH) high-producing strain (DL-18) is obtained, the temperature remains 37 DEG C, the speed remains 180 r / min, shake flask fermentation remains for 42 hours, the dry cell weight (DCW) in end products is 7.04 g / L, the total quantity of the GSH is 420.46 mg / L, and the DCW in the end products and the total quantity of the GSH are respectively improved by 1.73 times and 1.94 times compared with the original mutant strain. Hereditary stability of the DL-18 is detected through subculture, yatsushiro is cultivated, content changes of the GSH are not significant, and therefore, the hereditary stability of the mutant strain is high.

The present invention relates to the identification and isolation of a DNA polymerase and uses of this polymerase. In particular, the present invention describes the nucleotide sequence of the human gene for DNA polymerase lambda (Pol λ), the amino acid sequence of Pol λ, and the amino acid sequence of several isoforms derived from alternative splicing of its mRNA. The association of some of these isoforms with tumour samples makes Pol λ a marker for the diagnosis, prognosis and evolution of tumoral processes.

The invention belongs to the field of seed processing and relates to a method for processing seeds by using a physical method, in particular to a method for injecting roegneria kamoji seeds by utilizing ion beams. The method comprises the following steps of: selecting dry roegneria kamoji seeds and controlling the water content to be 5-14 percent; putting the seeds on a plate of an ion beam injection device; and then, putting the plate in a target chamber to carry out low-energy nitrogen ion beam injection, wherein the injection dose of the low-energy nitrogen ion beams is 500-4,200 dose units, energy in the target chamber is 25 kilo electron volts, the flow magnitude of the nitrogen ion beams is 12 milliamperes, intermittent pulse injection is adopted, the pulse dose each time is 100-500, and the injection spacing interval each time is 20-30s, and the injection vacuum degree is 1.6*10<-2>-2.6*10<-2>. In the method, the germinating ability of the roegneria kamoji seeds is obviously improved, the seeds thrive in a seedling stage, and plants are strong; and the output of a single plant is increased, and the crude protein and the crude fat of the plants are both increased. The method can obviously improve the output and the quality of the roegneria kamoji and has higher application value.

The invention discloses a method for constructing a pepper mutant library from ethylmethane sulfonate. The method is characterized in that a Zunla 1 is used as a mutagenesis object; on the premise ofpointing out the influence of treating fluid dose and excluding space of each seed on germination percentage, semi-lethal dose is determined by comparing the germination percentage of pepper seeds with EMS treating fluid with different concentrations at different mutagenesis time; the pepper seeds are treated by mutagenesis of the semi-lethal dose; mutation frequency and mutation types of M2 generation are investigated, a mutant capable of stably inheriting of M4 generation is identified and a mutant library is constructed; the mutation types of leaves, stems, fruits, growth period, flower organs, fertility and the like are obtained and create abundant materials for functional genomics reach of pepper; and meanwhile, partial beneficial mutation can be directly applied to breeding practice.

A process for modifying cotton variety by space induction includes such steps as hybridizing between the cotton as female parent with high specific strength and the early-mature, high-yield and a disease-resistant cotton as male parent, space induction treatment of hybridized seeds, then reproduction further, backcrossing for breeding, and choosing by multiple generations. Its advnatages are high mutation frequency, several variant types, broad various spectrum, high yield and high resistance to diseases.

The invention discloses a method for breeding high-producing strains through acid protease by means of ultraviolet mutation.The method comprises the following steps of a, preparation of spore suspension liquid, wherein tablet spores cultured in a constant temperature incubator are taken, shaken and then diluted with sterile distilled water, and the spore suspension liquid is obtained for use; b, ultraviolet mutation, wherein the spore suspension liquid is taken and put under an ultraviolet lamp for irradiation, culture is carried out under a dark condition, and finally the fatality rate is calculated; c, strain screening, wherein c1, based on strains with the fatality rate between 70% and 80% in the step b, variant strains with the HC value higher than the mean value are picked out as preliminarily screened strains for secondary screening; c2, a conical flask containing a potato culture medium is inoculated with the strains obtained in the step c1, and after culture in a shaker, the enzymatic activity of acid protease is measured through a foline-phenol method; the mutated variant strains with the enzymatic activity higher than the mean value are selected for six times of continuous passage, and the high-producing strains are obtained.

The invention discloses a method for producing high-activity beta-galactosidase bacillus circulans strain and a breeding method of the strain. The produced high-activity beta-galactosidase bacillus circulans strain is bacillus circulans QHT-310-M481 is preserved in the China center for type culture collection on October 22nd, 2018. The preservation number is CCTCC NO:M 2018699, and the preservation address is Wuhan university, Wuhan, China. The produced high-activity beta-galactosidase bacillus circulans strain is obtained from a bacillus circulans original strain through mutagenizing by meansof 1-methyl-3-nitro-1-nitrosoguanidine, and a beta-galactosidase solution high in fermentation activity can be produced and used for an industrial catalytic galactosyl transfer reaction to produce galactooligosaccharide.

The invention relates to a DNA multiple directed mutation method in the gene engineering technique field. The invention acquires directed multiple high-frequency DNA discontinuity by applying unidirectional PCR reaction, augmenting the discontinuity sequence specifically, increasing the discontinuity formwork quantity and associating the superimposed extension PCR, which changes the designing method of the primer in favor of the formwork compound and the superimposed extension among the fragment, and improves the burst frequency. The invention provides a simple and effective method for mutagenizing DNA directionally in the external.

The present disclosure relates to the co-expression of an endonuclease with an end-processing enzyme for the purpose of enhanced processing of the polynucleotide ends generated by endonuclease cleavage.

The invention discloses a method for preparing a high-oleic oilseed rape material by utilizing artificially synthesized brassica napus. The method comprises the steps of utilizing the artificially synthesized brassica napus for hybridizing with natural brassica napus, obtaining a great deal of variations through mass exchange and recombination among filial generation chromosomes, and deficiency, insertion, transposition and inactivation of gene sequences, preventing brassica napus oleic acid synthesis from transforming to linoleic acid and linolenic acid, and utilizing mutation accumulation to acquire a high oleic acid material. According to the method provided by the invention, in earlier generation, oleic acid characteristics have no need to be selected, so that the breeding period can be shortened through a generation-adding technique; in earlier generation, a planting group contains about 300 plants (strains), so that compared with thousands of single plants (strains) for mutation breeding, the work load is smaller; during orientation selection, the selective pressure is increased by successive generations, so that the frequency of acquiring different types of high oleic acid is increased, and the breeding efficiency is improved. According to the method provided by the invention, the breeding selection group is small, the work load is small, the variation range is large, and high oleic acid brassica napus genetic resources with the oleic acid content being higher than 80 percent are obtained.

The invention relates to a 60Co-gamma ray radiation mutation breeding method for iris sanguinea tissue culture seedlings. The method comprises the following steps of 1, tissue culture seedling selection; 2, 60Co-gamma ray radiation treatment; 3, tissue culture seedling mutant primary selection; 4, enrichment subculture of primarily selected mutants; 5, rooting culture of primarily selected mutanttissue culture seedlings; 6, domestication transplanting; 7, field seedling mutant screening; 8, cutting propagation, tissue culture propagation and artificial selection. According to the method, theiris sanguinea tissue culture seedlings are adopted as materials, 60Co-gamma rays are utilized for mutation treatment, the mutagenic effect of different radiation dosages on the iris sanguinea tissueculture seedlings is observed, the proper radiation dosage is determined, and the iris sanguinea tissue culture seedling 60Co-gamma ray radiation mutation breeding technology is set up; by means of the method, rich heritable variations can be created, the breeding period of iris sanguinea is effectively shortened, a simple, convenient and efficient method is provided for new variety breeding, andthe hereditary basis of iris sanguine genetic resources is widened.

A minicell display method has been developed which has significant advantages for screening peptide libraries for candidates that can bind and effectively modulate a particular biological process. The method, based on the small, anucleate minicell, has increased versatility in generating unique sequences to screen as well as increasing the size of the peptides to be screened. In vivo mutagenesis, at the level of protein synthesis, as well as DNA replication, increases diversification of the library to be screened and therefore substantially increases the number of potential peptides that can modulate a particular biological response or mechanism.

The invention relates to the technical field of mutation breeding of lactic acid bacteria, and provides a method for breeding weak post-acidification streptococcus thermophilus strains. The method comprises the following steps: S1, preparation of a bacterial suspension: culturing an original strain to obtain a bacterial solution, transferring and continuously culturing the bacterial solution to a logarithmic metaphase, centrifuging, washing and concentrating the bacterial solution to obtain the suspension, and placing the suspension in an empty plate for later use; S2, ultraviolet irradiation: placing the suspension under an ultraviolet lamp for irradiation; S3, rejuvenation culture: resuspending the bacteria obtained in S2 in a fresh culture medium for rejuvenation culture; S4, penicillin treatment: adding penicillin for treatment, performing dilution, and coating an MC culture medium with the solution for culture; and S5, screening weak post-acidification strains. According to the technical scheme, the problem that the weak post-acidification streptococcus thermophilus strain cannot be efficiently bred in the prior art is solved.

Described herein is a novel, mitochondrial encoded, open reading frame, that leads to the production of a new mitochondrial peptide. Residing within the ND-Two subunit, a specific small nucleotide polymorphism disrupts expression of this mitochondrial peptide, and is correlated with an increase in obesity and diabetes, particularly in certain ethnic populations. In vitro administration of the peptide increases insulin secretion, decreases fat accumulation and improves glucose uptake in muscle cell. Antibodies generated against the peptide can be used for detecting peptide deficiency, in addition to SNP detection, supporting diagnostic approaches. In vivo studies further revealed that administration of the peptide improves glucose tolerance, thereby providing a new therapeutic avenue for a novel diabetes therapy and decreases bodyweight, thus serving as a novel obesity therapy. Generation of synthetic analogs further enhance or abrogated activity relative to the natural peptide.

The invention relates to a method for changing patterns and colors of maidenhair. The breeding method of a new species of the maidenhair mainly contains natural variation, hybrid breeding, space breeding and induced mutation breeding by ion radiation, but the frequency of the natural variation is low, the hybrid breeding process is complicated, and the space breeding cost is high. Dry seeds of the maidenhair are vertically plugged onto flower mud of a culture dish, a germ faces upwards, and the germ part is exposed; the well-prepared sample is placed into an ion radiation vacuum system to face the radiation source; the vacuum system is vacuumized to 10 to 4Pa; the ion radiation source is started, and the ion radiation technical parameters are adjusted; and the ion radiation is carried out, the ion radiation source is closed after the ion radiation is completed, the vacuum system is inflated, and the maidenhair dry seeds are taken out to be bred. The method is low in cost, wide in variation frequency spectrum, high in variation rate, convenient to operate and wide in application prospect.

The invention discloses a method for widening tetraploid wheat genetic variation. The method comprises the following steps that parent tetraploid wheat AABB is utilized to hybridize with diploid aegilops tauschii DD, in-vitro culture is carried out on a hybrid young embryo ABD to obtain an ABD plant seedling, then chromosome doubling treatment is carried out to obtain low-generation artificially synthesized hexaploid wheat AABBDD, the low-generation artificially synthesized hexaploid wheat AABBDD is backcrossed with the same parent tetraploid wheat AABB to obtain a pentaploid wheat material AABBD, the pentaploid wheat material is used for selfing, chromosome behaviors such as chromosome exchange, translocation, inversion, deletion and insertion are generated through chromosome recombination in the selfing process, rich variation types are generated in genetic offspring, and a new tetraploid wheat material composed of different chromosomes is formed. According to the method, the chromosome of the tetraploid wheat can be induced to generate new structural variation in a relatively short time, and the method is high in beneficial mutation efficiency, rich in mutation type, environment-friendly, safe and low in cost.

The invention relates to a method for improving the degradation capability of Pseudomonas sp on petroleum hydrocarbons. Petroleum hydrocarbons can be effectively degraded by adopting Pseudomonas sp, and the main problems of high degradation cost, narrow variation frequency spectrum, low variation rate and complex operation exist at present. The method for improving the degradation capability of Pseudomonas sp on petroleum hydrocarbons is characterized in that ion beams generated by an ion radiation system are injected to the Pseudomonas sp to improve the degradation capability of the Pseudomonas sp on the petroleum hydrocarbons. The method adopting the ion radiation induced mutation breeding has the advantages of convenient adjustment and control of the ion energy and the injection dosage, strong adaptability, high variation frequency, wide variation spectrum and good variation stability.

The invention belongs to a breeding technique of agriculture and forestry industrial crops, and particularly relates to an induced-mutation breeding method for obtaining new mutant germplasm based on 60Co-gamma-ray irradiation for whole grafted seedlings of mangifera indica. The breeding method comprises the breeding steps of (1) collecting seeds of mellow mangifera indica, accelerating germination until plant height is 9-11cm, performing transplanting to a nutrition bag, and performing breeding as rootstock seedlings; (2) collecting branches of the mangifera indica of the selected variety and 0.5-1 aged old, and grafting the branched to the rootstock seedlings so as to obtain grafting seedlings; (3) performing grafting for about 1-3 weeks, and treating the grafted seedlings through 60Co-gamma-ray irradiation; and (4) screening out the grafted seedlings with obvious trait changes namely the new germplasm of the mangifera indica, performing field planting in land for growing field crops and preforming further breeding utilization. According to the breeding method disclosed by the invention, a great quantity of new good-quality germplasms of the mangifera indica, having individual gene variation and property mutation can be quickly and effectively created, besides, original heredity background and good properties are maintained, and the breeding method has positive significance in avoiding the present situation that excellent germplasm resources of the mangifera indica are in short, and improving the good variety breeding efficiency of the mangifera indica.

The invention discloses a multifunctional plasma mutation apparatus, and the mutation apparatus comprises a plasma generating system for generating plasma which mutates mutating objects; an ultraviolet mutation device for executing ultraviolet mutation to the mutating objects; a chemical mutation device for performing chemical mutation to the mutating objects; a multifunctional loading platform where appliances used for mutation experiment are placed; an automatic control system for automatically controlling the plasma generating system, the ultraviolet mutation device, the chemical mutation device and the multifunctional loading platform so as to realize multiple mutating modes. Through the multifunctional plasma mutation apparatus, single-factor mutation and compound mutation can be executed and there are wonderful mutating types, thus the microorganism is greatly improved in mutation frequency and mutation types.

The invention relates to a plant induced mutation breeding method for enhancing mutation frequency and mutation spectrum, belonging to the field of induced mutation breeding. The method comprises the following steps: (1) induced mutation: carrying out induced mutation on embryonic cells, and carrying out secondary amplification culture; (2) meiosis-like induction: carrying out meiosis-like induction on the cells subjected to induced mutation and secondary amplification culture; and (3) naturally doubling to obtain the mutant homozygote cells, and screening the mutant homozygote by combining the breeding objective and mutant characters. The invention can be used for screening a great deal of materials in vitro within a short time to obtain a unicell-derived homozygote stress-tolerant mutant, thereby obviously shortening the breeding period and greatly enhancing the selection efficiency.

The invention discloses a method for regulating saccharomyces cerevisiae oxygen stress through Y-family polymerase Rev1, and belongs to the technical field of bioengineering. A gene REV1 is knocked out through a homologous recombination method, a knock-out strain rev1 delta is constructed, thus the oxidation stress resisting ability of a yeast strain is lowered, specifically, under the stress condition, the biomass of the knout-out strain is decreased by 9%, the survival rate is decreased by 34.8%, the gene mutation frequency is decreased by 63.8%, and the yield of pyruvic acid is decreased by30.8%. The gene REV1 is over-expressed through pY26 plasmid, an overexpression strain rev1 delta / REV1 is constructed, thus the oxidation stress resisting ability of the strain is enhanced, specifically, the biomass is increased by 8.3%, the survival rate is increased by 11.5%, the gene mutation frequency is increased by 186.2%, and the yield of the pyruvic acid is increased by 57.7%. The resultsshow that Rev1 can improve the survival ability and fermentation performance of the yeast strain under the oxygen stress condition.

The invention provides a chemical mutagenesis method for increasing the mutation range of dwarf bananas through induction. The chemical mutagenesis method comprises the following steps of preparationof a proliferation culture medium, mutagenesis treatment of the bananas, washing treatment and proliferation culture. According to the chemical mutagenesis method, a lithium chloride solution is adopted to treat and inhibit the growth of seedlings, so that the growth vigor is obviously different, and single plants with special characters appear in form; through mutagenic agent treatment, the mutation frequency is increased, the variation range is expanded, and the mutation frequency can be increased to 8.89% at most and is 1000 times or above higher than the natural mutation frequency throughchemical mutagenic agent treatment; and the mutation types are intensively represented as leaf color mutation, leaf type mutation and plant type mutation, wherein 6.47% of plants are represented to have bright green leaves, 7.11% of the plants are represented to have yellow stripes, 1.96% of the plants are represented to have leaf edge curling, and 4.50% of the plants are represented to have shortleaves, so that the artificial mutation range is wide, and the mutation types are rich.

The invention belongs to the genetic engineering technology field, and specifically relates to a frequency controllable GC specific DNA mutation method. Sodium hydroxide firstly denatures a gene to be mutated to a single chain, hydroquinone and sodium bisulfite are further used for processing, thereby allowing C in the DNA chain to become U by deamidization; the modified DNA is taken as a template, U is matched with A by PCR amplification, and A is continually matched with T in the follow-up amplification, thereby allowing GC in the original DNA chain to be converted to the AT direction and obtaining mutant DNA molecules; and the mutant DNA molecules are loaded to an expression vector, thereby constituting a DNA molecular mutant library. As the processing time of the sodium bisulfite is prolonged, the final mutation frequency of DNA is gradually increased, and the DNA with the mutation frequency of about 5 percent can be finally obtained, thereby overcoming the problem of excessively low mutation frequency in a plurality of conventional random mutation methods. The frequency controllable GC specific DNA mutation method is designed against GC base groups in the DNA molecules, the total mutation rate of GC is more than 90 percent, and the frequency controllable GC specific DNA mutation method is particularly applicable to the mutation of genes which are rich in GC. The invention can be used for the in vitro improvement of DNA molecules.

The invention relates to a method used for increasing capsorubin content in capsicums. Breeding methods of plant new varieties are plenty, and mainly comprise natural variation, crossbreeding, space breeding and ion beam implantation mutation breeding, but the frequency of the natural mutation is low. The method of the invention comprises following steps: burying capsicum seeds vertically in the flower mud of a culture dish at equal intervals with germs upwards and exposed; placing the prepared capsicum seed culture dish in an ion beam implantation vacuum system with the culture dish facing an ion source; reducing the pressure of the vacuum system to 10 to 4 Pa; turning on the ion radiation source, adjusting technological parameters of ion radiation; and performing ion radiation, turning off the ion radiation when ion radiation is complete, inflating the vaccum system, and taking the capsicum seeds out of the vaccum system for culturing. Beneficial effects of the ion beam implantation mutation breeding method of the invention are that: the technological parameters, ion energy and implantation dosage are convenient to adjust and control, adaptability is high, variation frequency is high, variation spectrum is wide, and stabilization is high.

The invention relates to a method for the heavy ion irradiation mutagenesis of streptomyces avermitilis strains and the culture of irradiation strains, belonging to the field of microbe strain breeding. The fermentation titer of the streptomyces avermitilis strains achieves 4000 microgrammes / milliliter by the traditional mutagenesis method and is very difficult to enhance again. The method comprises the following steps of: carrying out mutagenesis by adopting 12C+6 heavy ion beam irradiation, wherein an irradiation dose is 40-70 Gy, and a preferred irradiation dose is 50 Gy; and screening direct mutation strains of a plating medium and a slant medium to obtain high-titer strains reaching the fermentation titer of 6000 microgrammes / milliliter. The invention has stable and fast mutation, easy mutation control and simple, convenient and easy operation of the method.