334 results about "Screening cultures" patented technology

The invention relates to a Cas9 mediated carnation gene editing carrier and application. The application comprises the following steps: firstly, establishing a CRISPR-Cas9 system of carnation-containing target gene sites, introducing the Cas9 expression carrier into an agrobacterium tumefaciens C58 strain, putting roots of carnation leaves into a pre-culture medium, culturing with light for 3-4 days at 22+/-2 DEG C, activating agrobacterium containing the Cas 9 expression carrier, dipping the pre-cultured explant into the activated agrobacterium solution for 20-30 minutes, completely absorbing the agrobacterium solution, transferring into a co-culture medium, performing dark culture for 3-4 days at 22+/-2 DEG C, further transferring into a screening culture medium to culture, performing light culture at 22+/-2 DEG C so as to differentiate regeneration buds, further transferring the regeneration buds into a multiplication medium for multiplication screening culture, detecting positive transgenosis regeneration plants, sequencing target sites, and detecting mutation strain systems of carnation target gene sites.

An interleukin-15 (IL-15) gene modified natural killing cell strain for immunotherapy of tumor is prepared through inserting the cDNA coding region of IL-15 in pcDNA3 eucaryotic expression carrier, configuring secretion-type recombinant eucaryotic expression carrier pc DNA3/IL-15, using liposom to transfecte cell NK-92, adding Geneticin (G418) to screening culture medium, screening, limited dilution to cloned cells, detecting activity, choosing positive cell clones, and amplification.

The present invention provides an adipose tissue-derived mesenchymal stem cell amplification culture method, which comprises: preparing a suspension containing adipose tissue-derived mesenchymal stem cells with an adipose-derived stem cell culture medium, and inoculating the suspension into a stem cell culture device being subjected to a fibronectin coating treatment to carry out amplification culture. According to the present invention, the solid-phase support for growth and adhesion of the fibronectin-coated adipose-derived mesenchymal stem cells is used, and the synergetic screening culture of the low-concentration fetal bovine serum and the high-concentration gentamicin is used, such that the adipose tissue-derived mesenchymal stem cells can be amplified by 400-500 times within two weeks, the purity is high, and the adipose tissue-derived mesenchymal stem cells can be subjected to directional induction differentiation to obtain the adipose tissue.

The invention discloses a rapid assembling method of multi-fragment DNA yeast. The rapid assembling method comprises the following steps: (1) performing co-transformation on a plurality of DNA molecules with homologous arms, and linearized yeast shuttle vectors, so as to obtain yeast; (2) eluting all the transformed yeast colonies on a whole screening culture plate, centrifuging, and discarding the supernate to obtain transformed yeast cells; (3) extracting plasmid DNA of the yeast cells obtained in step (2), transforming the competent cells of colon bacillus; and (4) screening colon bacillus, and cloning to obtain large-fragment DNA. The method for assembling large-fragment DNA molecules is fast in speed, simple, convenient and feasible, high in success rate, low in cost, high in efficiency, easy to operate, beneficial in enlarging the industrialization scale and wide in application, and a plurality of small-fragment DNA molecules can be assembled into one large-fragment DNA molecule.

The invention relates to Enterococcus faecalis HKF7 with lactic acid activity as well as a screening culture method and application thereof. The classified name of the bacterial strain is Enterococcusfaecalis HKF7, the Enterococcus faecalis HKF7 was preserved in China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO:M2017297, and the preservation date is May 31, 2017. The bacterial strain is separated and screened from Hainan tropical aquaculture seawater and the intestinal tracts of Hainan tropical living litopenaeus vannamei. The Enterococcus faecalis HKF7 provided by the invention does not have hemolytic activity, has high hydrophobic activity and high self-coagulation rate, high artificial gastroenteric tolerance, high cholate tolerance and high bacteriostatic ability. The litopenaeus vannamei is fed with the Enterococcus faecalis HKF7 serving as a feed additive; and through culture test verification, the Enterococcus faecalis HKF7 can improve the immunity of the organism, promotes the growth of the litopenaeus vannamei, has stable effect and is a bacterial strain with good application prospect.

The invention relates to an Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method. The method comprises: pre-cultured Stevia rebaudiana stem apex explant is immersed into an Agrobacterium rhizogenes solution of pRI101-ANDNA plasmid carrying target gene, treatments such as infection, co-culture and sterilization are performed, the hairy root is cultured in a kanamycin-containing screening culture medium, the anti-kanamycin hairy root is formed on the explant through induction, the hairy root is induced to form callus, differentiation of adventitious bud is performed, the adventitious bud continuously grows to form the test-tube plantlet, and a PCR amplification method is adopted to identify the transgene plant carrying out the target gene. With the technical scheme, the target gene can be transformed into the Agrobacterium rhizogenes, such that the steps of separation from the protoplast and culture are eliminated compared with genetic transformation of polyethylene glycol and other chemical methods; and compared with genetic transformation of gene gun and other physical methods, the method of the present invention has characteristics of no requirement of expensive equipment, and simple operation technology.

The invention provides a method for directionally screening a salt-tolerant body through peanut in vitro mutagenesis. The method mainly comprises the following steps: sterilizing the surface of peanut embryo; separating a leaflet, inoculating into a somatic embryo induction and mutagenesis medium added with 2,4-D and pingyangmycin, and performing mutagenesis culture treatment; and transferring a survival explant on which a somatic embryo is formed to a somatic embryo germination and screening culture medium added with bone alkaline phosphatase (BAP) and NaCl, and directionally screening the salt-tolerant body. The salt-tolerant body is directionally screened by combining in vitro mutagenesis and tissue culture, and plenty of manpower, materials and financial resources can be saved; and the salt-tolerant body is regenerated in an embryogenesis way, an somatic embryo is derived from a cell, and the chimerism of the salt-tolerant body can be avoided. A peanut mature seed is taken as a mutagenic material and cannot be limited by seasons, the operation is convenient, a novel salt-tolerant peanut germplasm can be created, the peanut genetic basis is enriched, and the difficulty that new high-yield salt-tolerant varieties are difficult to breed due to the lack of high salt-tolerant germplasm resources in peanut cultispecies is overcome.

The invention discloses a method for improving the winter resistance of Japanese lawngrass. This method includes converting transcription activation factor CBF1/DREB1b of quasi-mustard induction gene into Japanese lawngrass embryonal callus to improve the winter resistance of Japanese lawngrass, which can include the following steps: 1) disseminating the Japanese lawngrass embryonal callus with agricillin liquor carrying CBF1/DREB1b; 2) placing the disseminated Japanese lawngrass embryonal callus onto the coculture medium to perform coculture; 3) placing the cocultured embryonal callus onto the screening culture medium for screening; 4) placing the screened embryonal callus onto the differentiated culture medium for differentiation culture; 5) placing the differentiated and germinated young plant onto the rooted culture medium for radication; 6) placing the plant which can produce root onto strong-plant screening culture medium for screening the Japanese lawngrass strong-plant which has improved winter resistance. The invention has important theoretical and operation significance for culturing new species of Japanese lawngrass with improved cold resistance and extended green period and has a wide application foreground.

The invention relates to a method for carrying out bacteriostatic culture after genetic transformation of arabidopsis thaliana. The method comprises preparation of a bacteriostatic agent, culture vessel disinfection, agrobacterium tumefaciens transformation, preparation of a screening culture medium, screening culture and detection of seedlings with resistance. The method for carrying out bacteriostatic culture after genetic transformation of arabidopsis thaliana has the advantages that the culture vessel and the culture medium are unnecessary to undergo high temperature and high pressure sterilization, thus reducing the workload and the energy consumption, simplifying the links of culture after genetic transformation of arabidopsis thaliana and reducing the cost of culture after genetic transformation of arabidopsis thaliana; the method for carrying out bacteriostatic culture after genetic transformation of arabidopsis thaliana is simple to operate, is only characterized by adding the bacteriostatic agent after preparation according to different culture medium formulas, and has strong practicability and good popularization property; compared with the conventional methods, the method for carrying out bacteriostatic culture after genetic transformation of arabidopsis thaliana has the advantage that the cost can be reduced by more than 10%.

The invention relates to a method for carrying out gene transformation on manioca by using Agrobacterium tumefaciens mediated transformation, which comprises the following steps: preparing a manioca explant and agrobacterium liquid, and coincubating the mainoca explant after being soaked by the agrobacterium; inserting the coincubated explant into a screening culture medium C13PC for culture to obtain a resistance callus, wherein the screening culture medium C13PC contains 1-3mg/L of Glufosinate ammonium or 1-10mg/L of hygromycin; transferring the obtained resistance callus into a differential medium, and differentiating adventitious buds; and obtaining a transgenosis plant through rooting culture. The method can successively lead in functional genes to carry out genetic improvement on a wild manioca. The conversion efficiency can be about 10% or even more than 10%; the transgenosis inheritance is stable, and the cost is low; a manioca in vitro has good regenerative system stability; and the method has simple operation and high regeneration efficiency, and the time from the induction of the callus to the obtain of the regeneration plant is only 80-90 days.

The invention discloses bacillus radicicola capable of degrading pyridine as well as a breeding method and application thereof. The bacillus radicicola is bacillus radicicola (Rhizobiumsp.) by identification, and named (Rhizobiumsp.) NJUST18; the log-in number of GenBank is JN106368. The bacterial strain is preserved in the China center for type culture collection (CCTCC) on March 28th, 2013; the preservation number is CCTCC NO:M 2013110. Enrichment of pyridine degradation bacteria is carried out by directly adopting a culture medium taking pyridine as sole carbon source and nitrogen source; separation is carried out by adopting a screening culture medium taking the pyridine as the sole carbon source and nitrogen source; the screening process is quick and fast; sundry bacteria on the culture medium are few; the workload of affination is reduced. Compared with the other pyridine degradation bacteria, the bacterial strain has efficient pyridine degradation capability, good adaptive capacity and tolerance, and has a good application prospect in treatment of a high-concentration pyridine wastewater.

The invention discloses a Eupenicillium javanicum bacterial strain and a screening method thereof. The preserving number of the bacterial strain is CCTCC M208204. The bacterial strain of the Eupenicillium javanicum can be used for producing cellulose, the step of which comprises that: the bacterial strain is inoculated into a seed culture medium and is cultured for 48-84 hours in the temperature of 25-32 DEG C so as to obtain the seed liquid; the seed liquid is inoculated into a fermentation medium according to 3-10% of inoculums concentration; the initial pH value of the fermentation medium is 4.5-6.0; the fermentation medium is cultured for 96-12 hours in the temperature of 25-32 DEG C so as to obtain fermentation liquor; the crude enzyme liquid containing cellulase is obtained by the centrifugation of the fermentation liquor. The cellulose produced by the Eupenicillium javanicum bacterial strain has the advantages of high activity, low cost, etc.

The invention relates to a transgenic cultivation method of rice dedicated for a starch medium. The method comprises the following steps: a mature embro of a rice species with a low content of alpha amylose is taken as an explant and is induced to form calluses; the calluses are subcultured to enter a proliferation period and are preculutured under a dark condition; the calluses are co-cultivated with agrobacterium tumefaciens for transformation and then transferred into a screening culture medium for being screened for three tims; resistant calluses are taken to be induced and differentiated into seedlings, and histochemical stain and molecular identification are performed to resistant plants to identify the differentiated plants containing target genes; the starch gelanitization property of selected transgenic plants is identified, and the transgenic plants which have the starch gelatinized and can form semi-solid gel after being cooled are selected and reserved; and expression stability of the starch gelatinization property of the plants is further evaluated, seed propagation is performed to the excellent plants with a stable starch gelatinization property, and finally dedicated rice for the starch medium is cultivated. The dedicated rice for the starch medium can be taken as a novel biological medium and be widely applied to scientific researches, food, medicine and chemical engineering.

The invention provides a bifidobacterium LJM-001 strain. The collection number of the bifidobacterium LJM-001 strain is CGMCC No.4090. The LJM-001 strain is hypersensitive to dimethoate and is a working strain for detecting dimethoate residues in food. The invention also provides a microbiological method for screening dimethoate residues in food. According to the size of antibacterial rings on a screening culture medium, whether the residual quantity of the dimethoate in food samples exceeds the standard or not can be judged. The method for screening the dimethoate residues in food has the characteristics of simpleness and convenience for operation, low cost, high sensitivity and suitability for sample screening, and the screening requirement of a large number of samples in the pesticide residue detection department in China is met.

The invention discloses a method for separating anaerobic ammonia oxidizing bacteria. The method comprises the following steps of: collecting a raw material, namely, collecting particle sludge as a raw material and preparing bacterial suspension by using the particle sludge; performing preliminary screening enrichment culture, namely, performing preliminary screening enrichment culture on the prepared bacterial suspension to obtain a preliminary screening culture; and performing secondary screening identification culture, namely, performing secondary screening identification culture on the culture obtained by the preliminary screening enrichment culture, detecting a culture obtained after the secondary screening identification culture and determining that strain of the culture is the anaerobic ammonia oxidizing bacterium if the culture passes the detection. The separation method has the advantages of separating the anaerobic ammonia oxidizing bacteria simply and rapidly, obtaining a pure culture of the anaerobic ammonia oxidizing bacteria and contributing to theoretical research of microbiology and industrial application.

The present invention relates to the technical field of gene preparation and cell culture, particularly to a human umbilical cord-derived mesenchymal stem cell strain stably expressing exogenous GLP-1 gene, wherein human umbilical cord-derived mesenchymal stem cells are separated and cloned from human umbilical cord, an eukaryotic expression plasmid pCDNA3.1 is adopted as a vector to transform exogenous GLP-1 gene into the human umbilical cord-derived mesenchymal stem cells, and G418 pressurizing screening culture is performed to obtain the cell strain. With the human umbilical cord-derived mesenchymal stem cell strain stably expressing exogenous GLP-1 gene, the disadvantage that continuous administration of the GLP-1 protein can perform the 2 diabetes mellitus treatment due to easy degradation and short half-life is solved when use of the GLP-1 protein to treat 2 diabetes mellitus.

The invention belongs to the field of life sciences and food safety, and relates to bifidobacterium breve isolated from the intestinal tract of a healthy person and a method for detecting antibiotic residues in milk by means of the strain. The isolated strain is DSQH-1, and the preservation number is CGMCC No.8579; the DSQH-1 strain is sensitive to antibiotics such as penicillin, tetracycline and erythromycin. The method for detecting various antibiotic residues in milk by taking the strain as a working strain comprises the steps of strain activation, bacterial suspension preparation, screening culture medium preparation, quantitative detection culture medium preparation, standard tube preparation, standard curve drawing, sample preparation, sample screening and detecting, sample quantitative detection and the like. The method for detecting various antibiotic residues in milk is easy and convenient to operate, simple in equipment requirement, high in sensentivity, not prone to contamination and suitable for screening and quantitative detection of a large number of samples and can also be used for self inspection of producers.

The invention relates to a method for quickly obtaining a capsicum transgenic plant, which comprises the following steps: sampling; activating and infecting agrobacterium; preparing a regeneration culture medium and a screening culture medium; inoculating the immature embryo, and culturing; and forming adventitious bud and regenerating the plant, thereby efficiently obtaining the capsicum transgenic regenerated plant. In the invention, it usually takes 7 weeks from the immature embryo infected by agrobacterium to the acquisition of the regenerated plant, the formation rate of the regenerated plant in the culture process is up to 50% to the maximum, and the positive rate of the transgenic plant is up to 80%. In the invention, the immature embryo is used as a receptor material to infect theagrobacterium and carry out inoculation and culture so as to directly form the regenerated plant, thereby shortening the culture period and enhancing the culture effect and genetic transformation efficiency. If applied to genetic functional verification and breeding practice, the invention can greatly accelerate the research into capsicum functional genomics and the development of breeding practice.

The invention discloses a genetic transformation method of a Chinese fir. The genetic transformation method comprises the steps of: establishment of a transformation receptor, preparation of agrobacterium liquid, infection, bacterium removal culture and screening culture. According to the genetic transformation method of the Chinese fir disclosed by the invention, a Chinese fir suspension cell system is taken as the receptor and a b-glucuronidase (GUS) gene is used as a report gene to implement a genetic transformation through an agrobacterium mediated method, and the plant regeneration is realized through a somatic embryogenesis way, and a stable and efficient Chinese fir genetic transformation system is established. Due to the utilization of the technical system, genetic improvement of the Chinese fir with comparatively low cost can be achieved; and anti-disease, stress-resistant, wood formation and other related genes obtained from a basic and theoretical research are subjected to the genetic transformation so as to obtain a high-quality Chinese fir variety having characteristics of rapid growth, strong anti-disease and stress-resistant performances, and the like.

The invention discloses a method for screening a pluripotent cell and a special culture medium thereof. A pluripotent cell screening culture medium provided by the invention comprises PD0325901 shown in formula (I) and CHIR99021 shown in formula (II). The method for screening a pluripotent cell provided by the invention comprises the step of using the screening culture medium to culture a cell A, thus obtaining the pluripotent cell; and the cell A is a cell obtained by inducing an inducible factor encoding gene in a differentiation cell. The pluripotent cell obtained by applying the method of the invention has the protuberant cloned shape appearance of mESC; the symbol gene and the surface protein of an express embryonic stem cell have the activity of surface alkaline phosphatase and the capacity of automatically differentiating to form an Embryoid body as the embryonic stem cell;, and the pluripotent cell can be further differentiated into the cell types of an inner embryonic layer, a middle embryonic layer and an outer embryonic layer. The invention has important significance for the research field of the pluripotent cell.

The invention relates to a genetic transformation method using sweet sorghum young ear or young ear induced callus as an explant, belonging to the technical field of plant genetic engineering. In the invention, the agrobacterium rhizogenes meditation method is adopted to transform foreign genes for the sweet sorghum young ear explant induced callus; resistant healing is obtained by carrying out preculture, coculture and screening culture in sequence on the sweet sorghum young ear explant induced callus; and then the transformed plant is obtained by carrying out successive differentiation culture, budding, rooting and transplanting on the sweet sorghum young ear explant induced callus. In addition, the established genetic transformation system is used to transform the sweet sorghum plant to obtain the genetically modified pest-resistant plant. The method of the invention provides technical support for researches on using the biotechnology to improve quality of the sweet sorghum and increase the stress resistance and the like and lays very good foundation for accelerating the basic research on the sweet sorghum.

The invention provides a plant growth-promoting rhizobacteria strain Gxun-20 and application thereofin plant growth promotion. A bacterial strain with efficient IAA secretion, phosphorus dissolving and growth promoting capabilities is selected out through an ADF screening culture medium and an LB purification culture medium in a screening mode, 16S rRNA and physiological and biochemical characteristics of the bacterial strain are determined, the bacterial strain is determined to be brevibacillus brevis, the bacterial strain is gram-positive bacteria, and the bacterial colony morphology is round, smooth, convex and white. The strain is preserved in the Guangdong Microbial Culture Collection Center on June 13th, 2019. The fermentation condition of Gxun-20 is optimized, and the bacterial liquid obtained by fermentation is used for carrying out a growth-promoting test on plants. Compared with other strains, the strain Gxun-20 has higher IAA yield, and can be used for promoting the growth and development of plants, so that the use of chemical fertilizers and the damage of chemical fertilizers to soil are effectively reduced.

The invention provides a method for producing grease rich in essential fatty acid linoleic acid and alpha-linolenic acid through culturing microalgae by use of CO2. The method comprises the following steps: (1) screening and domesticating pollution-resisting algal species by utilizing a special screening culture system and a special culture medium under the high-efficiency high-CO2-concentration-resisting high-temperature-resisting conditions; (2) culturing domesticated microalgae by use of CO2; and (3) extracting the grease rich in linoleic acid and alpha-linolenic acid by taking the microalgae as a raw material. According to the invention, CO2 emitted by waste gas in a power plant is converted into a substance which can absorbed for growth of microalgae, so that algae cells with high biomass are obtained and high-value essential fatty acid linoleic acid and alpha-linolenic acid grease can be produced; and by using the method, the emission of CO2 is reduced, the greenhouse effect is alleviated, CO2 can be converted into high-value-added linoleic acid and alpha-linolenic acid grease, high yield of grease can be obtained, and the contents of linoleic acid and alpha-linolenic acid are maintained above 60%, thereby ensuring the quality of essential fatty acid grease and reducing the cost for producing the essential fatty acid grease.