178 results about "Protoplast" patented technology

The invention discloses a preparation method of fresh taxus chinensis stem and leaf protoplasts. The preparation method comprises a preparation process and a purification process. The preparation method of the taxus chinensis stem and leaf protoplast is simple and easy to operate, raw materials are easy to obtain and low in price, reagent components have good biocompatibility, and the taxus chinensis stem and leaf protoplast is free of harmful components, safe and environmentally friendly. The invention provides a simple and rapid taxus chinensis stem and leaf protoplast enzymolysis preparation method, which adopts vacuum filtration, accelerates the permeation of enzymatic hydrolysate and improves the enzymolysis efficiency, so that the protoplast can be rapidly obtained. Meanwhile, high-purity separation of the protoplast is carried out in combination with density gradient sedimentation of the iodixanol solution, so that the protoplast with uniform size and complete form is obtained, and convenience is provided for scientific researches such as subsequent conversion and single cell transcriptome sequencing.

The invention discloses a plant gene editing system and method for delivering CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 based on mobile RNA (ribonucleic acid). The system does not depend on tissue regeneration or protoplast culture, Cas9 and gRNA are remotely delivered from the root to enter the overground part by fusing a CRISPR / Cas9 gene editing carrier of a tRNA-like sequence (TLS) movable motif and combining a hairy root transformation system, target gene editing is completed, and heritable gene editing seeds can be obtained at the current generation. The method comprises the following steps: 1, constructing a movable CRISPR / Cas9 gene editing fusion vector, wherein the movable CRISPR / Cas9 gene editing fusion vector comprises a target vector, an intermediate vector and an editing vector; and 2, application of the agrobacterium rhizogenes mediated plant hairy root transformation system. And 3, detecting the mobility and editing type of the movable editing carrier. And 4, gene function verification application. The system greatly breaks through the bottlenecks of difficult plant genetic transformation and long time consumption of precise breeding, and provides theoretical basis and technical support for gene function research and germplasm innovation of important agronomic traits of plants.

The invention relates to an osmanthus protoplast preparation method and an instantaneous conversion method. The preparation method of the sweet-scented osmanthus protoplast comprises the following steps: collecting different tissues of fresh sweet-scented osmanthus, performing surface disinfection and sterilization, cutting into thin strips or slices, adding enzymatic hydrolysate for enzymolysis, filtering, centrifuging, washing with a W5 solution, and re-suspending. The instantaneous transformation method of the osmanthus protoplast comprises the following steps: adjusting the concentration of the osmanthus protoplast by using an MMG solution, adding plasmids for mixing, adding a PEG solution for transformation incubation, adding a W5 solution for washing and centrifuging, and detecting the instantaneous transformation efficiency of the osmanthus protoplast. According to the method, a high-quality protoplast separation system of the leaves, the stems and the petals of the sweet-scented osmanthus is successfully established, efficient conversion of mesophyll and petal protoplast is realized for the first time, and a reliable technical platform is provided for molecular biology research of the sweet-scented osmanthus.

The invention discloses application of novel reverse transcriptase and related fusion protein of the novel reverse transcriptase in plant-guided editing. Candidate RT protein is obtained through systematic mining from a public database, novel reverse transcriptase cl1 with activity is screened out in combination with a fluorescence report system, and a guided editing system suitable for plants is constructed. The system tests in wheat protoplast, and verifies the editing activity of cl1 to a plurality of endogenous targets. Further combining with protein structure prediction and rational design point mutation optimization, the cl1 mutant with higher editing efficiency is obtained, and the method can be applied to accurate and effective editing of plant genomes and plant breeding and improvement.

The invention discloses a method and a device suitable for CUAMP. The invention discloses a method for extracting liriodendron tulipifera cell nucleuses by a Tag technology and application of the liriodendron tulipifera cell nucleuses. Hybridized liriodendron tulipifera calluses are used as materials, and protoplasts are released through enzymolysis solution vacuumizing and mild enzymolysis for 1.5 h; the method comprises the following steps: sequentially purifying by using a W5 solution and mannitol, detecting the activity by FDA, cracking by using an NE buffer solution, and washing by using a Wash buffer solution in two steps, thereby obtaining 2 * 10 < 6 >-3 * 10 < 6 > high-purity and high-integrity cell nucleuses. The obtained core is clean in background and complete in membrane structure, and can be directly used for CUTamp; tag is used for building a library, so that the magnetic bead capturing efficiency is improved by 46%, and protein-DNA interaction high-throughput sequencing under the condition of low sample size is realized. The method disclosed by the invention is simple and convenient to operate and good in repeatability, and provides key technical support for epigenetic research of rare tree species such as liriodendron tulipifera.

The invention discloses a high-yield and high-activity pear protoplast extraction method based on light shielding and enzymolysis optimization, which comprises the following steps: by taking pear tissue culture seedling leaves growing for 30-40 days or field pear tender leaves as an extraction material, carrying out enzymolysis treatment and protoplast purification by adopting enzymatic hydrolysate to obtain protoplast; before enzymolysis of the pear tissue culture seedlings, the whole pear tissue culture seedlings are placed in a dark environment and subjected to dark pretreatment at 25 + / -2 DEG C for 48-72 hours. According to the method, the yield of the field pear leaf and tissue culture seedling pear leaf protoplast can be increased by more than or equal to 2 times (the highest protoplast yield is greater than or equal to 3.0 * 10 / g FW), the activity is increased to be greater than or equal to 95%, and the technical bottleneck of the existing patent is remarkably broken through. The method has the advantages of high yield, high activity, wide application range and the like, can provide a high-quality protoplast material for pear genetic regeneration, gene editing, single cell transcriptome analysis and metabonomics research, and has wide scientific research and industrial application potential.

The invention discloses a method for extracting sphagnum moss protoplast, which comprises the following steps of: culturing a sphagnum moss material obtained by tissue culture under a certain condition, and performing pretreatment and enzymolysis under a specific permeation condition to finally obtain the high-quality sphagnum moss protoplast. The method provided by the invention provides an important technical basis for molecular biology research of sphagnum moss transgenosis and the like.

The present application relates to the field of plant tissue culture technology, and particularly discloses a lily stem cell abundance ex vivo regulation method. The lily bulb is used as the starting material, stem cell magnitude amplification and regulation culture can produce lily tissue organ materials with high stem cell abundance. The present application provides sterile lily materials with high stem cell abundance, which has the characteristics of strong regenerative ability, high proliferation rate, etc., is the basis for protoplast preparation and high-efficiency regeneration, and is suitable for different lily varieties, and provides technical support for cell engineering breeding, seedling scale breeding, etc.

This application belongs to the fields of microbiology and cell biology, and provides a method for directly preparing germination-promoting fungal protoplasts from orchid protocorms, comprising: (1) physical homogenization; (2) filtration; (3) removal of plant contents and residual cell walls; (4) purification of mycelial clusters; and (5) release and purification of fungal protoplasts. Through the rational design of homogenization and purification steps, this application achieves high-yield, high-purity, and high-vitality preparation of germination-promoting fungal protoplasts, providing excellent materials for orchid gene variety breeding and genetic engineering operations.

The utility model discloses enzymolysis equipment and relates to the field of enzymolysis device.The enzymolysis equipment comprises a base and an enzymolysis tank, the lower portion of the enzymolysis tank is connected with a separation tank through a connecting pipe, a mounting opening is formed in the upper portion of the separation tank, a separation barrel is rotatably mounted in the mounting opening, and separation holes are formed in the side face of the separation barrel; leaking holes are formed in the outer side of the lower surface of the separating tank, a funnel is installed on the lower portion of the separating tank, and a discharging pipe is installed in the middle of the funnel. According to the device, an enzyme solution is discharged into the separation tank from the separation holes under the action of centrifugal force generated during rotation of the separation cylinder, the enzyme solution and protoplast can be rapidly separated, separation can be rapidly carried out in a rotating mode, separation is more comprehensive, the separation effect is improved, and the separation efficiency is improved. The problems that an enzyme solution and protoplast are inconvenient to separate in an existing separation mode, and separation is not comprehensive enough are solved.

The invention belongs to the technical field of microbial genetic transformation, and particularly relates to a method for high-yield production of pleurotus ostreatus transformants, a genetic transformation system and application of the genetic transformation system. Through optimization of protoplast preparation conditions, the number of the obtained protoplast is 4.95 * 10 < 8 > CFU.mL <-1 >, and the regeneration rate of the protoplast in an OM-YEPS regeneration culture medium is 12.7%. A method for producing high-yield pleurotus ostreatus transformants is researched by using a PEG-mediated protoplast transformation method, a set of efficient and stable genetic transformation and screening method is established, about 200-300 positive transformants with stably expressed exogenous genes are obtained, and the transformation efficiency is 65.38%. The genetic transformation system of the high-yield Pleurotus ostreatus transformant, constructed by the invention, lays a solid technical foundation for subsequent gene function verification, metabolic engineering transformation and molecular breeding with excellent characters, and further accelerates the biological research and industrial application process of Pleurotus ostreatus and even other macro fungi.

This invention discloses a gene editing method and application for *Trichoderma reesei* based on a ribonucleoprotein complex. It aims to overcome the problems of low editing efficiency, easy generation of heterozygotes, and risk of exogenous DNA integration in *Trichoderma reesei* using plasmid-based CRISPR / Cas9 systems. This invention combines a dual-site cleavage strategy guided by double sgRNAs with direct RNP delivery. The method involves: designing two guide RNAs spaced 50-700 bp apart for the target gene, pre-assembling them with Cas9 protein in vitro to form an active RNP complex; delivering this complex along with exogenous donor DNA to *Trichoderma reesei* protoplasts. The exogenous donor DNA contains a left homologous arm, an editing sequence, and a right homologous arm, wherein the left and right homologous arms are homologous to the genomic sequences outside the dual cleavage sites on the target gene determined by the double sgRNAs, respectively; upon entering the cell, the RNP complex introduces dual-site cleavage at the target site, achieving precise editing under the guidance of the exogenous donor DNA.

The invention belongs to the technical field of plant cytology, and particularly relates to a method for improving the transformation efficiency of plant protoplast, which comprises the following steps: preparing enzymatic hydrolysate: each liter of enzymatic hydrolysate contains 20 mmol of KCl, 5-20 g of cellulase, 2-6 g of macerozyme, 10 mmol of CaCl2, 1 g of BSA and the balance of water; performing leaf enzymolysis; obtaining a protoplast; preparing a competent protoplast; resuspending the MMG solution; and transforming and incubating the protoplast resuspension by using the target plasmid. According to the method, the high-quality protoplast can be obtained, and when the protoplast is applied to downstream experiments, the conversion efficiency and stability of the experiments can be remarkably improved.

This invention discloses the application of the FAD2 gene of *Botrytis cinerea* in regulating the growth, pathogenicity, and stress resistance of *B. cinerea*. The FAD2 gene of this invention is derived from *B. cinerea*. By constructing a knockout gene fragment of the FAD2 gene and introducing it into the protoplasts of *B. cinerea*, a knockout mutant of the FAD2 gene was obtained. Studies have found that the FAD2 gene plays an important role in regulating the growth of *B. cinerea*, influencing its ability to withstand high salinity and osmotic stress, and significantly reducing the pathogenicity of *B. cinerea* mycelium to tomato fruits and leaves. Therefore, FAD2 can serve as a key gene for studying the pathogenic mechanism of the disease and can be used as a target for fungicides. This can be used to develop fungicides to control *B. cinerea* or for gene function research on the pathogen, contributing to the breeding of new plant disease-resistant varieties and showing broad application prospects in plant fungal disease research.

The use of an LtPCL6 protein and a gene encoding same in regulating pathogenicity, mycelial growth, and stress resistance of Lasiodiplodia theobromae. The gene encoding LtPCL6 protein is derived from Lasiodiplodia theobromae, and a knockout mutant is obtained by constructing a knockout fragment of a Lasiodiplodia theobromae LtPCL6 gene, and introducing same into a Lasiodiplodia theobromae protoplast. Experiments indicate that the LtPCL6 gene has an effect in the growth, cell wall and cell membrane integrity, and stress resistance of Lasiodiplodia theobromae, and significantly reduces the pathogenicity of the Lasiodiplodia theobromae with regard to a host. Therefore, the LtPCL6, serving as a fungicide target and a key protein of a pathogenic mechanism of diseases, can be used for developing agents for diseases caused by Lasiodiplodia theobromae infection and breeding new resistant varieties, and has broad application prospects in the control of plant pathogenic fungal diseases.

The invention discloses a construction method and application of a basic group editor of industrial bacteria of cephalosporium acremonium based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9), and belongs to the field of gene engineering. The cephalosporium acremonium industrial bacterium base editor provided by the invention can be used for specific mutation of a specific site of the DNA level of the cephalosporium acremonium industrial bacterium. The cytosine base editor CBE constructed by the invention is a plasmid comprising a fusion protein A and an sgRNA expression element. The base editor CBE can successfully mediate C.G-T.A, is suitable for the base editor of the high-yield CPC cephalosporium acremonium industrial bacteria, utilizes the protoplast, is high in conversion efficiency, can mediate specific site mutation in the cephalosporium acremonium industrial bacteria, and has application value.

The invention belongs to the technical field of edible mushroom biological breeding, and particularly discloses a Stropharia rugoso-annulata breeding method based on Tricholoma giganteum and Stropharia rugoso-annulata protoplast fusion, which comprises the following steps: respectively preparing Stropharia rugoso-annulata protoplast and Tricholoma giganteum protoplast; performing thermal inactivation on the stropharia rugoso-annulata protoplast, and performing ultraviolet inactivation on the tricholoma giganteum protoplast; mixing the two inactivated protoplasts, and adding an induction reagent for protoplast fusion; coating the fusion product on a regeneration culture medium for culturing to obtain a fusion strain; screening a target fusion strain through an antagonism test and a molecular marker technology; fruiting management is carried out, and a new high-temperature-resistant stropharia rugoso-annulata strain is screened out. By optimizing protoplast preparation and PEG fusion promotion conditions, the distant fusion efficiency is remarkably improved, and a novel stropharia rugoso-annulata strain which can normally fruit at the high temperature of 30 DEG C and is excellent in agronomic character and good in flavor is successfully obtained in combination with an efficient screening and identification system.

This invention discloses a stable and efficient method for preparing protoplasts from *Pseudolarix amabilis*. The method directly uses etiolated *Pseudolarix amabilis* seedlings as material to prepare protoplasts, requiring no other aseptic procedures, and the entire process is simple and convenient. Furthermore, by setting different buffer component ratios and a specific mannitol concentration, this invention achieves stable maintenance of the osmotic pressure of *Pseudolarix amabilis* seedling cells during the separation process. Experimental verification shows that the protoplast yield using this method is 1.32 × 10⁻⁶. 5 The protoplasts were prepared at a density of 1 protoplast per ml, with 95% of them intact and undamaged, resulting in significantly improved quality and activity. Furthermore, the enzymatic hydrolysate described in this invention overcomes the difficulty of enzymatic hydrolysis caused by the high degree of leatheriness in *Pseudolarix amabilis* leaf tissue. This method is suitable for widespread application in the field of herbaceous plant biotechnology.

The present invention relates to methods of producing viable grapevine protoplasts with regenerative capacity, and methods of genome editing grapevine protoplasts to regenerate genome edited grapevines.

The invention discloses an extraction method and application of extracellular vesicles in camellia oleifera leaf protoplast lotion. The method comprises the following steps: (1) taking complete disease-free camellia oleifera leaves, cutting off petioles, cleaning with ultrapure water, and absorbing water with filter paper; (2) putting the cleaned leaves into a needleless injector cylinder added with an infiltrating liquid, infiltrating in a vacuumizing manner, and centrifuging the infiltrated leaves to obtain a camellia oleifera leaf protoplast washing liquid; and (3) carrying out graded ultracentrifugation on the protoplast washing liquid, and collecting supernate. (4) filtering out cell debris from the supernate through a needle filter to obtain filtrate; (5) centrifuging the filtrate again, removing large vesicles and / or microparticle precipitates, and collecting supernate; (6) adding the supernatant into an ultracentrifuge tube for centrifugation, and removing the supernatant to obtain extracellular vesicle precipitate; and (7) gently resuspending the extracellular vesicle precipitate by using the precooled PBS, centrifuging the resuspended solution, and removing the supernatant to obtain the washed extracellular vesicle precipitate.

The present disclosure provides methods for inducing callus formation and plant regeneration from isolated protoplasts of grape. This technology allows for the production of non-chimeric gene edits in grape plants by allowing the delivery of DNA through the plant cell membrane with the recovery of whole plants from a single edited cell.

The invention provides an application of a compound enzyme in preparation of a Chinese rose protoplast, and belongs to the technical field of plant protoplast preparation. According to the compound enzyme disclosed by the invention, the cellulase R-10 and the macerozyme R-10 are compounded for use, and the cellulase R-10 mainly acts on a cellulose component in a cell wall of a Chinese rose leaf, so that a cell wall structure begins to collapse; the macerozyme R-10 can act on components such as pectin in cell walls, and has a synergistic effect with cellulase R-10, so that the cell wall structure is further destroyed, and the release of protoplast is promoted. When the compound enzyme is used for preparing the rosa chinensis protoplast, the high activity of the protoplast is kept, and the number of the obtained protoplast is large. The method disclosed by the invention has outstanding beneficial effects in the aspects of keeping the high activity of the rosa chinensis protoplast and increasing the number of the rosa chinensis protoplast, and opens up a broader way for related research and application of the rosa chinensis protoplast.

The application discloses a chrysanthemum constitutive high-expression promoter and application thereof in gene editing. The nucleotide sequence of the promoter CmUbi is shown as SEQ ID NO. 4. Through double luciferase reporter experiments and stable genetic transformation experiments, it is proved that the expression activity of the CmUbi promoter in chrysanthemum protoplast and callus is significantly higher than that of commonly used CaMV 35S, AtRPS5a and corn ZmUbi promoters. A CRISPR / Cas9 gene editing vector is constructed by using the promoter, and a key gene of branch of hexaploid chrysanthemum is edited BRC1b , and a plurality of allele simultaneous mutation complete editing strains are successfully obtained, and the number of branches of the mutants is significantly increased. The endogenous high-activity promoter of the chrysanthemum provided by the application effectively solves the problems of low expression activity of an existing heterologous promoter in the chrysanthemum and poor gene editing efficiency of a polyploid, and provides an efficient and adaptive expression regulation element for chrysanthemum molecular breeding and functional genomics research.

The invention provides a method for improving the success rate of a wheat protoplast instantaneous transformation system, which comprises the following steps: S1, fully rinsing a cell culture container for incubation by using a 10% bovine serum albumin solution, so that all structures in the container are infiltrated by the 10% bovine serum albumin solution, and discarding waste liquid after rinsing; and S2, resuspending the wheat protoplast into which the exogenous gene is transferred by using 900-1200mu L of a W5 solution, transferring the wheat protoplast into the cell culture container rinsed in the S1, wrapping the cell culture container by using tin foil paper, and incubating for 12-16 hours at room temperature. Compared with a traditional incubation process, the method has the advantages that the integrity rate and activity of the protoplast are remarkably improved, and the risks of protoplast rupture and death in the incubation process are effectively reduced. According to the system, the instantaneous conversion efficiency can stably reach more than 50%, and the reliability and operability of gene function verification and regulation mechanism research are greatly improved.

Provided is a method for generating a plant having improved growth and / or yield. The method comprises selectively increasing the size of one or more guard cells in a plant wherein the guard cell size is increased compared to guard cell size in an independent wild type control plant of the same species under the same environmental conditions. The ratio of guard cell size to leaf epidermal cell size in the plant is increased as compared to the ratio in the control plant. Also provided is a plant obtainable by the method; a gene construct; a transformed plant part or plant progeny; and a plant part, plant tissue, plant organ, plant cell, plant protoplast, embryo, callus culture, pollen grain or seed, derived or obtained from the plant.

Disclosed is a use of an Ltvcx1-1 protein and an Ltvcx1-1 protein coding gene in regulating the pathogenicity and stress resistance of Lasiodiplodia theobromae. The Ltvcx1-1 protein coding gene is derived from Lasiodiplodia theobromae. By constructing a knockout gene fragment directed at the Ltvcx1-1 gene of Lasiodiplodia theobromae and introducing the knockout gene fragment into a protoplast of Lasiodiplodia theobromae, a knockout mutant is obtained. It has been found from experiments that the Ltvcx1-1 gene plays a role in the cell membrane integrity and stress resistance of Lasiodiplodia theobromae and significantly reduces the pathogenicity of Lasiodiplodia theobromae to a host. Therefore, Ltvcx1-1 can be used as a target for a fungicide and a key protein of a pathogenic mechanism of a disease to develop an agent for a disease caused by Lasiodiplodia theobromae infection and breed a novel disease-resistant variety, and has wide application prospects in the control of phytopathogenic fungal diseases.

The application belongs to the field of plant synthetic biology and plant gene expression regulation, and particularly relates to a vector for plant promoter double system quantitative characterization, a standardization method, a product and application. The method combines a protoplast transient expression system and an agrobacterium-mediated leaf transient expression system, uses a compact promoter characterization plasmid (pLIP) containing a firefly luciferase module driven by a to-be-detected promoter and a renilla luciferase internal reference module driven by a 35S promoter, and the two modules are located in a T-DNA interval. The ratio of the two luciferases is calculated, the RPU (relative promoter unit) method is introduced, a standard promoter (35S) is used as a reference, and normalization and direct comparison of promoter activities in different systems are realized. The scheme significantly improves the repeatability and cross-system compatibility of promoter activity data, and is suitable for efficient screening and standardized characterization of regulatory elements in plant synthetic biology.

Disclosed in the present invention is the use of a threonine dehydratase in the regulation of the growth of Didymella segeticola and as a bactericide target. The threonine dehydratase gene Td of the present invention is derived from Didymella segeticola, and a knockout mutant is obtained by means of constructing a knockout gene fragment targeting the Td gene and then introducing same into a D. segeticola protoplast. Experiments find that the Td gene plays a role in the growth, the in-vivo pyruvic acid content and the in-vivo ATP content of D. segeticola, and the sensitivity thereof to wuyiencin. By means of molecular docking analysis, molecular dynamics simulation and microscale thermophoresis experiments, it is verified that threonine dehydratase can be used as a bactericide target, and the bactericide wuyiencin is obtained by means of screening. The threonine dehydratase of the present invention can be used as a bactericide target and a key protein in the pathogenic mechanism of a disease, and has broad application prospects in the control of plant pathogenic fungal diseases.

The present application belongs to the field of plant biotechnology, and particularly relates to a Korean pine cell fusion method based on chemical induction combined with a magnetic bead method. The method comprises the following steps: S1, preparing first protoplasts and second protoplasts; S2, preparing passivated first protoplasts and passivated second protoplasts; S3, mixing the passivated first protoplasts with the passivated second protoplasts in equal volumes after the passivated first protoplasts are cultured with a sodium silicate aqueous solution to obtain mixed protoplasts; S4, performing electrofusion on the mixed protoplasts in a cell fusion instrument to obtain fusion products; and S5, screening hybrid cells from the fusion products by using a magnetic bead method. The present application adopts a double screening mechanism of chemical passivation and magnetic bead screening, inactivates parent cells by treating with iodoacetamide and rose Bengal B, and then specifically enriches hybrid cells by using the magnetic bead method, so that the acquisition rate and screening accuracy of hybrid cells are significantly improved.