245 results about "Protoplast" patented technology

The invention belongs to the technical field of leguminous plants, and particularly discloses a soybean stem protoplast preparation and transformation method, which specifically comprises the steps of soybean aseptic seedling culture, soybean stem protoplast preparation and protoplast transformation. According to the method, the protoplast is prepared from the stem part of the first internode of the soybean, the formula of an enzyme solution is optimized, the protoplast cleaned and collected by W5buffer is utilized for conversion, and PEG instantaneous conversion reaction conditions are utilized, so that the yield and the conversion rate of the soybean protoplast are improved, and the method is more beneficial to identifying the soybean gene function.

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 present invention relates to the field of plant-derived cellular vesicles, and in particular of nanovesicles obtained from plant protoplasts. A method for their preparation and uses thereof in the medical, cosmetic and nutraceutical fields are described.

The invention discloses a method for enhancing squalene synthesis through oil tea HMGR gene editing. According to the method, phosphorylation regulation and control sites Ser47 and / or Thr208 recognized by MAPK are targeted, and enzymatic activity inhibition is relieved and metabolic flux is enhanced by constructing phosphorylation resistance mutants (such as Ser-> Ala and Thr-> Val). The constructed editing vector can be introduced into camellia oleifera tissues in manners of PEG-mediated protoplast transient transfer, agrobacterium infection of calluses and the like, so that stable expression is realized. Experiments prove that the enzyme activity of HMGR in the editor is remarkably improved, the squalene accumulation amount is increased by 50% or above, the editing efficiency is high, expression is stable, and the method is suitable for high-value development of plant triterpenoid functional components and excellent strain breeding. The method also has the potential of popularization and application in other triterpenoid synthetic crops.

The invention discloses a genetic transformation method of PEG (Polyethylene Glycol) mediated penicillium sclerotiorum strain protoplast and application of the genetic transformation method. The method comprises the following steps: (1) culturing penicillium sclerotiorum to produce spores, germinating hyphae, and collecting and cleaning fresh penicillium sclerotiorum hyphae; (2) compounding Lysing Enzymes, Yatase, cellulase and pectinase to prepare a protoplast enzymatic hydrolysate, and then adding the protoplast enzymatic hydrolysate into the penicillium sclerotiorum hypha for enzymolysis to obtain a penicillium sclerotiorum protoplast; and (3) adding plasmids to be transformed into the penicillium sclerotiorum protoplast, and carrying out PEG (Polyethylene Glycol) mediated transformation and resistance screening to obtain a penicillium sclerotiorum protoplast transformant. According to the method, the number and regeneration rate of the protoplast can be remarkably increased, and the enzymolysis time can be effectively shortened.

The invention provides an efficient double-base editing system without PAM (Pulse Amplitude Modulation) limitation. According to the technology, different point mutations are introduced into SpRY, the SpRY is fused with efficient adenine deaminase and cytosine deaminase to construct a double-base editor, and efficient PAM-limitation-free double-base editing is achieved in the rice protoplast. According to the plant basic group editing method, efficient and PAM-less double basic group editing can be achieved, and the plant basic group editing technology is wider in target range, higher in efficiency and higher in double basic group editing product proportion.

As a main grain crop in the world, the yield and quality of rice are often threatened by bacterial leaf blight and rice blast. According to the invention, a recombinant expression vector for expressing STTM-miR169d / p is constructed by starting from regulation and control of the expression quantity of miRNA through an STTM technology. By adopting an agrobacterium tumefaciens-mediated rice protoplast transformation technology, a rice plant containing the STTM-miR169d / p transgenic fragment is obtained. Through field inoculation of xanthomonas oryzae pv. Oryzae and inoculation of rice blast spores on in-vitro leaves, it is verified that the STTM-miR169d / p transgenic rice has significantly improved resistance to xanthomonas oryzae pv. Oryzae and rice blast compared with wild type rice. The invention not only provides a new germplasm resource for cultivating disease-resistant rice, but also provides a new thought for regulating and controlling the disease resistance of rice from the aspect of miRNA.

The present invention relates generally to the field of molecular biology and concerns a method for high throughput screening and characterization of one or more candidate plant pathogen effect genes and / or one or more candidate plant disease resistance genes in a single plant cell, the method comprises: (i) introducing into a plant protoplast one or more expression cassettes comprising at least one candidate plant pathogen effect gene and / or at least one candidate plant disease resistance gene; (ii) transiently expressing the one or more candidate plant pathogen effect genes and / or the one or more candidate plant disease resistance genes; (iii) measuring reactive oxygen species (ROS) produced by the protoplast; then (iv) separating the plant protoplast producing the ROS; and (v) identifying the introduced at least one candidate plant pathogen effect gene and / or at least one candidate plant disease resistance gene. Alternatively, the protoplast is capable of expressing the selectable marker, thereby allowing for the measurement of modulated selectable marker expression as an alternative indicator for HR activation.

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 preparation method and an instantaneous transformation method of a mangrove plant bamboo joint tree protoplast, and relates to the technical field of plant protoplast preparation and transformation. According to the method, seedling leaves of the mangrove plant bamboo joint tree protoplast serve as experimental materials, the protoplast is obtained through an enzyme lysis method, and then exogenous genes are transferred into the mangrove plant bamboo joint tree protoplast through a PEG-mediated transformation method for transient expression. The method comprises the following steps: firstly, selecting a bamboo joint tree with fewer mangrove family secondary metabolites as a material, and inducing through spectra with different illumination intensities and wavelengths, so that accumulation of leaf secondary metabolites is reduced while the mesophyll cell structure is adjusted, and further, the purposes of promoting cell wall lysis and protoplast separation are achieved. The invention develops a composite enzymolysis formula adaptive to cell wall components of the mangrove tree, and establishes a set of system for maintaining activity and culturing protoplast of the mangrove tree, so that an exogenous gene can be successfully and transiently expressed in the system.

The application discloses a lithium acetate-polyethylene glycol mediated protoplast lipid nanoparticle delivery method and belongs to the technical field of biology. 2+ )concentration to improve the PEG formula, successfully delivers the lipid nanoparticle (LNP) to the protoplast of oil palm, coconut and other palm crops, the mRNA of the nanoparticle is wrapped with enhanced green fluorescent protein (eGFP), and the fluorescent protein can be directly expressed in the protoplast with the mRNA as a template without a promoter. After the LNP is delivered to the protoplast of the palm crops by the method, green fluorescent signals are detected by laser confocal microscopy. The method can be used for directly delivering target gene mRNA to the protoplast of the palm crops, and can also deliver CRISPR / Cas system protein and the like for gene editing technology research.

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.

The present invention discloses a fusion strain of Lactobacillus paracasei and Bifidobacterium adolescentis, as well as its preparation method and application, belonging to the field of strain breeding technology. A fusion strain of Lactobacillus paracasei and Bifidobacterium adolescentis was successfully prepared using the method provided by the present invention. The prepared fusion strain has a new morphology, a broadened carbon source utilization range, and significantly improved inhibitory effects on three pathogens. Furthermore, it is resistant to both vancomycin and mupirocin lithium salts. It also has good fermentation characteristics, with a pH of 4.05 and a titer of 114°T after 28 days of storage, and the viable cell count can still be maintained at 1×10 7 The protoplasts have a CFU / mL value of above 2260 mPa·s, a viscosity value of 37.462 g, a hardness of 37.462 g, and a consistency of 282.04 g·s, and can be used for the development and application of dairy products. The preparation method provided provides protoplasts with strong regeneration ability, high vitality, and high fusion rate, providing support for the selection of excellent lactic acid bacteria strains using protoplast fusion technology.

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.