99 results about "Protoplasm" 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 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 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.

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 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.

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.

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.

The invention discloses a preparation method and application of a pine resin canker pathogen protoplast, and belongs to the technical field of genetic transformation. According to the preparation method of the pine resin canker pathogen protoplast disclosed by the invention, the protoplast with the final concentration of 1-2 * 10 < 7 > / mL is obtained through operations of enzymolysis, filtration, centrifugal resuspension and the like. The invention also establishes a Fusarium circinatum NAUA-1 genetic transformation system, and the genetic transformation system comprises the steps of protoplast preparation, plasmid transformation and screening, so as to obtain a stable genetic transformation strain FcGFP-1. Results of the embodiment of the invention show that the protoplast preparation method is efficient, stable and simple and convenient to operate, a transformation system can realize stable integration and expression of an exogenous gene, and the biological phenotype and pathogenicity of a transformed strain are consistent with those of a wild type; and technical support is provided for research on gene functions and pathogenic mechanisms of the pine canker germs, and drug molecular target mining and disease prevention and control are facilitated.

The invention relates to the technical field of protoplast preparation, in particular to a preparation method of protoplast of sclerotium rolfsii. The method comprises the following steps: carrying out enzymolysis on sclerotium rolfsii by adopting protoplast enzymatic hydrolysate; a Miracloth filter membrane is adopted for filtering, a container for collecting filtrate is placed on ice, and a precooled 0.6 M sorbitol solution is used for flushing; centrifuging the collected filtrate, collecting precipitates, cleaning the precipitates by using a precooled 0.6 M sorbitol solution to remove enzymatic hydrolysate, and then carrying out resuspension centrifugation by using STC to collect precipitates so as to obtain protoplasts; according to the characteristics of sclerotium rolfsii, a corresponding enzymolysis system is established, the enzymolysis condition is mild and easy to control, the obtained protoplast is complete and stable in form and high in yield, and the quantity of the protoplast reaches up to 1 * 10 < 8 > / mL.

The invention provides a PEG / CaCl2 mediated Penicillium citrinum genetic transformation method, and belongs to the technical field of microbial genetic transformation, and the method comprises the following steps: adding a target gene segment into Penicillium citrinum protoplast suspension, and carrying out an ice bath reaction; the target gene segment is obtained by taking pJET-hph-02944 plasmid as a template and carrying out PCR (Polymerase Chain Reaction) amplification by adopting a hygromycin B resistance gene primer pair with gene sequences as shown in SEQ ID No. 1 and SEQ ID No. 2; adding a PTC (Positive Temperature Coefficient) buffer solution into the mixed system, uniformly mixing and standing; continuously adding a TB3 liquid culture medium containing ampicillin, and carrying out recovery culture; uniformly mixing the resuscitated protoplast with a molten TB3 solid culture medium, pouring a plate, and carrying out dark culture for the first time; then covering a TB3 solid culture medium containing hygromycin B and ampicillin, carrying out secondary dark culture to obtain a transformant, and transferring the transformant to a PDA plate containing hygromycin B for screening. According to the method, CaCl2-PEG is adopted for mediated transformation of Penicillium citrinum, the transformation efficiency is higher, and the transformation speed is higher.

This invention discloses a PEG-mediated genetic transformation method for *A. anthracnose* protoplasts. Through the preparation, transformation, and transformation of *A. anthracnose* protoplasts, and the screening of transformants, the hygromycin B resistance gene fragment was successfully transferred into the *A. anthracnose* genome, achieving successful genetic transformation of *A. anthracnose* protoplasts. More importantly, this invention establishes a new enzymatic digestion system and improves the culture mechanism in protoplast preparation. During the culture and screening of transformants, a more suitable resistance concentration is used, balancing the stringency of screening with the survival of transformants. This improves transformation efficiency, shortens the overall transformation process, and establishes a more efficient and convenient genetic transformation system for *A. anthracnose* protoplasts.

The invention relates to the technical field of plant genetic transformation, and discloses a PEG-mediated rubber tree leaf protoplast large plasmid instantaneous transformation method which comprises the following steps: S1, taking 1-2g of leaves from the later stage of a bronze stage to a color change stage of a rubber tree within two years old, soaking the leaves in 0.6 M mannitol for 10 minutes, removing main leaf veins by using a scalpel, cutting the leaves into long and thin strips with the width of 0.5-1mm, transferring the long and thin strips into a 30ml enzyme solution, and carrying out enzyme deactivation on the long and thin strips; putting into a dark culture room at 28 DEG C, and carrying out enzymolysis treatment on a shaking table at 60 r / min for 5 hours; according to the method, transformation steps including centrifugal rotating speed, PEG action time and culture conditions are optimized, and a transformation reagent with a specific formula is adopted, so that the plasmid size limitation which is difficult to exceed in a rubber tree protoplast instantaneous transformation technology is broken through, large plasmids of more than 15kb are efficiently introduced into cells, and finally, the gene editing efficiency of more than 15% is achieved.

The utility model belongs to the technical field of genetic gene experiment devices, and particularly relates to a protoplast preparation and transformation functional island. The filtering module is arranged at the top end of the integrating table, and the interior of the integrating table communicates with the outside through the filtering module; the transfer module is arranged in the center of the interior of the integration table, and the transfer module is used for transferring the protoplast and the consumables; the storage module is arranged in the integration table, and the storage module is used for storing the protoplast and the consumables; the preparation and conversion module is arranged in the integration table, and the preparation and conversion module is used for preparing and converting the protoplast; the preparation and conversion module and the storage module are located on the periphery of the transfer module. According to the utility model, the accurate control of key steps such as enzymolysis, counting, culture and conversion can be realized, the experiment efficiency and repeatability are obviously improved, the dosage of experiment reagents is reduced through miniaturization and parallel processing, and high-throughput parallel processing is realized.

The invention discloses a precise editing method of a gamma-PGA high-yield strain based on CRISPR-Cas9n, and relates to the technical field of high-yield strains. According to the invention, a CRISPR-Cas9n double-nickase system is adopted, and adjacent DNA nicks are generated through the synergistic effect of double sgRNA, so that the non-specific cutting risk is greatly reduced; the double sgRNA targeting design is combined with a homologous recombination template, so that directional editing of key genes is realized, the homologous recombination efficiency reaches 75 + / -5%, and target modification is ensured to be stably integrated into a genome; by virtue of a PEG-mediated protoplast conversion method, the limitation of low efficiency of traditional electrotransfection is overcome, and the CRISPR carrier is rapidly removed in combination with a temperature-sensitive plasmid elimination system, so that cytotoxicity caused by continuous expression is avoided; under optimized fermentation conditions, the gamma-PGA yield of the edited strain reaches 15 + / -2g / L and is increased by 200% compared with that of the original strain.

The invention discloses an application of FvSup1 protein and a coding gene thereof in regulation and control of pathogenicity of fusarium verticillium, and belongs to the technical field of biology. New functions of the fusarium verticillium unknown function protein FvSup1 and the coding gene FvSup1 thereof are researched and confirmed for the first time. The method comprises the following steps: constructing an FvSup1 gene knockout vector, and introducing the FvSup1 gene knockout vector into a fusarium verticillium protoplast to obtain a knockout mutant delta FvSup1; the method comprises the following steps: constructing a gene back-up vector, and introducing the gene back-up vector into a delta FvSup1 protoplast; and a complement mutant delta FvSup1-C is obtained. Pathogenicity determination shows that the pathogenicity of the knockout mutant delta FvSup1 is obviously reduced; and the pathogenicity of the complement mutant delta FvSup1-C can be recovered to a wild type level. The test proves that the FvSup1 is a pathogenic related gene of the fusarium verticillium, and a new target is provided for preventing and treating plant diseases caused by the fusarium verticillium.

The invention provides a PEG (Polyethylene Glycol)-mediated powdery parasitic spore protoplast genetic transformation method and application thereof, and belongs to the technical field of biology. The method comprises the following steps: performing enzymolysis on tender hyphae of powdery parasitic spore by adopting a combination of driselase and lyase to efficiently prepare protoplast; transforming the protoplast and plasmid DNA (deoxyribonucleic acid) under the mediation of a PEG4000 (polyethylene glycol 4000) solution; a stable genetic transformant is screened and obtained through a culture medium containing G418. According to the invention, a genetic transformation system of the sphaerospora fuliginea is established for the first time, and the transformant can stably express an exogenous gene with high strength. The method disclosed by the invention is simple and convenient to operate, high in transformation efficiency and good in repeatability, provides a key technical platform for researching gene functions and parasitic mechanisms of the sphaerospora fuliginea and constructing efficient engineering biocontrol strains, and has important application value in the technical field of biological prevention and control of powdery mildew.

The invention provides a hybrid potato seedling hypocotyl single cell extraction method and application thereof. According to the characteristics of potato seedling hypocotyl single cells, the extraction efficiency of the protoplast is remarkably improved by adjusting the dosage of different cellulase in the enzymatic hydrolysate and combining a slight extrusion mode. The method is simple and rapid, does not need complex treatment and expensive reagents, and is convenient to popularize and apply. Besides, experiments prove that the protoplast extracted by the method can be effectively used for detection such as subsequent sequencing and the like, so that the cells extracted by the method are relatively high in activity and have a wide application value.

The invention belongs to the technical field of plant puree preparation, and particularly relates to dendrobium puree as well as a preparation method and application thereof. According to the preparation method, firstly, a 1% sodium bicarbonate aqueous solution is used for conducting heat extraction on dendrobium nobile, the 1% sodium bicarbonate aqueous solution can neutralize the acid environment, and acidic hydrolysis of polysaccharide in the extraction process is inhibited; according to the method, an ethanol solution with the concentration of 50% is added into dendrobium residues extracted by a sodium bicarbonate aqueous solution with the concentration of 1% for re-extraction, gradient separation of components can be realized by using solvent systems with different polarities, and the ethanol solution with the concentration of 50% can dissolve acidic polysaccharide which is not dissolved by alkaline water in dendrobium and can effectively extract medium polar substances such as flavones and glycosides at the same time. In addition, a water-saturated n-butyl alcohol solution is continuously added into the extracted dendrobium residues for extraction, and residual polysaccharide and saponin components in the dendrobium are efficiently extracted. Through the steps, the raw stock components in the dendrobium nobile can be effectively reserved, and the dendrobium nobile has an excellent anti-inflammatory effect.

The application provides a preparation method of a schizochytrium sp. protoplast. The preparation method of the schizochytrium sp. protoplast comprises the following steps: fermenting and culturing schizochytrium to obtain a fermentation liquor; a first fermentation stage: the temperature is 26-28 DEG C, and the culturing time is 36-40 h; a second fermentation stage: the temperature is 30-32 DEG C; and no sugar is additionally supplemented in the process of the fermentation culture. A large amount of protoplasts can be obtained by controlling the culturing conditions in the fermentation culture stage.