238 results about "Protoplasm" patented technology

The invention discloses application of threonine dehydratase in regulation and control of growth of Didymeria segeticola and application of threonine dehydratase serving as a bactericide target in regulation and control of growth of Didymeria segeticola. The threonine dehydratase gene Td disclosed by the invention is derived from Didymeria segeticola, and a knockout mutant is obtained by constructing a knockout gene segment of the Td gene and introducing the knockout gene segment into a D.segeticola protoplast. Experiments find that the Td gene plays a role in growth of D.segeticola, in-vivo pyruvic acid content, in-vivo ATP (adenosine triphosphate) content and sensitivity to wuyiencin. Molecular docking analysis, molecular dynamics simulation and trace thermophoresis experiments prove that the threonine dehydratase can be used as a bactericide target, and the bactericide wuyiencin is obtained through screening. The threonine dehydratase disclosed by the invention can be used as a bactericide target and a key protein of a disease pathogenic mechanism, and has a wide application prospect in prevention and treatment of plant pathogenic fungal diseases.

The invention discloses a polygonatum cyrtonema transcription factor gene PcWRKY1 as well as an encoding protein and application thereof. According to the application, a WRKY transcription factor PcWRKY1 which is obviously inhibited by high-temperature stress is screened, the transcription level of the PcWRKY1 transcription factor gene responding to the high-temperature stress is further determined, a tissue expression mode and subcellular sites of the PcWRKY1 transcription factor gene are analyzed, and finally, the regulation effect of the PcWRKY1 transcription factor gene on the high-temperature tolerance of the polygonatum cyrtonema protoplast is determined. The PcWRKY1 transcription factor gene is located in a cell nucleus, codes a protein containing 519 amino acids and is expressed in roots, stems, leaves, tubers and seeds of polygonatum cyrtonema, the expression quantity in the seeds is the highest, and the PcWRKY1 transcription factor gene can inhibit the increase of MDA content and promote the improvement of CAT enzyme activity under high temperature stress. Further, the tolerance of the polygonatum cyrtonema protoplast under high-temperature stress is improved.

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 improving biocontrol photosynthetic bacteria through a far-source hybridization and genome rearrangement technology. The method comprises the following steps: inoculating a rhodopseudomonas palustris GJ-22 seed into a photosynthetic bacteria liquid culture medium for culturing; inoculating the bacillus subtilis KY-2 into an LB liquid culture medium for culturing; the cultured GJ-22 is taken and subjected to treatment and centrifugation, and then a GJ-22 protoplast is obtained; kY-2 is taken to be treated, and KY-2 protoplast is obtained after centrifugation; fusing the obtained GJ-22 protoplast and KY-2 protoplast, diluting a photosynthetic bacterium hypertonic liquid culture medium, and regenerating on a photosynthetic bacterium soft agar culture medium to obtain a fused strain; a fusion strain is screened out through an induced tobacco core leaf anti-TMV test to serve as a starting strain for genome rearrangement. Preparing protoplast from the original strain; dividing the prepared protoplast into two groups, and respectively carrying out ultraviolet inactivation and thermal inactivation; respectively fusing the inactivated protoplasts, and regenerating to obtain fusant strains; finally, a target strain with genetic stability is screened through a fusant strain induced tobacco core leaf anti-TMV test.

The invention discloses a method for site-specific replacement or insertion of DNA fragments in a plant genome, and belongs to the technical field of biology. The method disclosed by the invention comprises the following steps: editing a genome of a plant protoplast by adopting a CRISPR / Cas9 system and taking a double-stranded DNA repair template as a donor template to obtain an edited protoplast; and carrying out culture regeneration on the edited protoplast by utilizing a plant protoplast transformation method to obtain a site-directed mutagenesis plant in the genome so as to realize site-directed mutagenesis of the target plant genome. The method disclosed by the invention can be used for accurately replacing DNA in a plant genome at a fixed point. According to the invention, a recognition sequence of recombinase can be accurately inserted into a genome at a fixed point through homologous recombination, and then accurate fixed-point replacement of a gene large fragment is realized by virtue of a specific recombinase system.

The invention relates to the field of microbial genetic transformation, and discloses a preparation method of Ilyonectria robus fungus protoplast and a construction method of a genetic transformation system.The preparation method comprises the following steps that S11, I.robus hyphae are prepared, after I.robus is cultured on a PDA solid culture medium for 14 days, the I.robus hyphae are inoculated to a CMC liquid culture medium to be subjected to shake-flask culture, and spores are filtered and collected; diluting the spore concentration, inoculating into a YEPD liquid culture medium, carrying out shake-flask culture, filtering and collecting hyphae; and S12, preparing enzymatic hydrolysate, namely adding the hyphae prepared in the step S11 into the enzymatic hydrolysate prepared in the step S12, carrying out shake flask enzymolysis, filtering, centrifuging filtrate, discarding supernate, adding a 0.7 M NaCl solution, centrifuging, and discarding the supernate, so as to obtain the I.robus fungus protoplast. The protoplasts with proper quantity and good quality can be efficiently obtained, and the transformation efficiency of a subsequent genetic transformation system is improved; and the constructed genetic transformation system can be used for construction of a mutant library of the I.robusta strain, interaction of a host plant and the I.robusta strain and research on the pathogenesis of the I.robusta strain.

The invention discloses a mining method and application of a new functional gene of asexual reproduction filamentous fungi, and relates to the technical field of applied microorganisms, and the preservation number is CGMCC No.12166. The preservation unit is China General Microbiological Culture Collection Center; the preservation date is April 11, 2016. The invention discloses a mining method of a new functional gene of asexual reproduction filamentous fungi. The mining method comprises the following steps: S1, constructing a uridine synthesis defect type trichoderma strain A with hygromycin resistance; s2, obtaining a random mutant strain B; s3, constructing a temporary simulated diploid through a protoplast fusion method; s4, carrying out random complementation on chromosome genes of the random mutants through homologous recombination of the two sets of chromosomes in the nucleus, and screening through phenotypic change to obtain phenotypic complemented mutants; and S5, determining a key mutation site for back-complementing by using a re-sequencing technology, and reversely verifying the gene function by using a gene editing method. The method disclosed by the invention provides a new method for researching functional genes of asexual reproduction type filamentous fungi.

The invention provides an efficient and stable nori pythium gene transformation method which comprises antibiotic concentration screening, protoplast preparation, transformation system optimization, positive transformant screening and transformant phenotype and gene expression stability analysis, and the evaluation method is the nori pythium gene transformation method disclosed for the first time in China. The method provides a valuable tool for researching gene functions and pathogenic mechanisms of pythium aphanidermatum in the future.

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 discloses an application of a protoplast CUAmp; the invention discloses a high-throughput method for detecting a transcription factor recognition motif by using a Tag technology. The method comprises the following steps: 1) cutting up plant leaves, soaking the cut plant leaves in enzymatic hydrolysate, and separating cells, so that rice powder is completely soaked in the enzymatic hydrolysate; 2) after enzymolysis, wrapping filter residues with gauze, and extruding the filter residues to collect protoplast cells; (3) adding the protoplast cells into a centrifugal tube containing plasmids, instantaneously transferring the plasmids into the cells, and expressing genes transferred by the plasmids; (4) adding a GFP antibody, a secondary antibody and a Tn5 transposon, recovering the DNA combined with the GFP antibody, and carrying out subsequent NGS library building; 5) performing high-throughput sequencing to obtain a transcription factor sequence, and performing biological information analysis to obtain a transcription factor combined motif; and 6) verifying the interaction of the transcription factor and the recognition motif by using an EMSA method.

The invention discloses an application of DspckA in regulation and control of the pathogenicity of Didymeria segeticola and application of the DspckA as a bactericide target. The DspckA gene disclosed by the invention is derived from D.segeticola, and a knockout mutant is obtained by constructing a knockout gene segment of the DspckA gene and introducing the knockout gene segment into a D.segeticola protoplast. It is found through experiments that the DspckA gene plays a role in the pathogenicity of D.segeticola and the sensitivity of the DspckA gene to ningnanmycin. Molecular docking analysis and trace thermophoresis experiments prove that the DspckA protein can be used as a bactericide target and has interaction with ningnanmycin, and amino acids at T264, T265 and E306 are key binding sites. The DspckA can be used as a bactericide target and a key protein of a disease pathogenic mechanism, and has a wide application prospect in prevention and treatment of plant pathogenic fungal diseases.

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 is applicable to the field of protoplast extraction application, and provides an enzymatic hydrolysate for Leymus chinensis protoplast extraction, which comprises the following components: 2.5-5% of cellulase R-10, 0.5-1% of macerozyme R-10, 0.7-1 M of mannitol, 5-9 mM of potassium chloride, 10-20 mM of MES buffer solution, 10-20 mM of calcium chloride, 0.05-0.2% of BSA, and the balance of ultrapure water. The invention also provides a preparation method and an application method of the enzymatic hydrolysate. In conclusion, according to the particularity of the leymus chinensis cell wall, an enzymatic hydrolysate formula is optimized, extraction conditions are adjusted, cell damage is effectively reduced, higher protoplast activity and quantity are obtained in leymus chinensis extraction, the extraction efficiency of the leymus chinensis protoplast is remarkably improved, and further conversion and gene expression research requirements are met.

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 provides a wheat powdery mildew resistance gene TaGAP1 as well as an encoding protein and application thereof. The TaGAP1 is silenced through a BSMV-mediated instantaneous silencing technology, after powdery mildew E09 is inoculated, the number of powdery mildew colonies of a silenced plant is obviously increased, the powdery mildew grows and develops faster than that of a control plant, and it is indicated that the silent TaGAP1 reduces the resistance of wheat to powdery mildew; the bacterial colony area and biomass of powdery mildew of overexpressed TaGAP1 are remarkably reduced, and the resistance of wheat to powdery mildew is remarkably enhanced, so that TaGAP1 can be used as an effective gene resource for improving the powdery mildew resistance of wheat. Tobacco transient expression and wheat protoplast localization show that the TaGAP1 protein is localized in cytoplasm and cell nucleus. The wheat powdery mildew-resistant gene TaGAP1 provided by the invention provides a new gene resource for cultivating powdery mildew-resistant wheat varieties, and has important application value for widening the genetic basis of wheat and genetically improving the powdery mildew resistance of wheat.

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 female-breeding primary pulp composition based on a seed circulation method and a preparation method and application thereof, and relates to the technical field of functional foods.The female-breeding primary pulp composition is prepared from, by weight, 15%-25% of organic flaxseeds, 15%-25% of organic black beans, 10%-20% of figs, 10%-20% of soybeans, 5%-15% of the root of kudzu vine, 5%-15% of pumpkin seeds and 2%-8% of lycium ruthenicum. The invention also provides a preparation method of the female-nourishing protoplasm composition and application of the female-nourishing protoplasm composition in functional food for regulating the balance of female estrogen and progestational hormone. According to the female-nourishing puree composition based on the seed circulation method, various medicinal and edible raw materials are selected and scientifically compounded, a special processing technology is adopted for wall breaking and effect releasing, and the ready-to-drink puree which can be drunk fixedly every day so as to realize synchronous balance adjustment of estrogen and progesterone is prepared; the pain points of high cycle dependence, low absorption rate and inconvenience in use of the traditional method are fundamentally 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.

According to the method, engineering microorganisms are immobilized on stationary phases such as gelatin by applying a microorganism immobilization technology, cell walls of the microorganisms are removed by applying lysozyme, and 6s-5 methyltetrahydrofolic acid in cells can be easily extracted through cell membranes under the action of a surfactant. A culture solution and an eluent are made into mobile phases, the mobile phases flow through a stationary phase containing engineering bacteria, so that the 6s-5 methyltetrahydrofolic acid is continuously extracted from microorganisms, and after the 6s-5 methyltetrahydrofolic acid is collected, the treated mobile phases such as the culture solution and the eluent are circularly input into the stationary phase to form a continuous flow production process. The continuous flow process shows a milk squeezing effect, and the concentration balance of 6s-5 methyltetrahydrofolic acid in microorganisms is broken, so that the 6s-5 methyltetrahydrofolic acid is continuously biosynthesized and output. The technical problem of low concentration of biological fermentation final products at present is solved.

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 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 relates to the field of genetic transformation, in particular to a protoplast genetic transformation method of white-rot fungi Irriga lactea. According to the invention, the preparation and genetic transformation method of the white-rot fungus Irpex lacteus protoplast is established for the first time, the transformation efficiency is 6-7 transformants / [mu] g DNA, the transformation period is 1-2 weeks, the method has the characteristics of high transformation efficiency, short transformation time period and the like, and powerful support is provided for research work of gene functions and strain improvement of Irpex lacteus in the future.

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 discloses a method for establishing an achnatherum inebrians protoplast transient protein expression system. The method comprises the following steps: cutting achnatherum inebrians leaves into thin strips along veins, carrying out enzymolysis treatment on the strips in an enzymatic hydrolysate for several hours, washing, separating to obtain mesophyll protoplast, introducing an exogenous gene into the achnatherum inebrians protoplast by using a polyethylene glycol mediated transformation method, washing, incubating and the like to obtain the successfully transformed and expressed protoplast. The method provided by the invention does not need to apply vacuum conditions, is simple to operate, provides optimal achnatherum inebrians protoplast preparation conditions, can prepare and obtain the protoplast with successfully converted exogenous genes in a short time, and the established achnatherum inebrians protoplast transient protein expression system has the advantages of high expression efficiency and high yield. Important basic data is provided for gene function characterization and molecular breeding.

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.