204 results about "Transformation efficiency" patented technology

The invention discloses a pichia pastoris engineering bacterium for synthesizing bakuchiol from de novo by converting methanol as well as construction and application of the pichia pastoris engineering bacterium. A heterologous p-coumaric acid synthesis pathway is introduced to neutral sites of different chromosomes of pichia pastoris, a high-yield p-coumaric acid strain is obtained through gene knockout, overexpression or heterologous expression of tyrosine and phenylalanine synthesis pathway key genes of a shikimic acid pathway, a heterologous bakuchiol biosynthesis pathway is introduced on the basis, and a high-yield p-coumaric acid strain is obtained. After overexpression of bakuchiol synthetase, endogenous overexpression or heterologous expression of MVA pathway key genes and exogenous acetyl coenzyme A supply pathway genes, methanol concentration optimization and bacterial strain His4 gene supplementation, the yield of bakuchiol is effectively increased to 91.2 mg / L and is increased by 59.8 times compared with that of an initial bacterial strain, and the yield of bakuchiol in a 15L fermentation tank reaches 692.8 mg / L. The method has the characteristics of high conversion efficiency, low production cost, convenience in preparation, wide industrial application prospect and the like.

The invention provides an AbAIL5 gene and application of the AbAIL5 gene in improvement of genetic transformation efficiency of amorphophallus bulbifer, and belongs to the technical field of biology. The invention provides an AbAIL5 gene. The nucleotide sequence of the AbAIL5 gene is shown as SEQ ID NO: 1. Genetic transformation is performed on the amorphophallus bulbifer somatic embryo by constructing an overexpression vector of the AbAIL5 gene, so that the genetic transformation efficiency of the amorphophallus bulbifer callus can be improved, and candidate genes are provided for application of a large-scale breeding technology and a genetic transformation technology of the amorphophallus bulbifer callus in good varieties.

The present application relates to the technical field of elicitor peptide MsPep and its application, and discloses the use of elicitor peptide MsPep in improving the genetic transformation efficiency of alfalfa, wherein the application takes the leaf of alfalfa as an explant, introduces an exogenous gene through Agrobacterium tumefaciens, and adds the elicitor peptide MsPep in each stage of genetic transformation, so as to significantly improve the dedifferentiation rate, the out-growth rate, the differentiation rate, the out-bud rate, the rooting rate and the transformation efficiency, and shorten the transformation period, and the present application has application prospect in improving the genetic transformation of alfalfa and other plants.

The invention belongs to the technical field of plant biology, and discloses a wild soybean genetic transformation method which comprises the following steps: S1, disinfecting wild soybean seeds; s2, the backs of the disinfected wild soybean seeds are cut once, the cut wild soybean seeds are placed on a culture medium, and imbibition seeds are obtained; s3, seed coats of the wild soybean seeds are removed, growing points of embryo tips are exposed through dissection, and explants are obtained; s4, putting the explant into an agrobacterium infection solution containing target plasmids, and carrying out vacuum infiltration in a vacuum environment; and S5, after releasing vacuum, standing and infecting in an agrobacterium infection solution for 3-16 hours. The glycine soja genetic transformation method can effectively improve the transformation efficiency of glycine soja.

The invention discloses a rubus cochinchinensis efficient genetic transformation method based on RUBY reporter gene and agrobacterium rhizogenes mediation, which realizes that the transformation period is shortened to be within 15 days and the transformation efficiency is improved to be more than 60% through direct explant treatment under a non-tissue culture condition. The method comprises the following specific steps: 1) preparing an explant material; (2) constructing an RUBY carrier and transforming agrobacterium rhizogenes; (3) infecting the explant by utilizing agrobacterium rhizogenes carrying a target gene; (4) co-culturing after infection and carrying out RUBY visual screening; 5) positive root molecular biology verification; (6) inducing positive roots into buds; according to the method, induction of the positive callus, the positive root and the positive bud of the rubus coreanus stem segment under the non-tissue culture condition is successfully achieved, a stable and efficient genetic transformation system is constructed, the method has the remarkable advantages of being easy and convenient to operate, low in cost, wide in applicability, high in reproducibility and the like, and a key technical support is provided for molecular breeding of the rubus coreanus.

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 application discloses application of SbWOX gene and SbBBM gene in corn breeding and a corn genetic transformation method, and relates to a bioengineering technology.The application provides a nucleic acid molecule with co-expression of SbWOX protein gene and SbBBM protein gene, and corresponding gene co-expression vectors and microbial transformants.The application also provides application of the co-expression of the SbWOX gene and the SbBBM gene in the corn genetic transformation method.The application obtains corresponding genes and mutant proteins based on the SbWOX gene and the SbBBM gene of sorghum, the two genes are co-expressed in the corn genetic transformation process, even without adding plant growth hormones in the antibiotic screening medium in the tissue culture process, the plant genetic transformation process can be normally completed, and the transformation efficiency and the regeneration efficiency of the corn can be remarkably improved, the transformation rate reaches 10.42%, and the differentiation rate reaches 32.89%.

This invention discloses the application of the GhATG18a gene in accelerating callus induction in cotton. The invention constructs a pCAMBIA2300-GhATG18a overexpression vector from the full-length CDS fragment of this gene, then introduces it into Agrobacterium, and transforms cotton using Agrobacterium-mediated transformation to obtain transgenic lines. qRT-PCR results show that, compared with the CCRI24 control material, the expression level of the GhATG18a gene in the overexpression lines OE-GhATG18a-1 and OE-GhATG18a-2 is significantly upregulated. Phenotypic observation and histological sections both show that the OE-GhATG18a line overexpressing this gene accelerates the process of somatic cell dedifferentiation and callus induction in cotton, thereby improving the genetic transformation efficiency of cotton. Therefore, overexpressing this gene and applying it to commercial cotton varieties that are difficult to genetically transform can help accelerate the callus induction process, thereby improving the genetic transformation efficiency and providing technical support for breeding high-yield and stress-resistant high-quality cotton germplasm.

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 genetic transformation and gene editing method applicable to multiple sesquiterpene species, belonging to the field of plant genetic transformation and gene editing technology. The method uses the cotyledonary nodes of sterile seedlings of the sesquiterpene genus as explants, and introduces an optimized pScEF1α-Cas9 gene editing transformation vector using Agrobacterium infection. Regenerated plants are obtained through recovery culture, selection culture, and rooting culture. The vector contains four gene expression units: pScEF1α:Cas9, pGmU6:sgRNA, pGmUBI:DsRed2, and pCaMV35S:HygR. In vivo screening is further performed using the DsRed2 fluorescent reporter gene, and the gene-edited plants are identified by molecular detection. This method is applicable to common sesquiterpene, stem-nodled sesquiterpene, and spiny sesquiterpene, and has advantages such as strong species versatility, high transformation efficiency, short cycle, visualized screening, and high positive rate, providing efficient technical support for gene function research and genetic improvement of sesquiterpene species.

The present disclosure provides an application method in rice callus differentiation based on an Oryza sativa Leafy Cotyledon 1 (OsLEC1) gene, including the steps of: selecting guide ribonucleic acid (gRNA) target sites; cloning tandem fragments including gRNA; ligating each gRNA fragment; performing polymerase chain reaction (PCR) amplification on a ligation product; performing enzyme digestion on the purified product and a target vector; transforming the ligated vector; performing Agrobacterium-mediated genetic transformation of rice; and screening and identifying transgenetic plants. In the present disclosure, the differentiation of callus directly affects the emergence efficiency of transgenic plants. The knockout of OsLEC1 can promote the differentiation of rice callus, suggesting that OsLEC1 can serve as an important target gene for improving the transformation efficiency of rice and even gramineous crops. OsLEC1 can be used as a starting point to construct various molecular tools to enhance transformation efficiency.

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 invention discloses an application of an OsPLT1 gene in promotion of rice callus regeneration. It is found for the first time that overexpression of OsPLT1 can improve the regeneration and transformation efficiency of rice calluses, including rice varieties difficult to differentiate and regenerate. Furthermore, the OsPLT1-rZmG2-rZmW2 is used in a combined manner, and a rice callus specific promoter is used for driving gene segment transformation, so that the transformation efficiency of the rice is further improved.

The invention belongs to the technical field of genetic transformation, and particularly relates to a method for improving apple leaf disc regeneration and genetic transformation efficiency. And culturing the apple leaf disc in the apple leaf disc regeneration culture medium to obtain a leaf disc regeneration adventitious bud. The amino acid sequence of the MdREF1 small peptide is as shown in SEQ ID NO. 1. The apple homologous peptide MdREF1 is identified in an NCBI (National Center of Biotechnology Information) database on the basis of AtPROPEP1-8 and AtPep1-8 sequences of arabidopsis thaliana. The synthetic peptide of the apple MdREF1 is added into a basic regeneration culture medium, so that the leaf disc callus growth amount and the adventitious bud differentiation number can be remarkably increased. Wherein by adding 50nmol / L of MdREF1 peptide, the regeneration frequency can be improved by 1.74 times, the average regeneration bud number can be increased by 2.16 times, and the conversion efficiency can be improved by 3.14 times.

The invention discloses a recombinant genetically engineered bacterium for producing a micafungin precursor FR901379 and an application of the recombinant genetically engineered bacterium. The recombinant genetically engineered bacterium for producing the micafungin precursor FR901379 is obtained by performing overexpression on an epigenetic modification factor in a phomopsis sheathing genome and performing screening to obtain the recombinant genetically engineered bacterium for producing the micafungin precursor FR901379. The epigenetic modification factor comprises a histone methyltransferase (Dot 1), a histone methyltransferase (Set2) or a histone deacetylase (Rpd3). The yield of FR901379 produced by the engineering strain is increased by 40% compared with that of an original strain. The method disclosed by the invention has the characteristics of simplicity and convenience in operation, high transformation efficiency and good genetic stability.

The application discloses a method for improving genetic transformation rate of high-altitude meadow grass, comprising the following steps: (1) taking mature seeds of high-altitude meadow grass as explants to induce callus, and obtaining the callus; (2) adding a surfactant Silwet L-77 with a mass fraction of 3‰ of the bacterial liquid to the agrobacterium bacterial liquid carrying a hygromycin resistance gene, and obtaining a culture bacterial liquid; (3) placing the regenerated plant in a screening culture medium containing 40 mg / L of hygromycin resistance for culture, screening the survived plants, and obtaining the genetically transformed plants. The method can improve the transformation efficiency of the high-altitude meadow grass by about 4.54 times through adding the surfactant in the genetic transformation process and cooperating with the screening of the obtained differentiation culture medium.

The present application relates to the field of microbial technology, and particularly relates to a gene editing method of propionibacterium acnes VB208 and application thereof. Propionibacterium acnes ) on February 20, 2023, and is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.26576. The propionibacterium acnes screened by the present application has the characteristics of skin surface occupation, high conversion efficiency, gene modification potential and efficient expression of exogenous proteins, and can be used as an engineering strain. Furthermore, a genetic operation system suitable for the strain can be established by using the propionibacterium acnes, and the application prospect is wide.

The invention belongs to the technical field of synthetic biology and enzyme engineering, and particularly relates to a multi-enzyme in-vivo space co-localization carbon metabolic flux strengthening method, a genetically engineered bacterium and application of the genetically engineered bacterium. According to the invention, a recombinant expression vector is constructed, key enzymes RHLA and RHLB participating in synthesis of a target product are respectively subjected to fusion expression with protein scaffold modules RIAD and RIDD through (G4S) 3 flexible connecting peptide, and are introduced into host chassis bacteria, so that a stable multi-enzyme composite system is formed in cells, the transmission distance of a metabolic intermediate is shortened, and the expression efficiency is improved. Spatial co-localization and concerted catalysis of key catalytic enzymes are realized, and the conversion efficiency of a carbon source to a target product is improved. Compared with the prior art, the genetically engineered bacterium constructed by the invention has the advantages of stable structure, no need of complex regulation, reasonable metabolic flow distribution and the like in the fermentation process, the synthesis efficiency and yield of the target biosurfactant are remarkably improved, and the feasibility of industrial amplification application is enhanced.

The present application relates to the field of molecular biology, in particular to a recombinant human heparin N-deacetylase encoding gene, a recombinant vector, a recombinant strain and application thereof, the recombinant human heparin N-deacetylase constructed in the expression process shows excellent stability and activity, has the ability of specific catalysis of deacetylation of acetyl group in heparin precursor, and can effectively generate deacetylated heparin precursor. This property significantly improves the conversion efficiency and reduces the generation of by-products. The present application optimizes the codon of the recombinant human heparin N-deacetylase, and uses the promoter P AOX1 or P GAP to regulate the expression, so that the expression level of the recombinant human heparin N-deacetylase in Pichia pastoris is significantly improved, and the obtained GS115-AOX1-hNDase and GS115-PGAP-hNDase strains successfully realize the secretory expression of the target gene, providing a new strategy and method for the biosynthesis of heparin.

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 gene engineering, and particularly relates to an efficient electrotransformation method of lactococcus lactis BL19. According to the method disclosed by the invention, the electric shock conversion efficiency of the lactococcus lactis BL19 is remarkably improved, and a foundation is laid for genetic engineering transformation in the future.

The invention relates to the technical field of biology, in particular to an SNP molecular marker related to the Ningxiang pig feed conversion efficiency character and application of the SNP molecular marker. The molecular marker comprises SNP1 and SNP2; the SNP1 molecular marker is A or G corresponding to the 97685244th site from the 5'end on a chromosome 1 of which the reference genome is Sus Scrofa Build11.1; the SNP2 molecular marker is A or C corresponding to the 97782417th site from the 5'end on the chromosome 1 of which the reference genome is Sus Scrofa Build11.1. The invention also discloses a method for detecting the Sus Scrofa Build11.1 in the invention, and the Sus Scrofa Build11.1 in the invention. The molecular marker remarkably related to the feed conversion efficiency of the Ningxiang pigs is obtained, guidance is provided for breeding of the Ningxiang pigs by utilizing the molecular marker, the feed conversion efficiency and typing can be accurately and efficiently predicted, pig breeds with high feed conversion efficiency and excellent meat quality can be identified and screened, and the breeding efficiency is improved.

The invention discloses a LeRho1 gene expression vector and a method for improving heat resistance and trichoderma resistance of lentinus edodes. The LeRho1 gene expression vector is a recombinant expression vector, a target gene for coding the LeRho1 protein is inserted into the recombinant expression vector, and the amino acid sequence of the LeRho1 protein is shown as SEQ ID NO. 1. The invention finds that the LeRho1 gene has a positive regulation effect on the heat resistance of the shiitake mushroom strain, and the LeUSPA and LeUSPH genes mediate the thermal response of the shiitake mushroom strain through negative regulation. Therefore, according to the method, the LeRho1 gene overexpression vector or LeUSPA and LeUSPH gene silencing vectors are constructed and transferred into the shiitake mushroom strain for genetic transformation, the heat resistance and trichoderma resistance of the shiitake mushroom strain can be remarkably improved, and the transformation efficiency is high.

The invention discloses a method for regulating and controlling efficient conversion of methanol based on (p) ppGpp signal molecules. Specifically, by regulating synthesis and hydrolysis balance of (p) ppGpp signal molecules in microbial cells and combining promoter engineering and nutrition condition optimization, methanol metabolic pathway key gene expression and amino acid metabolic flux are enhanced, and the methanol utilization efficiency and target product synthesis capacity of microorganisms are remarkably improved. The regulation and control mechanism of the (p) ppGpp on methanol conversion under the stress of a low nitrogen source is defined for the first time, efficient conversion of methanol to organic acids such as acetic acid and butyric acid is realized, a brand new strategy is provided for preparation of high-added-value chemicals through methanol bioconversion, and the method has the advantages of being economical, environmentally friendly and high in conversion efficiency and has a wide application prospect.

The invention belongs to the technical field of bacterial plasmid editing, and particularly relates to miniature DNA nuclease as well as a preparation method and application thereof. The preparation method of the micro DNA nucleotidase comprises the following steps: carrying out expression on a p15A-IscB-2. 0 plasmid, so as to obtain the micro DNA nucleotidase. Wherein the nucleotide sequence of the p15A-IscB-2 plasmid is shown as SEQ ID NO.2, and the p15A-IscB-2 plasmid is obtained by performing synonymous mutation on a 5 '-AACNNNNNNGYGC-3' sequence in an IscB gene in p15A-IscB. The p15A-IscB-2 plasmid disclosed by the invention is non-toxic to escherichia coli, can express an IscB protein, and is high in conversion efficiency, so that the p15A-IscB-2 plasmid can be directly used for gene editing of escherichia coli.

The invention discloses a construction method of a cabbage efficient genetic transformation regeneration system. The method comprises the following steps: (1) culturing aseptic seedlings; (2) preparing an explant; (3) preparing agrobacterium dip dyeing liquid carrying a target carrier; (4) carrying out dip dyeing and co-culture on the explants; (5) callus differentiation and bud induction; and (6) growing seedlings and rooting, and carrying out PCR (Polymerase Chain Reaction) and Western blot identification on the obtained plants. By optimizing key parameters and vector selection of the cabbage genetic transformation method, compared with the prior art, the method has the advantages that antibiotics are not needed for preliminary screening in the regeneration process, hormone is not needed in the rooting process, the cost is low, the transformation period is short (greater than or equal to 45 days), the regeneration frequency is high, the transformation efficiency is high, and subsequent related protein research work can be conveniently carried out; the important value is realized on shortening the cabbage gene function research period.

This invention relates to a genetic transformation method for Ningxia wolfberry using germinating seeds as explants, belonging to the field of plant genetic transformation technology. The invention mainly includes the following steps: (1) After disinfecting Ningxia wolfberry seeds, inoculate them onto a germination medium, and obtain germinating seeds through vernalization and dark culture treatment; (2) Activate and culture Agrobacterium rhizogenes and Agrobacterium tumefaciens strains carrying target gene expression frames to obtain a bacterial solution for infection; (3) Take the germinating seeds obtained in step (1) and mix them with the bacterial solution obtained in step (2) for infection, then transfer the infected germinating seeds to a co-culture medium or substrate for co-culture; (4) Cultivate the material after co-culture in step (3) to induce root growth, and detect the expression level of the target gene or reporter gene on the roots or plants to obtain transgenic positive plants. This invention has the advantages of simple operation, significantly shortened transformation cycle, and high transformation efficiency.

The present application relates to a method for transforming corn, comprising: the primary callus obtained by the method for inducing corn callus, using the method for obtaining corn suspension cell line to obtain corn suspension cell line; agrobacterium infection corn suspension cell group in the corn suspension cell line. The method for transforming corn of the present application first induces the primary callus from corn proembryo, quickly obtains the suspension cell line, shortens the experimental period to 40-50 days, is simple and convenient to operate, has good repeatability, simultaneously makes the corn genetic transformation not affected by the season, meets the needs of annual stable transformation; then uses agrobacterium infection to transform the corn suspension cell line, and regenerates seedlings again; the transformation efficiency can reach about 30%, the transgenic positive rate can reach about 90%, and the single copy rate is about 50%; since the pressure on corn explant (proembryo) supply is significantly reduced, the human and material resources consumption required for culturing the explant is greatly reduced, and large-scale genetic transformation is realized.

The invention discloses a non-tissue-culture-dependent pearl millet injection transformation method, and belongs to the technical field of plant genetic engineering. The method comprises the following steps: selecting germinated seeds of pearl foxtail millet with the primary bud length of 0.8-1.0 cm as receptors; preparing agrobacterium liquid, and adjusting the concentration to OD600 = 0.5; injecting the bacterial liquid into the embryo along the coleoptile direction of the seed until the bacterial liquid seeps from the end part of the coleoptile; co-culturing the transformed receptor for 24 hours under a dark condition after injection, and transplanting the transformed receptor into a seedling culture pot for cultivation; and finally, carrying out PCR (Polymerase Chain Reaction) screening. By optimizing key parameters such as the receptor development stage, the agrobacterium strain type and the bacterial liquid concentration, efficient genetic transformation of the pearl millet without tissue culture is achieved, and the T0 transformation positive rate reaches 63%. According to the invention, the transformation process is simplified, the transformation period is shortened, the cost is reduced, the transformation efficiency and stability are improved, and an effective technical support is provided for gene function research and molecular breeding of the pearl millet.

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.