155 results about "Gene gun" patented technology

The invention discloses an expression system and constructing PHB transgene algae method of rhine chlamydomonas exogenesis gene, which comprises the following parts: promotor, exogenesis-goal gene, screening badge and rhine chlamydomonas acceptor algae strain, wherein the method comprises the following: A, selecting and cultivating acceptor algae strain; B, cloning key enzyme gene by PHB biosynthesizing; C, expressing carrier of PHB biosynthesizing key enzyme rhine chlamydomonas; D, using PHB biosynthesizing key enzyme to lead in rhine chlamydomonas from fungus Bacillus alcaligenes with bead-milling method, gene-gun method or electrization; obtaining bivalence or transgene Chlamydomonas of producing PHB by screen selecting and molecule detecting. The poly-hydroxybutyric acid is compounded by acting in conjunction of PHB compounding key enzyme of phbA, phbB and phbC gene coding, which uses acetylcoenzyme A as substrate by photosynthesis. The method simplifies the operating, which reduces cost.

The invention relates to chitosan (CS) vehicles with chitosan nanoparticles, as well as methods for making such chitosan vehicles and for using them to carry a DNA or proteins by forming CS-DNA or CS-protein complexes. The present invention also relates to CS-DNA or CS-protein complexes being useful for transdermal delivery of DNA or protein with a low-pressure gene gun. In another aspect, the present invention also relates to CS-DNA or CS-protein complexes being useful for transcutaneous delivery of a DNA or protein with a skin patch. Further aspects of the present invention relate to methods for making CS-DNA or CS-protein complexes and for using them for diagnostic, therapeutic and biological industrial applications.

A targeting method is described that allows precise cassette replacement at a previously characterized genetic locus. A target DNA construct containing a pair of incompatible FRT sites flanking a target cassette was introduced into soybean by regular biolistic transformation. Transgenic events containing a single complete copy of the target site were then selected and retransformed with a donor DNA construct containing the identical pair of incompatible FRT sites flanking a donor cassette. Precise DNA cassette exchange happened between the target cassette and the donor cassette via recombinase mediated cassette exchange (RMCE) so that the donor cassette was introduced at the exact genomic site previously occupied by the target cassette. Through repeated RMCE using additional incompatible FRT sites, multiple groups of transgenes can be stacked at the same genomic locus.

The invention belongs to the field of genetic engineering, relating to a method for increasing rice yield and yield traits by using lrk1 gene transformation. By using a gene gun microprojectile bombardment method, after recovery cultivation, a rice plant expressing the lrk1 gene is obtained from rice callus genetically transformed by the lrk1 gene. Results show that, compared with a control sample, the successfully transformed rice plant has faster seed germination potential, better seedling stage tillering potential, as well as generally superior seedling stage growth potential and plant ripeness spike index. The invention provides the new method for increasing the rice yield with low cost and high benefit.

The invention discloses a transgenic method using wheat anthers as receptors. The method provided by the invention comprises the following steps: 1) using plant anthers as receptors to carry out gene transformation with a gene gun method to obtain transformed anthers; 2) culturing the transformed anthers obtained in step 1) to obtain embryoid bodies, The embryoid bodies are then cultured to obtain haploid regenerated shoots. The present invention establishes a wheat transgenic technology system with anthers as receptors, in vitro moisturizing of anthers, adhesion and fixation of anthers during gene gun bombardment, gold powder particle size for preparing bullets, bullet coating effect, bombardment distance and haploid Chromosome doubling and other processes have been systematically studied, and transgenic plants with homozygous genotypes can be obtained in contemporary times.

The invention discloses an artificial synthesized Bt insecticidal gene for transgenic anti-insect plants, further, the invention relates to the optimization and transformation systematically to a coding frame and a codon of a CrylAc anti-insect gene, according to an activity analysis to Bt protein in different regions and in accordance with the coding characteristics of the monocotyledons, obtaining anti-insect gene CrylAc-M which can be expressed in a monocotyledons efficiently, with the optimized codon and coding frame. The CrylAc-M gene after being transformed is connected on an expression carrier with CaMV35S-Adhl as promoter, and then the Bt gene CrylAc-Adhl is transferred into a maize inbred line with highly efficient conversion rate by a gene gun co-transformation method. A transgenic progeny is positive through Bt protein testing and a field anti-insect test of the field is tested to be highly resistant.

Disclosed is a transformation method whereby an ability to produce a useful substance of a stramenopile can be improved. The method for transforming a stramenopile comprises transferring a foreign gene into the stramenopile which is a microorganism belonging to the class Labyrinthula, more specifically, to a genus Labyrinthula, Altornia, Aplanochytrium, Schizochytrium, Aurantiochytrium, Thraustochytrium, Ulkenia, etc. Said foreign gene, which is a gene relating to tolerance against an antibiotic, a colorimetric protein and / or a fatty acid desaturase (Δ5 desaturase gene, Δ12 desaturase gene and / or ω3 desaturase gene), is transferred by using the electroporation or gene-gun technique.

The invention refers to a method for reconstructing the cloned and constructed human body tumor death factor TRAIL gene expression carrier into spirulina gene group to be expressed stably with gene gun. The special components of the reconstructed TRAIL expression carrier include resistant mark gene expression box CAT, chlorophyll body photosynthetic gene promotor and ender, spirulina homologous segment, TRAIL gene segment without coding film combining part, the expression system also includes characters of primary nucleus and eukarya. The invention sets suitable striking parameter, uses gene gun to guide the reconstructed expression carrier into spirulina silk body, and through several times of sifting of chloramphenicol with incremental density and molecular biology testing, the invention gets spirulina converter of TRAIL gene. The invention applies to spirulina reconstruction and human body gene expression, it also applies to reconstruct and express other primary nucleolus and eukarya.

The invention discloses a method for constructing a Dunaliella salina chloroplast transformation vector, in particular a method for constructing a dual-exchange dual-selection marker Dunaliella salina chloroplast transformation vector by using a light dependence free protochlorophyllide reductase chlN, chlB or chlL gene as a homological segment, chloramphnicol acetyltransferase (CAT) gene and a resistance gene bar of phosphinothricin (PPT) as a weedicide as screening markers and an atpA promoter and a rbcL terminator as expression elements. The Dunaliella salina is transformed through a gene gun method or electrization method and then subjected to two-step screening to realize homogenization, fixed point integration of an exogenous gene in a Dunaliella salina chloroplast genome is ensured, and finally the chloroplast transformation Dunaliella salina strain for stably expressing the exogenous gene is obtained. In the invention, the chlN, chlB or chlL gene is selected as the homological segment and can be used as an auxiliary screening marker of a transformant; and the chlorampenicol resistant CAT gene and the resistance gene bar of the PPT are selected to be used as screening marker genes together, thus the stable transformation strain can be obtained within shorter time.

The invention relates to a triterpene synthase of tripterygium wilfordii TwOSC3 protein and an encoding gene thereof. A recombinant expression vector with a Twosc3 gene is constructed by cloning cDNAof the Twosc3 gene onto a eukaryotic expression vector pYES2, and then the recombinant expression vector is shifted to yeast expression host bacteria, so as to acquire friedelin. A mutation research result shows that amino acid 482 encoded by the Twosc3 gene is a key site, and the yield of friedelin or amyrin can be increased or reduced; a gene gun mediated interference experiment shows that the interference of the Twosc3 gene has an obvious inhibiting effect on the synthesis of celastrol in tripterygium wilfordii; the TwOSC3 protein and the encoding gene thereof provided by the invention canbe used for biosynthesizing plant triterpenoids and cultivating high-quality tripterygium wilfordii.

The invention discloses a composition of plant direct gene transformation, transforming method and appliance, which is characterized by the following: making the component include goal gene and free nucleic acid component of adjusted and controlled element, transforming buffer liquid and transforming assistance component; co-culturing; soaking; surface-daubing; injecting; dripping; spraying; transforming with dip flower method or through genetic gun bombardment, supersonic wave and electric shock method. This component can be used to receptor plant cell direct transformation of fertilized ovum, suspended cell, protoplast, single-layer cell and diachyma cell and can be used to seed, receptor plant pollen, pistil, flower pillar, pollen pipe and ovary, which possesses impotent meaning for plant genetic seeding, variety improvement and gene functional analysis.

An immunotherapeutic strategy is disclosed that combines antigen-encoding DNA vaccine compositions combined with siRNA directed to pro-apoptotic genes, primarily Bak and Bax, the products of which are known to lead to apoptotic death. Gene gun delivery (particle bombardment) of siRNA specific for Bak and / or Bax to antigen-expressing DCs prolongs the lives of such DCs and lead to enhanced generation of antigen-specific CD8+ T cell-mediated immune responses in vivo. Similarly, antigen-loaded DC's transfected with siRNA targeting Bak and / or Bax serve as improved immunogens and tumor immunotherapeutic agents.

The invention relates to a tetrahymena cell line containing luciferase gene, a construction method and applications thereof. According to the present invention, a PCR technology is adopted to optimize a N-terminal sequence and a C-terminal sequence of firefly luciferase gene into tetrahymena preference codons, the optimized firefly luciferase gene LUC is linked to a carrier pBX to obtain firefly luciferase gene-containing recombinant plasmid pBX-LUC, a gene gun particle bombardment method is adopted to transform the firefly luciferase gene-containing recombinant plasmid pBX-LUC into tetrahymena thermophila B2086 cells, intracellular homologous recombination and paromomycin resistance screening are performed, and MTT1 gene in the cells are replaced by the LUC gene to obtain the engineered cell line B2086-LUC, wherein the engineered cell line B2086-LUC expresses luciferase under specific heavy metal induction, coloration is performed after a fluorescent substrate D-Luciferin is added, and the enlarged fluorescence intensity is detected on so as to reflect conditions of heavy metal pollutants in a reaction environment through quantitative data, such that the detection method is rapid and sensitive, and easy to operate. The tetrahymena cell line containing luciferase gene has a wide application prospect in biological monitoring of water body environment heavy metal pollution.

The invention discloses a wheat mature embryo callus gene gun genetic transformation method. By pretreatment of wheat mature embryo culture, addition and organic matters, and optimization of cultivation and transformation technology processes, technical effects which are close to wheat young embryo callus induction, regeneration and transformation efficiencies are obtained. Compared with reported mature embryo callus gene gun genetic transformation technologies, the method has higher transformation efficiency, the induction, differentiation and transformation capabilities of the wheat mature embryo callus are close to that of young embryo callus, and technical problems of low induction rate, regeneration frequency and transformation efficiency of callus in previous wheat mature embryo callus gene gun genetic transformation are solved. Wheat genetic transformation is free from seasons and periods of recipient material selection. High-efficiency transformation study on mature embryos of seeds harvested in the current year or stored for one year in any time is possible. Wheat genetic transformation and molecule breeding efficiencies are raised.

The present invention discloses a safe and environmentally friendly cichorium intybus chloroplast transformation system establishment method, which comprises: (1) establishing a high frequency regeneration system of a cichorium intybus leaf explant; (2) constructing a safe and environmentally friendly cichorium intybus chloroplast site-specific integration expression vector; and (3) adopting a gene gun bombardment method to transform the cichorium intybus explant, and carrying out three rounds of resistance screening to obtain the chloroplast transgene strain. According to the present invention, the novel safe selection marker gene PMI is adopted to replace the traditional antibiotic marker gene aadA to construct the chloroplast site-specific integration expression vector and establish the cichorium intybus chloroplast transformation system, wherein the system has advantages of high screening efficiency, high exogenous gene expression level, strong transgenic plant bio-safety and the like; and receptor types of chloroplast transformation are broadened, and technical platforms are built for genetic improvement on cichorium intybus, or application of cichorium intybus chloroplast as a bioreactor to produce animal oral vaccines, medical proteins and the like.

The invention discloses a wheat anther callus gene gun genetic transformation method, which comprises the steps of anther callus induction, hyperosmotic cultivation, gene gun bombardment transformation, renewal cultivation, differentiation and screening cultivation, strong seedling and rooting cultivation, chromosome doubling and molecular detection and screening. Compared with the conventional somatic cell callus gene gun transformation, the anther callus has high regeneration frequency after the bombardment of the gene gun, and transgenosis homozygous stable plants can be obtained directly after the chromosome of the regenerated plant is doubled, so the technical shortcomings that the callus has low regeneration frequency and transformant gene heterozygosis and homozygote is selected via selfing two generations in the conventional somatic cell callus gene gun transformation are overcome, the wheat genetic transformation and molecular breeding efficiency is increased, and the transformation efficiency is above 8 percent.

The invention relates to a method for preparing transgenic large scale sea weed, wherein the reportedly exogenesis gene or functional exogenesis gene and antibiotic or weedicide genes are recombined to the promotor downstream originated from advanced plant or algae for constructing carrier for transportation, the large-scale sea weed spores are used as transportation receptor, and gene guns are used to direct the recombined plasmid DNA into the spores of large-scale sea weed, new plants can be grown through the approach of natural growth. The invention can realize the stabilized expression of exogenesis genes.

The invention relates to a triterpene synthase of tripterygium wilfordii TwOSC1 protein and an encoding gene thereof. A recombinant expression vector with a Twosc1 gene is constructed by cloning cDNAof the Twosc1 gene onto a eukaryotic expression vector pYES2, and then the recombinant expression vector is shifted to yeast expression host bacteria, so as to acquire some triterpenoids, such as friedelin and amyrin. A mutation research result shows that amino acids 486 and 502 encoded by the Twosc1 gene are key sites, and the yield of friedelin or amyrin can be increased or reduced; a gene gun mediated interference experiment shows that the interference of the Twosc1 gene has an obvious inhibiting effect on the synthesis of celastrol in the tripterygium wilfordii; through further experiments, a gene gun mediated overexpression experiment shows that the overexpression of the Twosc1 gene can increase the yield of the celastrol in tripterygium wilfordii suspension cells; the TwOSC1 proteinand the encoding gene thereof provided by the invention can be used for biosynthesizing plant triterpenoids and cultivating high-quality tripterygium wilfordii.

The invention belongs to the technical field of plant transgenosis, and particularly relates to a Brassica napus transgenic method. A chloroplast transformation tissue culture system suitable to take the rape cotyledons as an explant is constructed, and a chloroplast transformation transgenic plant is obtained through a gene gun transformation method. The invention also discloses a method for constructing a rape chloroplast specificity expression vector, transforming the rape chloroplast and obtaining the transgenic plant by taking rape chloroplast fragments as homologous recombination fragments. The invention provides a novel pathway for transgenosis of Brassica napus.

The present invention discloses a method for obtaining transgene wheat by using a gene gun, and a special culture medium. According to the present invention, wheat immature embryo is adopted as explant, a hypertonic culture medium provided by the present invention is directly used to carry out a hypertonic treatment without pre-culture, gene gun bombardment is performed, and the special culture medium provided by the present invention is adopted to carry out a hypertonic treatment, callus induction, differentiation and rooting culture after bombardment, wherein a screening agent is used during the differentiation and rooting stages and is not used during the callus induction state so as to significantly improve the wheat gene transformation efficiency, wherein the wheat gene transformation efficiency can be up to 36%; the method has advantages of short transformation period (the time from immature embryo taking to obtaining of the regenerated plant having root, stem and leaf is only 8-10 weeks), high transformation stability and high repeatability; and the effective approach is provided for wheat genetic improvement industrialization, and the broad application prospects are provided.

The invention relates to the technical field of bioengineering, in particular to an FsCYP51 gene and an application thereof. According to the FsCYP51 gene and the application thereof. An FsCYP51 gene full length and a CDS full length are obtained by using Fusarium sacchari as a test material and designing a specific primer and cloning the specific primer according to genome sequencing data. Meanwhile, a multivalent HIGS plant expression vector is constructed according to the cloned FsCYP51 gene full length and CDS full length, and an interference fragment is successfully transformed into a sugarcane receptor material by utilizing a gene gun mediated genetic transformation method, so that a foundation is laid for researching the FsCYP51 gene function and creating the tip rot resistant sugarcane germplasm.

The invention discloses an onion epidermal cell transformation method mediated by agrobacterium. Onion monolayer endocuticula is dipped and floated in an infiltration medium containing with agrobacterium and is fully contacted with agrobacterium for 5-30min for transformation and then is picked up and laid in an MS solid culture medium for culture for 16-24 hours under a temperature of 25 DEG C, 16-hour lighting and 8-hour darkness. The invention needs no expensive instrument such as a gene gun, etc, no expensive experimental carrier materials such as gold powder, etc. but a super clean working platform and relative agrobacterium strains can realize high effective transformation of onion epidermal cell in the simplest experimental environment. The whole course only requires four days with low cost and the transformation rate thereof can reach 80 percent.

The invention discloses an optimization method of a wheat pollen cell electric-shock-method transformation system and application of the optimization method. The optimization method takes a wheat ripe pollen cell as a transformation acceptor; electric shock conditions of electric shock liquid and germination liquid are screened and optimized so that the vitality and in-vitro germination rate of the wheat ripe pollen cell are improved; by using an electric shock transformation method, transcription factor genes related to dry, high-salt and low-temperature induced expression are transformed to obtain transgenic plants. Compared with current main transgenic methods including an agrobacterium transformation method and a gene gun transformation method and the like, a pollen electric shock transformation method is simple to operate and has relatively low cost, a tissue culture process is omitted and the problems that the reproducibility is not strong and a chimera is easy to generate are solved; the acceptor has no gene type limitation and the transformed plants have the advantages of stable later generations, rapidness in homozygosis and the like; a resistance screening process in the tissue culture process does not need to be carried out and the safe transgenic plants without screening markers can be obtained.

The invention provides a method for inducing somatic embryos of corn and belongs to the technical field of biological engineering. The method comprises the following steps of: inducing corn explants in a callus inducing culture medium; transferring the explants undergoing inducing culture to a subculture medium; propagating the somatic embryos in the subculture medium; and putting the propagated somatic embryos into differentiation culture medium for differentiation and seedling. By the method, a plurality of somatic embryos of corn can be induced with high efficiency, and the induced somaticembryos of corn have the characteristics of realizing the differentiation of both poles at the same time by long-time subculture infinite propagation and without the control of exogenous hormones, along with high differentiation rate and seedling rate; and because influence of exogenous hormones is avoided in the differentiation process, the mutation rate of somatic cells is relatively lowered atthe seedling stage of corn. The invention can provide an excellent and practical acceptor system for various trans-genetic methods (such as an agrobacterium-mediated method and a gene gun method).

A method for inducing the Th1-type immune response for cells after the gene gun is used to inoculate DNA vaccine features that when the plasmid DNA vaccine is inoculated by gene gun, the synthetic oligonucleotide CpG DNA (CpG ODN) as adjuvant is proportionally introduced. A process for preparing the plasmid DNA-CpG oligonucleotide coated gold particle is also disclosed. It can be used for treating tumor and durable virus infection.

The present invention relates to a gene gun and the application of the gene gun for gene transformation. A low pressure gas is used in the gene gun to directly accelerate the biological material containing solution, so that the biological materials penetrate through the cell membrane / wall or the skin of an animal, without using metal particle carriers, for gene transformation.

The invention discloses a method for genetic transformation of purple alfalfa chloroplast. The method is characterized in that purple alfalfa leaves can be bombarded through a particle gun, the bombarded leaves are screened on a selective medium containing spectinomycin, the transgenic plant of the purple alfalfa chloroplast is obtained after three times screening. The invention is capable of providing a carrier and a method for chloroplast transformation of purple alfalfa and realizing the expression of green fluorescent protein in the purple alfalfa chloroplast.

The invention discloses a method for constructing a particle gun mediated liriodendron hybrids transformation system. The method comprises the following steps: 1) taking embryonic callus of liriodendron hybrids, and performing high osmotic treatment; 2) extracting plasmids, preparing gold dust, and preparing micro-particles; 3) performing particle bombardment; 4) transferring the callus to restoreto a normal level to perform dark recovery culture; 5) performing embryoid induction culture, performing light culture after embryoid grows out, and inoculating into a rooting medium to perform screening culture after seedlings grow out; and 6) performing molecular detection, thereby obtaining the liriodendron hybrids transformation system. According to the method for constructing the particle gun mediated liriodendron hybrids transformation system, disclosed by the invention, the feasibility of the particle gun mediated liriodendron hybrids system is explored, and a reliable experimental foundation is provided for transforming liriodendron hybrids by mediating target genes with a particle gun in future, so that a novel technology is created for functional gene transformation of the liriodendron hybrids, and a technical support is provided for researching the effects of various functional genes in the liriodendron hybrids.

A plant transgenic method based on silica nanoparticles as a gene carrier includes gene gun and ultrasonic transformation methods. Silicon nanoparticles of different sizes are surface-modified with polylysine (PLL) to construct nano-gene carriers. The modified nanoparticles can effectively combine with genes through electrostatic interaction to form silica nanoparticles-gene complexes , respectively transformed plant receptor materials by gene gun method and ultrasonic method, and successfully realized the efficient and stable expression of exogenous genes in plant cells. The method of the invention has the advantages of simple operation, low price, high conversion efficiency, and the conversion material is not limited by plant species type and tissue organ type. The method of the invention lays a good foundation for the wide application of the silicon dioxide nanometer gene carrier in plant transgenesis.

The invention discloses a litchi leaf specific expression gene LcGRX promoter and application thereof. The invention provides (1) a method for obtaining a primer sequence of the litchi leaf specific expression gene LcGRX promoter, (2) a construction method of a recombinant expression vector which takes pCAMBIA1304 as a basic expression vector and contains the LcGRX promoter and an LcTLP gene segment, and (3) a method for verifying the function of the promoter by using biolistic transformation and GUS transient gene expression. An experiment shows that the litchi leaf specific expression promoter is cloned from litchi and is verified to be a promoter for tissue specific expression, so that a basis is supplied to further transgenic breeding of litchi.