50 results about "Ti plasmid" patented technology

A method for expressing insecticidal protein structural genes in plant genomes is provided. In the preferred embodiments this invention comprises placing a structural gene for the Bacillus thuringiensis crystal protein under control of a plant or a T-DNA promoter and ahead of a polyadenylation site followed by insertion of said promoter/structural gene combination into a plant genome by utilizing an Agrobacterium tumefaciens Ti plasmid-based transformation system. The modified Ti plasmid is then used to transform recipient plant cells. Also provided are the plants and tissues produced by this method and bacterial strains, plasmids, and vectors useful for execution of this invention.

The invention discloses a site-specific insertional inactivation method mediated by agrobacterium tumefaciens and CRISPR/Cas9. Endogenous snRNA promoter of Ustilago scitaminea (u6 promoter of Ustilago scitaminea) is used to drive sgRNA expression cassette. CRISPR/Cas9 system integrates with Ti plasmid of agrobacterium tumefaciens to construct the Ustilago scitaminea site-specific insertional inactivation system mediated by agrobacterium tumefaciens with hygromycin as a selection marker. Specific sequence of the objective gene is cloned into sgRNA expression cassette for transformation of Ustilago scitaminea basidiospores, thus the mobile DNA fragment is exactly inserted into the objective gene sequence of Ustilago scitaminea, achieving the goal of damage to gene function. The invention provides an important tool for study of Ustilago scitaminea functional genomics. The system has the advantages of high efficiency and accuracy, and is convenient to use to conduct gene function researches in Ustilago scitaminea.

This invention relates generally to technologies for the transfer of nucleic acids molecules to eukaryotic cells. In particular non-pathogenic species of bacteria that interact with plant cells are used to transfer nucleic acid sequences. The bacteria for transforming plants usually contain binary vectors, such as a plasmid with a vir region of a Ti plasmid and a plasmid with a T region containing a DNA sequence of interest.

The invention relates to the technical field of plant genetic engineering, and discloses an agrobacterium-mediated sugarcane genetic transformation method with vacuum infiltration assistance. The agrobacterium-mediated sugarcane genetic transformation method includes: (1) induction and multiplication of embryonic callus of sugarcane core leaves, (2) activation and cultivation on agrobacterium tumefaciens with target genes, (3) agrobacterium infestation with vacuum infiltration assistance on embryonic callus of sugarcane core leaves, (4) resistant material screening and germination, and (5) resistant material verification. With the agrobacterium-mediated sugarcane genetic transformation method, the agrobacterium can pass through the gaps among callus cells and enter deep cells on vacuum, and tiny wounds can be generated on the surfaces of the cells, so that phenolic substances are secreted by plants to activate shear and transfer of T-DNA (transferred deoxyribonucleic acid) in Ti plasmids. The agrobacterium-mediated sugarcane genetic transformation system is perfected. Genetic transformation rate of sugarcane is remarkably improved as compared with that of the prior art.

The invention provides a Ti plasmid aspergillus niger gene replacement expression vector and application thereof, and belongs to the technical field of molecular biology. The T-DNA (Triple helix Deoxyribose Nucleic Acid) region elements of the Ti plasmid aspergillus niger gene replacement expression vector are arranged in the following sequence: an aspergillus niger target gene promoter, a multiple cloning site, an aspergillus niger target gene terminator, an aspergillus nidulans 3-phosphoglyceraldehyde dehydrogenase gene promoter PgpdA, an aspergillus niger selection marker gene and an aspergillus niger target gene terminator. According to the invention, a target gene is integrated at the site of the aspergillus niger target gene through homologous recombination, and the target gene is regulated and controlled by a target gene promotor of high expression; therefore, the position effect of transgenosis is eliminated and the expression level is improved.

This invention relates to several plasmids which are useful for genetically transforming plant cells. A first plasmid, such as pMON120, contains a T-DNA border, one or more marker genes, a unique cleavage site, and a region of Ti plasmid homology. A gene which is expressed in plant cells may be inserted into this plasmid to obtain a derivative plasmid, such as pMON128 which expresses neomycin phosphotransferase in plant cells. The derivative plasmid is inserted into a suitable microorganism, such as A. tumefaciens which contains a Ti plasmid. The inserted plasmids recombine with Ti plasmids to form co-integrate plasmids. Only a single crossover event is required to create the desired co-integrate plasmid. A. tumefaciens cells with co-integrate plasmids are selected and co-cultured with plant cells. The co-integrate Ti plasmids enter the plant cells and insert a segment of T-DNA which does not contain tumorigenic genes into the plant genome. The transformed plant cell(s) may be regenerated into a morphologically normal plant which will pass the inserted gene(s) to its descendants.

The invention is about the Cephalosporium acremonium transformation leaded by the plasmid Ti of the Agrobacterium tumefaciens. Compared to the 1-4 transformants it can get above 100 transformants from the 107 receptor cells. So the transforming efficient has improved greatly.

The invention relates to a plant stress resistance enhancement related protein named as ZmHDZIV14. The protein is derived from a maize (Zea mays L.) inbred line B73, the amino acid sequence of the protein is represented as a sequence table SEQ ID NO:2, and an encoding gene sequence of the protein is a nucleotide sequence represented as SEQ ID NO:1. The invention further provides an application method of an encoding gene of the plant stress resistance enhancement related protein. According to the application method of the encoding gene of the plant stress resistance enhancement related protein, an expression vector carrying the ZmHDZIV14 gene is used for converting plant cells or tissue by the aid of conventional biological methods including application of Ti plasmids, Ri plasmids and plant virus vectors, direct DNA transformation, microinjection, electric conduction, agrobacterium tumefaciens mediated transformation and the like, a plant is cultured from the converted plant through tissue culture, and the plant with improved drought resistance and stress resistance is obtained. The expression vector carrying the ZmHDZIV14 gene is used for converting the plant cells or tissue by the aid of the conventional biological methods including application of Ti plasmids, Ri plasmids and plant virus vectors, direct DNA transformation, microinjection, electric conduction, agrobacterium tumefaciens mediated transformation and the like, the plant is cultured from the converted plant through tissue culture, and the plant with the improved stress resistance is obtained.

The invention discloses a standard molecular for specifically detecting a transgenic rice strain Kefeng No.6 and an application thereof. The standard molecule disclosed by the invention is an annular carrier which comprises a cowpea trypsin inhibitor coding gene segment shown by the 10th-101st sites of a sequence 1, a bacillus thuringiensis insecticidal crystal protein coding gene segment shown by the 256th-336th sites of the sequence 1, a rice endogenous gene GOS segment shown by the 191th-258th sites of the sequence 1 and a border sequence segment of Ti plasmid T-DNA shown by the 108th-184th sites of the sequence 1. Experiments prove that the standard molecule disclosed by the invention has high sensitivity, wide linear range, good uniformity and strong stability, and is of important significance to the qualitative and quantitative detection of the transgenic rice strain Kefeng No.6; and the lowest detection line of the standard molecule can reach 2 copies.

The invention discloses an ultrasonic-assisted agrobacterium-mediated plant germination seed gene transformation method. Plant germination seeds are directly used as agrobacterium infection receptors, and tissue culture and plant regeneration processes are not needed. The method comprises the following steps of: performing scratch and ultrasonic treatment on growing point parts of receptor plant germination seeds in the seed germination process by using the plant germination seeds as the receptors and using agrobacterium Ti plasmids carrying exogenous gene fragments as gene vectors, and then culturing the seeds and the agrobacterium together, transferring exogenous genes to a receptor genome, and further determining the transformed plants by direct screening of the seeds or seedlings and polymerase chain reaction (PCR) amplification and Southern hybridization of the plant leaf DNA.

A transformation method, including the following steps: preparing an Agrobacterium Ti-plasmid, Escherichia coli plasmid, or other DNA vectors carrying exogenous genetic fragments as a genetic donor; collecting a male gamete (pollen) of the plant as a recipient; preparing a 5-50% sucrose solution after aeration and low temperature pretreatment; mixing the pollen with the exogenous genetic fragments in the 5-50% sucrose solution; transferring the exogenous genetic fragments into the pollen in the presence of ultrasonication; pollinating a pistil stigma of the plant with the treated pollen; harvesting seeds at maturity; sowing the seeds in a subsequent growing season; screening a germinating seed and a seedling; and performing PCR amplification and Southern hybridization using DNA samples of plants to further determine transformants.

The invention relates to a dual-elemental Ti plasmid carrier for the transformation of T-DNA (Transferred-Deoxyribose Nucleic Acid) of plants and partial fungi and the construction of a T-DNA insertion mutant library and a construction method of the dual-elemental Ti plasmid carrier. The method comprises the following steps: firstly, performing module trapping on a promoter integrated with sGFP (Green Fluorescent Protein) reporter genes at two ends of a T-DNA area of a plasmid; optimizing and adjusting a nucleotide sequence before opening reading frames for the sGFP at two ends of the nucleotide sequence; secondly, integrating a functional module rescued by the plasmid into a target plasmid through an enzyme cutting site. Finally, the plasmid is integrated with a hygromycin resistance gene which is used for screening a transformant; the hygromycin resistance gene and the promoter thereof can be replaced freely through a Sac I enzyme cutting site so as to adapt to different screening purposes and species. The convenience in the efficiency, the gene cloning and the related gene function verification of transgene is seriously affected by the selection and the module configuration and optimization of the dual-elemental Ti plasmid carrier. The application range of the plasmid is enlarged and the screening and cloning efficiency of target functional genes is improved by organically integrating various plasmid functional modules into one plasmid. Thus, a good molecular tool for finding beneficial genes and verifying the functions of the beneficial genes is provided.

This invention relates to the Agrobacterium bacterium to be used in plant transformation method comprising three types of plasmids. The Agrobacterium bacterium of this invention comprises plasmids of (1) to (3) shown below:

• • (1) a plasmid comprising the following components:
    • (i) the virB gene, the virC gene, the virD1 gene, the virD2 gene, the virD3 gene, the virG gene and the virJ gene of pTiBo542, and
    • (ii) an origin of replication;
  • (2) a disarmed Ti plasmid or a disarmed Ri plasmid of Agrobacterium bacterium; and
  • (3) a plasmid having a T-DNA region consisting of a desired DNA;

wherein each of the plasmids of (1) to (3) has a replication mechanism that enables a mutual coexistence with each other.

The invention discloses a method for improving the transgenosis efficiency of cotton. The method comprises the following steps: after mixing restriction enzyme digestion DNA with a plasmid DNA protective agent, introducing a target gene into cotton through the pollen tube pathway method by using lipidosome packaging to carry toxic protein genes on Ti plasmids. The method can effectively improve the transgenosis efficiency.

An hrf 2 gene of rice Xanthomonas is disclosed. It can be used as the target gene to be constructed on the Ti-plasmid dual-element vector pBI121 expressed in plant, so obtaining its recombinant vector pBI121:hrt2. Said recombinant vector is transferred to EHA105 to transform plant cells. The transformed regenerative plant (To generation) is screened in the culture medium and detected. The resultant transgenic plant has the improved broad-spectrum power to resist diseases and pests, and the improved other productive characteristics.

The invention relates to a gene for enhancing plant drought resistance, and provides a related protein for enhancing plant stress resistance. The gene has a is named as ZmHDZIV13, and derived from maize (Zea maysL.) inbred line B73, and has an amino acid sequence shown in a sequence table SEQIDNO:2. A coding gene sequence of the protein is shown in SEQIDNO:1. The invention also relates to an application method of the encoding protein of the related protein for enhancing plant stress resistance. The method is as below: transforming an expression vector carrying the ZmHDZIV13 gene into plant cells or tissues through conventional biological methods including Ti plasmid, Ri plasmid, a plant virus vector, direct DNA conversion, micro injection, conductance and Agrobacterium mediation, and culturing the transformed tissue into plants to obtain plants with improved drought resistance. The ZmHDZIV13 gene can be constructed into the existing plant expression vector by the method used in the prior art, and the encoding gene of the related protein for enhancing plant stress resistance can be converted into other plants by conventional methods to enhance the drought resistance of plants.

The present invention discloses a novel Ti plasmid pElrLCG for saving embryos from lethal mutation and a preparation method of the Ti plasmid pElrLCG. Connect 5.211kb long bearer fragment recovered from enzyme cutting of original bearer pJawoh18 with synthesized XhoI-loxP-NheI-NcoI-Nos-BsiwI-KpnI-StuI-BgIII-MFeI-AatII-1xMyc-loxP-SpeI fragment, and then insert Gr-Cre, Gateway Cassettee fragment between two loxP loca, to construct a new plasmid vector pElrLCG. The plasmid vector can be used to obtain homozygote of embryonic lethal mutant or study the development of homozygote of embryonic lethal mutant in late stage, and therefore is of great significance for study on lethal mutations of embryos, hybridization breeding, and special biologic behaviors of lethal mutants of embryos.

Described herein are viral amplicon-based protein expression systems and methods useful for producing heterologous proteins, such as enzymes, by agroinfiltration. The methods involve producing an Agrobacterium with a Ti plasmid encoding a heterologous protein, infecting plant cells with the Agrobacterium, allowing expression of the heterologous protein, and recovering the heterologous protein from the plant cells. In one embodiment, the protein produced is an endoglucanase.

The invention discloses a construction method and application of an agrobacterium Ti plasmid vector PCHF1302 and belongs to the technical field of plant genes. Double enzyme digestion is simultaneously conducted on enzyme digestion sites ECORI and HindIII on an agrobacterium Ti plasmid Pcambia 1302, wherein the PCHF3300 comprises a 35S promoter, a NOS terminator and a complete gene expression frame of MCS, a homologous arm is added through PCR, an expression frame started by the 35S promoter is successfully connected to the plasmid Pcambia 1302, and the PCHF1302 is constructed; the target genecloned by PCR and other methods can be conveniently connected to a plurality of cloning enzyme digestion sites (MCS), so that the target gene can be stably expressed in plants; a TDNA region containing hygromycin resistance gene and mGFP5 green fluorescent protein reporter gene can significantly improve the screening effect of transgenic plants.