137 results about "Cloning genes" patented technology

A process for producing lyophilized competent cells wherein competent cells are that can be stored or shipped as freeze-dried cells at temperatures between 0° C. and 8° C. and remain suitable for cloning genes or DNA fragments. The process includes culturing cells, rending the cells competent, and lyophilizing the cells. Once lyophilized, the cells can be stored or shipped as freeze-dried cells. The lyophilized cells are prepared for transformation protocols by being re-hydrated in a solution of dimethyl sulfoxide. Once re-hydrated, the transformation efficiency of the competent cells is at least 5x105 transformations per microgram of DNA.

The invention clones and identifies the gene of control rice stem fragility from rice brisk stem brittle culml(bcl), the gene is named as BCl. Transgenics function complementary experiment testifies that BCl has the function of control rice stem mechanism intensity. The gene series analysis testifies that the protein has 60.7 homologisation with the COBRA protein encoded by COB. The observation and the physical standards also testify that the cloned gene has function of adjusting the thickness of the plant cell wall and the supporting quality of plant stem.

The present invention relates to production of polyketides and other natural products and to libraries of compounds and individual novel compounds. One important area is the isolation and potential use of novel FKBP-ligand analogues and host cells that produce these compounds. The invention is particularly concerned with methods for the efficient transformation of strains that produce FKBP analogues and recombinant cells in which cloned genes or gene cassettes are expressed to generate novel compounds such as polyketide (especially rapamycin) FKBP-ligand analogues, and to processes for their,preparation, and to means employed therein (e.g. nucleic acids, vectors, gene cassettes and genetically modified strains).

The invention discloses rice root development and stress resistance related OsSAUR11 genes which are isolated and cloned from rice DNA fragments. Protein encoded by the genes contains SAUR family conserved structural domains. The rice root development and stress resistance related OsSAUR11 genes belong to the field of rice cloning genes. The sequence of the OsSAUR11 genes is as shown in followingsequences: a DNA sequence as shown in SEQ ID NO: 1, a DNA sequence at least 90% homologous to SEQ ID NO: 1 and a subfragment functionally equivalent to the sequence as shown in SEQ ID NO: 1; the expression of the OsSAUR11 genes is the root development and stress resistance. The rice root development and stress resistance related OsSAUR11 genes disclosed by the invention have the beneficial effectsthat the OsSAUR11 genes are in inducible expression of osmotic stress, abscisic acid and auxins, and overexpression of OsSAUR11 can enhance the deep root ratio of rice and enhance drought resistanceof the rice, is related to rice stress resistance, and has obvious response to adverse environments, thereby being applied to plant drought-resistance breeding, improving the root configuration and enhancing plant stress resistance.

The invention belongs to the technical field of cotton molecule breeding, particularly relating to a method for building a cotton fiber transcription genetic linkage map by EST-SSR signs. The method comprises the following steps: taking gossypium barbadense Pima 3-79 as a male parent and Gossypium hirsutum Emian 22 as a female parent, and hybridizing to obtain F1; planting F1, and causing F1 to be inbreeded to obtain F2; taking the F2 single plant as a starting material for the fiber transcription map to be plotted; extracting the RNA of the fiber of the field F2 single plant in 5 days after the single plant blooms, and carrying out reverse transcription of the RNA into cDNA to be served as a plotting group of the transcription map; utilizing the reported EST-SSR primer and the primer shown by a self-designed sequence table SEQ ID NO:1-90 to carry out the plotting group analysis to the F2 single plant by a denatured polyacrylamide gel electrophoretic analysis; and finally, analyzing data by MAPMAKER/ EXP.3.0 mapping software, and manufacturing and obtaining the transcription map. Compared with the existing method, the method of the invention is simple and practical, has accurate QTL positioning and is convenient for cloning genes relevant to cotton fiber development.

The invention provides a method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer in the field of biotechnology. The method includes the steps: cloning genes of artemisinic aldehyde delta 11(13) DBR2 from the sweet wormwood, constructing a plant expression vector containing the DBR2 genes, using agrobacterium tumefaciens for mediation to transfer the DBR2 genes into the sweet wormwood so that plants regenerate, using PCR (polymerase chain reaction) to detect integration conditions of the exogenous targeted DBR2 genes, determining the artemisinin content in the transgenic sweet wormwood by means of HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector), and screening the transgenic sweet wormwood plants with the improved artemisinin content. The artemisinin content in the obtained transgenic sweet wormwood can be remarkably increased and is 2.83 times maximally of that of a non-transgenic control plant, and accordingly the method for increasing the artemisinin content in the sweet wormwood lays a foundation for large-scale artemisinin production by the aid of the transgenic sweet wormwood.

The invention discloses a common mussel C type lysozyme and a preparation method thereof. The amino acid sequence of the common mussel C type lysozyme is shown by a sequence table SEQ ID No.1. The preparation method of the common mussel C type lysozyme comprises the following steps of: extracting common mussel gross RNA (ribonucleic acid), purifying mRNA (Messenger ribonucleic acid), preparing a cDNA (complementary deoxyribonucleic acid) template solution, cloning gene, building recombinant plasmid, expressing expression plasmid and purifying protein to obtain the C type lysozyme. The common mussel C type lysozyme is a new C type lysozyme which has strong inhibitory activity on various gram positive and negative microorganisms, and has application value in the aspects of developing food preservatives, feed additive, etc.

The present invention relates to an isolated major gene GS3 which regulates grain weight and grain length in the rice and the cloning of said gene. The DNA sequence of GS3 gene is as shown in SEQ ID NO. 1 and is 7883 bp in length. GS3 gene comprises 5 exons and encodes 232 amino acids. It is predicted based on bioinformatics analysis that said protein contains conserved domains including a PEBP-like domain, a transmembrane domain, a cysteine-rich domain of TNFR/NGFR and a VWFC domain. cDNA sequence of said gene is as shown in SEQ ID NO. 2. By sequence alignment between three large grain species and 3 small grain species of rice, it is revealed there is only one common single nucleotide mutation in a 7.9-kb region between the two different grain-length groups. Said nucleotide mutation is located at the second exon of the GS3 gene, in which a cysteine codon (TGC) in the small-grain group is mutated to a termination codon (TGA) in the large-grain group. This mutation causes a premature termination in the large-grain group, which leads to a 178-amino acids truncation (including part of the PEBP-like domain and all the other three conserved domains). The present invention also provides methods of producing transgenic plants comprising sequences disclosed herein.

The invention discloses a molecule cloning method. The molecule cloning method comprises the following steps: amplifying a vector and a target gene to be inserted by DNA polymerase with 3'-5' exonuclease activity, destroying a mehylated DNA template by restrictive endonuclease DpnI, and directly transforming a DpnI digestion product to competent cells in order to obtain a cloned gene. Experiments prove that the molecule cloning method has the advantages of simplicity, reliability, high efficiency, no DNA fragment purification and recovery, no consideration to the enzyme site of the vector, no linkage reaction, and realization of the insertion of any DNA fragment into a plasmid vector only through a PCR-based cloning technology.

The invention relates to the cloning of a transforming growth factor-beta (TGF-beta) superfamily type I receptor gene Tgfbrl1 of chlamys farreri in a molecular genetic marker technology, a screening and typing technology of a single nucleotide polymorphism (SNP) locus which is relevant with the weight of adductor muscles in the gene and a method for the application of the gene to the breeding of the high-yield chlamys farreri. A total-length complementary deoxyribonucleic acid (cDNA) sequence of the Tgfbr1 gene of the chlamys farreri is obtained by utilizing a homology-based cloning strategy; an SNP lotus is discovered by blastn comparison, and primers and a probe are designed for the locus; and SNP typing is performed in a natural population of the chlamys farreri by using a high-resolution melting curve technology, and the weight of the adductor muscle of an individual is measured. Statistic analysis displays that the loci c.1815C>T are obviously relevant with the weight of the adductor muscles of the chlamys farreri, and the weight of the adductor muscles of individuals with TT genetypes is obviously higher than that of the adductor muscles of individuals with CC and CT genetypes. In the selective breeding process of the high-yield chlamys farreri, the breeding candidate colonies of the chlamys farreri can be subjected to c.1815C>T typing, and the individuals of which the loci c.1815C>T are TT genetypes are used as a breeding parent preferably by combining typing information of other loci which are relevant with growth properties.

The invention discloses a coding sequence of an NAC family transcription factor expressed in rice and application thereof in pollen growth, belonging to the technical fields of molecular biology and genetic engineering. In the invention, the following processes of cloning of the NAC family transcription factor, analysis of a gene expression pattern, establishment of a transgenic carrier, phytotransformation, obtainment of a transgenic plant, and identification of a corresponding phenotype and a cellular level are comprised. The gene presented in the invention is expressed in Arabidopsis, and the obtained transgenic plant is abnormal in pollen growth and has a decreased ripening rate.

The invention belongs to the technical field of plant genetic engineering. Specifically, it relates to the separation and application of the PtAGL24 gene that can regulate the early flowering of plants. The nucleotide sequence of the PtAGL24 gene is shown in SEQ ID NO: 1, the sequence of the protein encoded by the gene is shown in SEQ ID NO: 2; the nucleotide sequence of the promoter PtAGL24P is shown in SEQ ID NO: 3. The ectopic overexpression of the gene can significantly shorten the childhood period of the plant and promote the early flowering of the plant. The gene and promoter of the invention provide biological resources for the breeding of perennial woody fruit trees and the shortening of flowering process. The role of PtAGL24 in the whole development process of the plant is further revealed through the spatial expression of the promoter, which shows that the cloned gene of the present invention is also induced by the low temperature of the environment, and has a certain response to the external environmental stress. The gene and promoter of the present invention can be used in regulating plant flowering period and low temperature stress.

The invention discloses a method for improving the content of verbascoside in rehmannia by virtue of transformation of RgPAL1 gene, and belongs to the field of biotechnology. The method comprises the following steps: cloning gene RgPAL1 from rehmannia, constructing a plant expression vector containing the RgPAL1, transforming the RgPAL1 into the rehmannia by virtue of mediation of agrobacterium tumefaciens and promoting regeneration of a plant, detecting integration of an exogenous target gene RgPAL1 through polymerase chain reaction (PCR), quantitatively analyzing the content of verbascoside in the transgenic rehmannia by virtue of HPLC (High Performance Liquid Chromatoraphy)-PDA, and screening to obtain a transgenic rehmannia plant having improved verbascoside content. By virtue of the method disclosed by the invention, the content of verbascoside in the transgenic rehmannia is significantly improved, and the content of verbascoside in the RgPAL1 transgenic rehmannia is 2.57 times as much as that in non-transgenic rehmannia, so that a feasible method is provided for scaled production of verbascoside from the transgenic rehmannia. The method disclosed by the invention provides an innovative and practical related method and technology for improving the verbascoside content.

The invention discloses a collagen gene functional fragment cloned from a turtle and shown in sequence SEQ ID NO.1 in a sequence table and a recombinant protein obtained by the expression of the clone gene functional fragment and shown in sequence SEQ ID NO.2 in the sequence table, wherein the recombinant protein is a recombinant turtle collagen gene functional peptide fragment. The whole length of the recombinant turtle collagen gene functional peptide fragment includes 252 amino acids, 27 specific Gly-X-Y repeating fields of collagen are contained and the molecular weight is 38kDa. The preparation method comprises the steps of constructing a colibacillus genetic engineering bacterium for expressing the recombinant turtle collagen gene functional peptide fragment; culturing the colibacillus genetic engineering bacterium; inducing and expressing the recombinant turtle collagen gene functional peptide fragment; performing purification renaturation on the recombinant turtle collagen gene functional peptide fragment. The experiment shows that the recombinant turtle collagen gene functional peptide fragment prepared by the method provided by the invention has a high antioxidant activity and also has a value of application as an antioxidant.

The invention discloses a method for sequencing clone genes with recessive mixed pools. The method comprises the following steps of constructing segregation populations by hybridizing parents; allocating pools for the segregation populations according to individual target properties; obtaining dominant and the recessive specific loci of the parents through high-throughput sequencing; comparing with the sequencing results of the recessive mixed pools to obtain candidate genes; gathering the candidate genes in the recessive mixed pools and re-sequencing clone target genes; The method is on the basis of sequencing, can be used for any species, and directly clones genes themselves. With the method, workload is decreased greatly, speed is substantially accelerated; and risks are reduced.

The invention discloses a coding gene of haemaphysalis qinghaiensis aquaporins (AQPs) and an application of the coding gene. The coding gene of the AQPs can be used for preparing novel anti-tick medicines and anti-tick vaccines. A sequence of the coding gene of the AQPs is shown as SEQ ID NO:1. The invention also provides specific primers cloned by the coding gene of the AQPs, wherein sequences ofthe specific primers are shown as SEQ ID NO:2 and SEQ ID NO:3; the invention also provides a method for cloning the coding gene of the AQPs, and the cloned gene product can be also applied to in-vitro recombinant protein expression and antibody preparation; and an obtained antibody can be used for preparing the novel anti-tick medicines and the anti-tick vaccines. The invention has important theoretical significance an potential application value on prevention and control of ticks. The coding gene of the haemaphysalis qinghaiensis aquaporins (AQPs) and the cloning method of the coding gene provided by the invention are applicable to systematic researches of subtype, varieties, expression distributin, protein structures, biological functions and the like of the AQPs.

A method for cloning the virus host factor gene by use of dual-chain RNA low-poison virus (chesnut blight bacterium system) includes such steps as light irradiating to induce the generation of conidia of chestnut phytophthora disease bacteria, inducing to obtain mutant, purifying, discriminating the virus carried by said mutant, testing the activity of its mRNA precursor, transforming and complementing of said mutant, discriminating, sequentially the exogenous DN'A fragment of the transformed plasmid with complem entary power, and configuring gene map.

The invention discloses a surface lipoprotein of streptococcus suis type-2, a preparation method thereof and application. The surface lipoprotein of streptococcus suis type-2 is numbered as gi|146320941 in genebank; the preparation method of the surface lipoprotein comprises the following steps: cloning gene of the surface lipoprotein; connecting the gene and expression plasmid of Escherichia coli to transform the Escherichia coli; inducing expressing; separating and purifying expression product; and the like. The surface lipoprotein can be used as a diagnostic target, and a specific antibodyof the lipoprotein can be detected in animals and people infected with the streptococcus suis; moreover, the surface lipoprotein is also an important protective antigen, and can be used for preparingvaccines.

The present invention relates to a shuttle BAC vector for facilitating the cloning, transfer and heterologous expression of streptomycete secondary metabolite biosynthetic gene clusters. The invention also relates to a plasmid rescue method using this vector for enhancing the process of cloning biosynthetic gene clusters for secondary metabolites from streptomycetes without sophisticated generation and screening of cosmids or BAC libraries. The cloned DNA can then be used for sequencing or heterologous expression of putative secondary metabolic gene clusters.

The invention belongs to the field of gene engineering, and particularly relates to a transgenic expression vector for regulating seed color of brassica napus L., and a construction method and application of the transgenic expression vector. The transgenic expression vector is RNAi-BnMYB47 transgenic expression vector, a nucleotide sequence of the RNAi segment of the RNAi-BnMYB47 transgenic expression vector is shown as SEQ ID NO:1, and the RNAi-BnMYB47 transgenic expression vector is obtained by the steps of cloning a RNAi segment of a BnMYB47 gene, connecting the RNAi segment with a pFGC5941M plasmid, performing conversion and plasmid extraction. The RNAi segment of the BnMYB47 gene and the expression vector can be applied to regulate the seed color of the brassica napus L., the difficulty of obtaining stable yellow-seeded brassica napus breeding due to instable seed color property of the brassica napus L. is overcome, conditions for obtaining a stably inherited new seed material oftransgenic yellow seeds of the brassica napus L. are created, and it is of great guiding significance for the brassica napus L. yellow seed breeding and genetic engineering for performing genetic improvement of brassica napus L. quality and breeding practice.

The invention belongs to the technical field of plant genetic engineering, and in particular relates to over-expression GhJAZ1 genes capable of enhancing low temperature stress resistance of plants. JAZ (JASMONATE-ZIM DOMAIN) family protein genes GhJAZ1 are cloned from cotton, a cDNA sequence of the genes GhJAZ1 is shown in SEQ ID NO: 1, and the coded protein sequence is shown in SEQ ID NO: 2. An over-expression vector of the genes is constructed, and a transgenic plant is obtained according to a genetic transformation method. The cloned genes in the invention are proved to be capable of improving the tolerance of the cotton on low temperature stress.