201 results about "Cloning" patented technology

The invention relates to a CHO-S cell strain capable of stably expressing H5N1 hemagglutinin protein and a construction method of the CHO-S cell strain, and belongs to the field of bioengineering.H5N1 hemagglutinin protein expression plasmids are obtained in an in-vitro synthesis and seamless cloning mode, 293T cells are transfected through the H5N1 hemagglutinin protein expression plasmids, and it is proved that the H5N1 hemagglutinin protein is expressed; the CHO-S cell strain capable of stably expressing the H5N1 hemagglutinin protein is obtained by transfecting CHO-S cells by using the H5N1 hemagglutinin protein expression plasmids and carrying out multiple rounds of cloning and screening, so that a basis is provided for obtaining H5N1 recombinant protein influenza vaccines.

The invention belongs to the technical field of pepper planting, and discloses a pepper multi-branch gene CaBr1 linked KASP molecular marker as well as a primer, a kit and application thereof, the KASP molecular marker takes pepper S8 (zhangshuang) as a reference genome, and the single nucleotide polymorphism at the 103175415th basic group on the No.8 chromosome is subjected to basic group G-to-A replacement. A chromosome region closely linked with multiple branches of the pepper is obtained by using a BSA (Bovine Serum Albumin) population positioning method, a molecular marker is developed in a candidate region to determine a candidate gene CaBr1, a KASP molecular marker is screened according to base mutation of the candidate gene, and the marker is used for carrying out genotype identification on 90 randomly sampled single plants of an F2 population, and the coincidence rate reaches 100%. The result not only contributes to pepper branch phenotype identification and assistant breeding, but also lays a foundation for map-based cloning of the branch gene and analysis of a molecular mechanism of pepper plant type regulation.

The invention relates to the field of plant gene map-based cloning and molecular breeding. Specifically, the invention provides a nucleic acid molecule and a polypeptide, and also provides a recombinant vector, a host cell and a plant or a part, a seed, a cell or a progeny thereof containing the nucleic acid molecule. Furthermore, the invention also provides a method for preparing the transgenic plant, and a method for regulating and controlling the nitrogen utilization efficiency, nitrate absorption or transport, biomass and / or yield of the plant.

The present invention belongs to the field of biotechnology, and specifically relates to a GmSGT2 gene related to plant height and branch development, and a mutant thereof and the use thereof. A Glycine max mutant having fewer branches and decreased plant height is obtained by means of EMS mutagenesis, and a target gene thereof that is located by means of a map-based cloning technique is a GmSGT2 gene of Hedou 12. It is found through searching that the gene encodes soyasapogenol B glucuronide galactosyltransferase, and can galactosylate soyasapogenol B monoglucuronide, thereby affecting the plant height of Glycine max and reducing the branches of Glycine max. Therefore, the GmSGT2 mutant can be used for cultivating dwarf high-yield Glycine max varieties, and is of great significance in the breeding of ideal plant types of Glycine max and research on important agronomic traits of plants. The mutant has broad application prospects and great research value with regard to understanding the plant height and branch regulation mechanism of Glycine max and improving the process of Glycine max breeding; and can provide an excellent germplasm resource stock for regulating the close planting and high-yield breeding of Glycine max.

The invention discloses application of wheat Rht1-D1b protein in regulation and control of a wheat tillering angle, and belongs to the technical field of biological breeding. The technical problem to be solved by the invention is how to increase the tillering angle of plants. Therefore, the invention provides the application of the protein or a substance for regulating and controlling the expression of a protein coding gene or a substance for regulating and controlling the activity or content of the protein in regulating and controlling the tillering angle of the plant, wherein the protein can be Rht1-D1b protein; the Rht1-D1b protein can be a protein of which the amino acid sequence is as shown in SEQ ID No. 3. According to the application, the Rht1-D1b gene is finally localized through gene localization and map-based cloning. The gene significantly increases the tillering angle of wheat. The wheat gene Rht1-D1b can be widely applied to the plant fields of wheat germplasm resource improvement, wheat plant type genetic breeding and the like, and has an important application value for improving the wheat yield.

The invention provides helper nucleic acid enhanced seamless cloning. The present invention provides products, methods and uses related to helper nucleic acid enhanced seamless cloning.

The invention discloses a method for quickly rescuing bovine coronavirus epidemic strains and application of the method. The bovine coronavirus SHZ isolate is subjected to efficient segmented cloning, the obtained segment and a Linker sequence are subjected to a cyclic polymerase extension reaction, and the obtained reaction product can be directly transfected to 293T cells for virus packaging without purification. Different from a traditional method, the method does not need to amplify full-length virus cDNA clone by means of bacteria and yeast, but directly obtains a sufficient amount of preliminary products through PCR, and avoids the problems of instability and low efficiency when partial sequences of a virus genome proliferate in a bacteria or yeast host. By adopting the reverse genetic system, the recombinant bovine coronavirus expressing the foreign protein is successfully rescued, a brand new technical platform is provided for dynamic visualization research of in-vivo and in-vitro replication of the virus, and an efficient and flexible tool is also provided for virology research, development of virus vector vaccines and screening of antiviral drugs.

The invention discloses a rice seed dormancy gene OsPHS1 and application of a protein encoded by the gene OsPHS1, and belongs to the technical field of plant genetic engineering, and the gene and the protein encoded by the gene are used for regulating the dormancy of rice seeds. The nucleotide sequence of the gene is shown as Seq ID No: 1, the CDS sequence is shown as Seq ID No: 2, and the coded protein sequence is shown as Seq ID No: 3. The function of the rice seed dormancy gene OsPHS1 is verified by cloning and identifying the rice seed dormancy gene OsPHS1, and research finds that the dormancy of rice seeds can be remarkably reduced by knocking out or mutating the OsPHS1 gene, and the dormancy of the rice seeds can be improved to a certain extent by overexpressing the gene; the invention plays an important role in developing rice varieties with moderate dormancy.

The invention relates to a recombinant expression cat interferon-omega gene and a preparation method and application thereof, the nucleotide sequence of the cat interferon-omega gene is as shown in SEQ ID NO.1, and the cat interferon-omega gene is obtained by introducing an Fc fusion fragment to the C terminal and / or N terminal of the natural gene sequence of the cat interferon-omega. XTEN or PAS is introduced to prolong a peptide fragment, and original glycosylation modification sites on a natural sequence are reserved. The method comprises the following steps: cloning a cat interferon-omega gene into an expression vector containing double promoters, further transfecting into a cell, carrying out stable cloning and screening, establishing a high-expression cell strain, and carrying out fermentation culture in a bioreactor. According to the invention, the cat interferon-omega gene is subjected to multiple modification and is efficiently expressed in a CHO-K1GS system, so that the protein yield, the stability and the half-life period are remarkably improved; the obtained fusion protein is high in purity and strong in activity, can obviously enhance immune response and protection effect when being matched with cat vaccines, and is good in safety.

The invention discloses a molecular marker closely linked with a wheat powdery mildew resistance gene PmCWI16926. The molecular marker is YTU2BS-P12, and the molecular marker is a molecular marker PmCWI16926. The nucleotide sequence of an upstream primer of the molecular marker is as shown in SEQ ID NO: 1; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO: 2; the molecular marker is used for carrying out PCR (Polymerase Chain Reaction) amplification on to-be-detected wheat genome DNA (Deoxyribose Nucleic Acid) to obtain a corresponding amplification product with the molecular weight According to the molecular marker YTU2BS-P12 closely linked with the wheat powdery mildew resistance gene PmCWI16926, provided by the invention, a genetic mapping group of the PmCWI16926 can be more efficiently detected, and map-based cloning of the PmCWI16926 is facilitated; when the marker is used for molecular marker-assisted selection of PmCWI16926, the breeding period can be shortened, the breeding efficiency can be improved, and the marker can be better applied to wheat breeding for disease resistance.

The invention relates to the field of plant gene map-based cloning and molecular breeding. Specifically, the invention provides a nucleic acid molecule and a polypeptide, and also provides a recombinant vector, a host cell and a plant or a part, a seed, a cell or a progeny thereof containing the nucleic acid molecule. Furthermore, the invention also provides a method for preparing the transgenic plant, and a method for regulating and controlling the nitrogen utilization efficiency, nitrate absorption or transport, biomass and / or yield of the plant.

The invention discloses a rape waterlogging-resistant gene BnA07ERF151 and an application thereof. The nucleotide sequence of the gene BnA07ERF151 is as shown in SEQ ID NO. 1. The invention also discloses a method for preparing the rape waterlogging-resistant gene BnA07ERF151. According to the invention, a brand new stain-resistant gene BnA07ERF151 is cloned from rape for the first time, and is transferred into arabidopsis thaliana to construct a pure line plant and complete stain resistance identification. Experimental results show that the growth state of a transgenic BnA07ERF151 gene strain in a waterlogging environment is remarkably superior to that of a wild type, bolting development is normal, the survival rate is increased to 43.75% from 12.5%, and the key effect of the gene on enhancing the waterlogging resistance of plants is clearly confirmed; successful cloning of the waterlogging-resistant gene provides a core gene resource for cultivation of new crop varieties, a variety of crops can be endowed with stronger waterlogging tolerance through a transgenic assisted breeding means, and the yield stability under extreme climate is effectively guaranteed, which has great strategic significance in improvement of global agricultural disaster resistance and guarantee of grain safety, and has a wide application prospect. And the method has remarkable social and economic benefits and industrial application prospects.

The invention relates to a key gene CbLTP63 for regulating and controlling salt stress response of catalpa bungei and application of the key gene CbLTP63, and belongs to the technical field of plant genetic engineering and biology. The nucleotide sequence of the CbLTP63 gene is as shown in Seq 1, and the coded amino acid sequence is as shown in Seq 2. The invention relates to cloning of a catalpa bungei salt stress response key gene CbLTP63. Identifying that the CbLTP63-GFP subcell is positioned on a cell membrane through an agrobacterium injection method; through qRT-PCR analysis and identification, the gene responds to salt stress on the transcriptional level and is down-regulated by H2O2, ABA and SA; through transgenosis identification, the gene is a catalpa bungei salt stress response negative regulation gene, and the salt tolerance of catalpa bungei calluses after gene silencing is obviously enhanced; the invention also relates to utilization of the gene in regulating stress response of catalpa bungei. The invention discloses a catalpa bungei salt stress response key gene CbLTP63, which has important application value in the fields of catalpa bungei gene engineering and clonal forestry.

Provided is a high-throughput microbe smart cloning and information analysis device, comprising a machine body, a strain feeding mechanism, a petri dish feeding mechanism, a lid opening mechanism, a lid opening and photographing mechanism, an inoculation mechanism, an inoculation needle replacement mechanism, a liquid level detection system, a pressure detection system, a high-performance computing module, and a control management system. The lid opening mechanism and the lid opening and photographing mechanism open the upper lids of strains and empty petri dishes that are transported to an inoculation position by the strain feeding mechanism and the petri dish feeding mechanism, respectively, and photograph the strains; the high-performance computing module receives image information, and locates and identifies the strains by means of a target detection algorithm; the inoculation mechanism moves an inoculation needle to inoculate the strains, while the liquid level detection system and the pressure detection system prevent the inoculation needle from pressing or damaging the petri dishes, and the inoculation needle replacement mechanism is used to replace the inoculation needle. By means of an automated device replacing manual operations to implement strain cloning actions, inoculation efficiency is improved, the safety of operators and strains is assured, and it is possible record and analyze strain information.

The invention discloses an anti-disease gene, an anti-disease protein, a molecular marker and application of tomato brown crinkled fruit virus disease, and belongs to the technical field of gene engineering. According to the invention, through a map-based cloning method, a new anti-disease gene TBR3 of the tomato brown fruit wrinkling virus disease is identified in a tomato chromosome 2 for the first time. The TBR3 gene is over-expressed in a plant body, so that the disease resistance of the plant to the tomato brown crinkled fruit virus disease is remarkably improved. Aiming at the disease-resistant gene TBR3, the invention also designs a molecular marker related to tomato brown crinkled fruit virus resistance, and tomato plants carrying a disease-resistant genotype and a susceptible genotype can be accurately distinguished, so that the molecular marker can be widely applied to early disease-resistant breeding and screening, and the problems of difficult early judgment and low breeding efficiency in a breeding process are solved.

The invention belongs to the field of plant biotechnology breeding, and relates to an application of a ZmGalAT1 gene and an encoding protein thereof in corn fertility control. According to the invention, a corn male sterility mutant galat1 is obtained by EMS mutagenesis, and a gene ZmGalAT1 for controlling corn male flower development is identified through map-based cloning. The gene is subjected to site-directed mutagenesis in wild type corn through a CRISPR / Cas9 gene editing technology, three galat1 allelic corn male sterility mutants are created, corn male flowers of the mutants are completely aborted, sterile mutants without transgenic ingredients can be obtained through offspring screening, and the mutants are further applied to corn sterile breeding and seed production. The invention also designs functional molecular markers for three allelic male sterile mutants of GalAT1, and has important application value in maize male sterile line cultivation, sterile hybrid seed production and molecular marker-assisted selection.

The invention relates to the technical field of plant genetic engineering, and particularly provides cloning of a GbGELP25D gene of gossypium barbadense and application of the GbGELP25D gene of gossypium barbadense. According to the invention, the GbGELP25D gene with a full length of 1092 bp is cloned from the sea island cotton variety Xinhai No. 7 for the first time, and the gene encodes a secretory lipase protein with a signal peptide and is positioned in an extracellular gap. Through bioinformatics analysis, phylogenetic classification, protein structure modeling and signal peptide function verification, it is clear that the gene belongs to a plant GELP family. Furthermore, a virus-induced gene silencing technology is utilized to prove that the silent GbGELP25D can obviously enhance the resistance of cotton to verticillium wilt, and the mechanism of the silent GbGELP25D is closely related to activation of ethylene synthesis and signal channels and induction of expression of disease-resistant related genes. The resistance gene provided by the invention enriches gene resources of cotton verticillium wilt resistance breeding, and has important theoretical significance and application value.

The invention provides a DNA library screening method and application of an intracellular mRNA sensor switch. The DNA library screening method comprises the following steps: designing a DNA library containing a switch sequence of a degenerate basic group; preparing a circular expression vector library; preparing a minimum linear expression vector library; performing seamless cloning; carrying out RCA rolling circle amplification; carrying out telomerase digestion; carrying out cell transfection; extracting RNA (Ribonucleic Acid) and carrying out reverse transcription; carrying out library building of next-generation sequencing by taking the cDNA as a template; carrying out next-generation sequencing; analyzing data and comparing to obtain the DNA library of the mRNA sensor switch. According to the DNA library screening method provided by the invention, more sensor switch sequences can be provided for a single site on mRNA in cells, and more selection possibilities are further provided for subsequent targeted therapy.

The invention relates to a DNA (Deoxyribose Nucleic Acid) for regulating and controlling a resistance gene LTPL160 of rice bacterial blight and breeding utilization of a protein of the DNA. According to the invention, the gene OsLTPL160 for regulating and controlling the resistance of the rice bacterial leaf blight is cloned by virtue of a non-gel quantitative proteomics analysis and transcription analysis method. The OsLTPL160 knockout and overexpression material is obtained by adopting a gene editing and transgenic technology. The OsLTPL160 is knocked out, so that the bacterial blight resistance of the rice is remarkably improved; and overexpression of the OsLTPL160 obviously reduces the resistance of the bacterial blight of the rice. The cloning of the OsLTPL160 gene and the construction of the material provide material and technical support for the bacterial blight resistance of the rice.

The invention discloses an application of an overexpressed CrOASTL1 gene in improving the soluble protein content of leguminous forage, and belongs to the technical field of biology. 1, and the CDS sequence of the CrOASTL1 gene is as shown in SEQ ID NO. 2. The method comprises the following steps: cloning a CrOASTL1 gene segment, constructing a plant overexpression vector, carrying out plant transgenosis operation, and introducing into leguminous forage plant cells to obtain a leguminous forage transgenosis line with the overexpressed CrOASTL1 gene. The CrOASTL1 gene is over-expressed in medicago sativa, and the soluble protein content of leaves of the medicago sativa is remarkably increased by about 19%. The CrOASTL1 gene of chlamydomonas reinhardtii is applied to quality improvement of leguminous forage for the first time, the technical obstacle that the genetic relationship of species is relatively far is overcome, a brand new gene resource and an efficient technical approach are provided for cultivating a new variety of leguminous forage with high protein and high nutritional value, and the method has important significance in promoting sustainable development of grass and animal husbandry.

The application relates to the technical field of biological medicine, and particularly discloses a fish rhabdovirus recombinant adenovirus vector vaccine and a preparation method and application thereof. The recombinant adenovirus vaccine carrying the M gene of the SCRV virus is prepared by using a replication-defective recombinant adenovirus vector, and the preparation method comprises the following steps: mandarin fish rhabdovirus M gene cloning, construction of a Pshuttle-CMV-ECMV vector with a target gene SCRV-M, obtaining of a SCRV-M recombinant adenovirus plasmid, screening of the SCRV-M recombinant adenovirus, and preparation of the SCRV-M recombinant adenovirus vaccine. The SCRV-M recombinant adenovirus vaccine prepared by the application can be used for immunoprophylaxis of SCRV virus infection in the juvenile fish stage through a soaking mode, the SCRV-M recombinant adenovirus vaccine has passed an animal safety test, and has a wide application prospect in the prevention and control of fish rhabdovirus.

The invention encompasses methods for increasing genetic progress in livestock, and for genetic dissemination, including the use of amniocentesis to obtain fetal amniocytes for use in genomic evaluation and cloning.

The invention discloses genetic engineering application of a rice cellulose synthesis gene OsCesA9, belongs to the field of genetic engineering, and discloses cloning of the gene OsCesA9 participating in regulation and control of rice grain length and application of the gene OsCesA9 in regulation and control of rice grain length. The CDS sequence of the gene is as shown in SEQ ID NO.1, and the cDNA sequence is as shown in SEQ ID NO.9. Overexpression of the rice gene OsCesA9 can increase the grain length of rice without influencing the panicle number of the whole plant, and is beneficial to genetic improvement of rice.

The invention discloses an application of CsTON2 protein and a coding gene thereof in regulating and controlling the length of cucumber fruits, and belongs to the technical field of biology. The CsTON2 protein is derived from cucumbers, the amino acid sequence of the CsTON2 protein is as shown in SEQ ID NO.2, and the nucleotide sequence of a gene for coding the protein is as shown in SEQ ID NO.1. The South China type short fruit mutant sf5 is obtained through EMS mutagenesis, a candidate gene is determined to be CsTON2 through map-based cloning, amino acid is changed into leucine from proline due to mutation (C is changed into T) of the gene, and the fruit length is remarkably shortened; and after the overexpression vector is constructed and the mutant is transformed, the CsTON2 expression quantity of the overexpression strain is obviously increased, and the fruit length is obviously increased. The positive regulation effect of the CsTON2 gene on the cucumber fruit length is defined, a key target gene and a molecular marker are provided for molecular breeding of the cucumber fruit length, and the CsTON2 gene has important theoretical significance and application value.

The application discloses a corn kernel size related protein SSRP1 and application thereof. The technical problem solved is how to regulate the size of corn kernels. Specifically disclosed is the application of a protein, a substance for regulating the expression of a coding gene of the protein, or a substance for regulating the activity or content of the protein in regulating the size of plant kernels and / or preparing a product for regulating the size of plant kernels and / or plant breeding and / or preparing a plant breeding product, the protein being a protein as shown in sequence 3 in the sequence listing. The application provides a new genetic resource for the study of the size of plant kernels, in particular corn kernels, and will play an important role in the application in the field of corn seed production and breeding.

The present application belongs to the field of biotechnology, and particularly relates to a carboxypeptidase B (CPB) precursor gene sequence, a recombinant carboxypeptidase B precursor and a preparation method of carboxypeptidase B. The carboxypeptidase B precursor gene sequence is shown in SEQ ID No. 6, the amino acid sequence is shown in SEQ ID No. 5, the gene sequence of the CPB enzyme is shown in SEQ ID No. 2, and the amino acid sequence is shown in SEQ ID No. 4. The preparation method of CPB comprises the following steps: (1) cloning and construction of a CPB enzyme expression vector; (2) expression of CPB precursor protein by using an engineering bacterium to form an inclusion body; (3) washing, crushing and centrifugation of the bacterium, collection of the inclusion body, denaturation and dissolution; (4) renaturation of the inclusion body; and (5) CPB activation and purification. The process steps of the present application are simple, animal pancreas is not used, and the possibility of infection is reduced; the specific activity of the obtained CPB enzyme is high, the yield per liter of fermentation liquid is high, and the present application is suitable for large-scale production and application.

The application belongs to the field of plant biotechnology and particularly relates to a CYP714A1 application of a promoter in marking petal cells, the CYP714A1 nucleotide sequence of the promoter is shown in SEQ ID NO. 1. The application uses CYP714A1 the promoter to mark and identify specific cell populations of petals, which proves that CYP714A1 the promoter can identify and mark the differentiated petal cells. The application provides CYP714A1 a method for cloning and application of the promoter, comprising: CYP714A1 CYP714A1 CYP714A1 CYP714A1 CYP714A1 CYP714A1 CYP714A1 CYP operably connecting the promoter to an expression vector to construct a recombinant vector, introducing the recombinant vector into a plant, starting expression of a downstream gene, and tracking the claw cell population in Arabidopsis petals.

The invention discloses a polyA tail element, a construction body and application of the polyA tail element and the construction body. The polyA tail element is inserted into a sequence capable of mutually complementarily pairing basic groups behind A at any one of 27th to 33rd and 85th to 91st in the direction from 5'to 3 '; or the basic group at the multiple site of the 12th, 13th, 14th or 15th of the polyA tail element is C. The polyA tail element disclosed by the invention is high in correct cloning rate and good in passage stability, and can be used for fermentation production of mRNA template plasmids; a construct or a recombinant expression vector containing the gene has strong stability and high translation efficiency, and can efficiently express a target protein; and a new template plasmid can be quickly transformed and constructed for transcriptional production of new mRNA, so that the production time and cost are saved.

Provided here are methods directed to the T cell receptor cloning of heat shock protein 60 (HSP60), the generation of HSP60-specific regulatory T cells (Tregs), and adoptive cell transfer of HSP60-specific Tregs for the treatment of autoimmune and other disorders. Methods of treatment also include administering a therapeutically effective amount of an inhibitor of expression or activity of nucleus accumbens-associated protein- 1 (NAC1) along with the HSP60-specific Tregs.

The present invention relates to a method of selecting a polypeptide for display on a genetic display system according to activity, comprising the steps of: displaying the polypeptide on the genetic display system, screening a system for binding to a target, and selecting a member binding to the target; cloning a nucleic acid encoding the polypeptide from a member of the display system, and expressing the nucleic acid in an expression system to produce the polypeptide, or sequencing the polypeptide from a member of the display system, and cloning and expressing the polypeptide or a library of polypeptides using synthesis-based DNA; and determining the activity of the polypeptide in an assay.