72 results about "Rapid amplification of cDNA ends" patented technology

The invention discloses a lycium chinense miller lycopene beta-cyclase gene, a recombinant vector containing the gene, a host cell and application. Total RNA (Ribonucleic Acid) of fresh lycium chinense miller leaf is extracted, and lycopene beta-cyclase gene LmLycB is cloned by using 3' RACE (Rapid Amplification of cDNA Ends) technology to obtain a complete gene sequence of 1,506bp. An Escherichia coli expression vector pMON-LmLycB is constructed; an escherichia coli heterogenous expression system is applied; the activity of cloned LmLycB gene-coded enzyme is identified; and two beta-lonone rings can be added at the tail end of lycopene by LmLycB to produce beta-carotene. A maize inbred line immature embryo genetic transformation system is optimized; a plant expression vector pCAMBIA2300-LmLycB-Bar containing a target gene is transformed into maize callus to finally obtain a transgenic positive plant; through HPLC (High Performance Liquid Chromatography) detection, the total carotinoid content of transgenic maize leaf is 165.70mug/g FW and is 12.76 percent greater than that of a wild maize; and beta-carotene content is 80.17mug/g FW and is 54.83 percent greater than that of the wild maize.

The invention discloses cloning, expression and application of an alpha-L-rhamnosidase gene. A coding gene is obtained from aspergillus tubingensis and is expressed in pichia pastoris GS115; specifically, total RNA (Ribonucleic Acid) of the aspergillus tubingensis is used as a template and cDNA (complementary Deoxyribonucleic Acid) of alpha-L-rhamnosidase is cloned through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and RACE (Rapid Amplification of cDNA Ends) technologies; pPIC9k is used as an expression vector and is subjected to secreting expression in the pichia pastoris GS115. The alpha-L-rhamnosidase can be used for efficiently hydrolyzing naringin; when the naringin is used as a substrate, Km and Vmax values respectively are 1.27mu.mol/mL and 3445mu.M min<-1>; the thermal stability at 60 DEG C is good and the alpha-L-rhamnosidase gene has a good application prospect in food and biopharmaceutical industries.

The invention relates to a cotton GbSTK gene and encoding protein thereof. Sea island cotton Pima90-53 is taken as a material, a cDNA-AFLP technology is used for screening to obtain differential fragments related to verticillium wilt resistance, and then, a reverse transcription-polymerase chain reaction (RT-PCR) and a RACE (rapid-amplification of cDNA ends) technology are used for amplifying to obtain a resistance gene of cotton verticillium wilt, namely a serine/threonine protein kinase (GbSTK) gene. The functional studies of the gene and the encoding protein thereof show that the GbSTK gene has certain effect on resisting verticillium wilt.

The invention discloses an anchor primer for efficient and rapid amplification of cDNA 5' and 3' ends. The two anchor primers can be specifically bound to a target nucleic acid sequence and trigger reverse transcription elongation reaction, thereby efficiently and rapidly amplifying cDNA 5' and 3' ends, and especially can be applied to rapid amplification of the 5' and 3' ends of cDNA in low abundance transcripts. In addition, the invention also provides a kit and an amplification method for efficient and rapid amplification of cDNA 5' and 3' ends.

The invention relates to the technologies of clam ferroprotein gene cloning and in vitro recombinant expression in molecular biology. In the invention, the expression sequence tag (EST) technology and rapid amplification of cDNA ends (RACE) 3' and 5' are utilized to clone ferroprotein gene cDNA with the total length of 798bp from a clam larva, the cloned ferroprotein gene contains an open reading frame with the length of 513bp, 171 amino acids are coded, the length of a non-coding region 5' is 119bp, the length of a non-coding region 3' is 154bp, a tailing signal is contained, and the gene plays an important role in formation of a shell during development of the clam larva. In the invention, the in vitro prokaryotic recombinant expression technology is utilized to obtain recombinant clam ferroprotein; and the ferroprotein has antioxidant activity, can be used for regulating and controlling formation and growth of shells of shellfish larvae such as clams and the like, and can be applicable to development regulation and immunological enhancement in the shellfish larva production process.

The invention discloses sequences of a Saccharum officinarum sucrose phosphate synthase B (SofSPSB) gene and an encoded protein thereof. The sequences are obtained by the following steps: carrying out evolution analysis of full-length sequences of sucrose phosphate synthase genes of all plants, designing primers based on the conservative regions of the same family of sucrose phosphate synthase gene sequences, extracting total RNA from Saccharum officinarum tender leaves, carrying out reverse transcription, amplifying with a conventional PCR (polymerase chain reaction) method, and cloning into the full-length cDNA sequence of SofSPSB gene in combination with the RACE (rapid amplification of cDNA ends) technique. The invention not only studies the transcription and expression mechanisms of sucrose phosphate synthase so as to lay a foundation for further discussion of a sucrose accumulation mechanism; and can obtain a purified protein with biological activity through an amino acid sequence so as to provide a basis of studying the biological function of sucrose phosphate synthase and improving the crop variety by using the SofSPS gene.

The invention discloses a grass carp thymosin extrasin beta11 gene sequence. A full expression sequence of a grass carp thymosin extrasin beta11 gene is obtained with a PCR (Polymerase Chain Reaction) method by designing primers with a thymosin extrasin beta11 EST sequence of a grass carp intestinal tract cDNA library preserved in the local laboratory used as a template, and combining with a RACE (Rapid Amplification of cDNA Ends) technology. The invention provides important theoretical basis for researching the structural constitution of the grass carp thymosin extrasin beta11 gene, and probing into the function of the grass carp thymosin extrasin beta11 gene in the disease resistant mechanism of grass carps, the position of the grass carp thymosin extrasin beta11 gene in a functional area, and the difference with other species, and plays a pathbreaking role of exploration on establishing a bond between a grass carp genome project and a research on the grass carp disease resistant mechanism.

The invention discloses a siniperca chuatsi IFN-alpha 3 (interferon alpha-3) gene, recombinant protein, a preparation method and application thereof. The preparation method of the siniperca chuatsi IFN-alpha 3 recombinant protein includes the steps of: cloning the full-length cDNA (complementary deoxyribonucleic acid) sequence of the siniperca chuatsi IFN-alpha 3, as is shown in SEQ ID NO:1 by using rapid amplification RACE (rapid amplification of cDNA end) technology of cDNA ends; cloning the ORF (open reading frame) sequence of the cDNA sequence by using a primer with a restriction site; cloning the ORF sequence into a pMD19T vector and then conducting double enzyme digestion with EcoR I and Xho I to obtain a target fragment; performing double enzyme digestion of an pET32a(+) vector with EcoR I and Xho I, connecting the pET32a(+) vector going through the double enzyme digestion with the target segment to obtain a recombinant expression vector pET32a(+)-IFN-alpha 3 to be transferredto DH5 (competent cells) alpha competent cells, selecting positive clones, and extracting preservation plasmids; transferring the plasmids into an expression bacterium BL21 (electroporation) and adopting an IPTG (isopropyl-beta-d-thiogalactopyranoside) induced expression to obtain IFN-alpha 3 recombinant protein, as is shown in SEQ ID NO. 2. The IFN-alpha 3 recombinant protein has the advantages of being capable of being used for immersion immunity to prevent viral diseases of the siniperca chuatsi, effectively activating the siniperca chuatsi into an anti-virus state, and reducing mortality.

The invention discloses trichoderma viride alkali protease as well as an eukaryotic expression method and application thereof. The coding sequence of the trichoderma viride alkali protease is obtained through an RACE (Rapid Amplification of cDNA Ends) method, the length of an amino acid sequence is 410 amino acids, and the length of the coding sequence is 1233 nucleotides. The coding nucleotide sequence of the trichoderma viride alkali protease is constructed in an eukaryotic expression vector to perform eukaryotic expression, and trichoderma viride alkali protease capable of inhibiting growth of aflatoxin can be obtained. The trichoderma viride alkali protease has the function of inhibiting the growth of the aflatoxin.

The invention belongs to the technical field of molecular biology, in particular relates to a Portunus trituberculatus anti-lipopolysaccharide factor PtALF-3 gene and encoding proteins and application thereof. In the invention, a PtALF-3 gene cDNA is amplified from a Portunus trituberculatus by utilizing a cDNA (Complementary Deoxyribonucleic Acid) library and an RACE (Rapid-amplification of cDNA Ends) technology and plays an important role in immune defense of the Portunus trituberculatus. A recombinant PtALF-3 protein has obvious antibacterial activity to Gram-negative Vibrio alginolyticus, P. Aeruginosa, Edwardsiella tarda, Gram-positive Staphylococcus aureus, Micrococcus luteus and fungus Pichia pastoris, and the minimal inhibitory concentrations are respectively 0.58 mu M, 0.58 mu M, 9.26 mu M, 74.08 mu M, 18.52 mu M and 9.26 mu M. The invention lays a foundation for disease prevention and control, gene-assisted selection and development of feed additives of the Portunus trituberculatus.

The invention relates to the biotechnology field and in particular relates to a cloning and functional expression method of a gene AhAP2ER related to drought stress of peanuts. The gene AhAP2ER in drought stress response is synthesized through preparation and treatment of materials, extraction of RNA (ribonucleic acid), synthesis of cDNA (complementary deoxyribonucleic acid) and RACE (rapid-amplification of cDNA ends) cloning technology. Expression of the gene AhAP2ER under the condition of drought stress is also analyzed by using fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction), thus finally drawing a conclusion that the gene AhAP2ER plays an important role in the adaptability of the peanuts to drought stress. The transgenic plants have obvious drought resistance characteristic compared with a control group by transferring the gene to the peanuts by transgenic means, indicating that the gene AhAP2ER can conduce to obviously improving the drought resistance of the peanuts.

The invention belongs to the technical field of molecular biology, in particular relates to a Portunus trituberculatus anti-lipopolysaccharide factor PtALF-2 gene and encoding proteins and application thereof. In the invention, a PtALF-2 gene cDNA is amplified from a Portunus trituberculatus by utilizing a cDNA (Complementary Deoxyribonucleic Acid) library and an RACE (Rapid-amplification of cDNA Ends) technology and plays an important role in immune defense of the Portunus trituberculatus. A recombinant PtALF-2 protein has obvious antibacterial activity to Gram-negative Vibrio alginolyticus, P. Aeruginosa, Edwardsiella tarda, Gram-positive Staphylococcus aureus and Micrococcus luteus, and the minimal inhibitory concentrations are respectively 1.02 mu M, 2.04 mu M, 4.07 mu M, 16.30 mu M and 65.19 mu M; but the recombinant PtALF-2 protein has no obvious inhibitory action to fungus Pichia pastoris. The invention lays a foundation for disease prevention and control, gene-assisted selection and development of feed additives of the Portunus trituberculatus.

The invention provides a method for extracting potato tuber total RNA (ribonucleic acid) by means of improved Trizol. The method is characterized by comprising the steps of adding phenol/chloroform/isoamyl alcohol (25:24:1) for performing primary extraction after extracting RNA by the regular Trizol (Invitrogen Company), then transferring liquid supernatant to a new tube, adding 0.7 time of isopropyl alcohol in comparison with the volume of the liquid supernatant and 0.2 time of 1mol/L sodium acetate in comparison with the total volume to precipitate RNA, respectively washing the obtained RNA once with 1mL of 3mol/L sodium acetate (pH=5.2) and 1mL of 75% alcohol, finally, drying, and dissolving with DEPC (diethylpyrocarbonate) water. Such method for extracting potato tuber total RNA by means of improved Trizol has high yield and high capacity; moreover, the obtained total RNA has excellent quality and high purity, and can meet the requirements of molecular biology researches, such as inverse transcription, Northern hybridization, gene in vitro translation, cDNA (complementary deoxyribonucleic acid) library construction and rapid amplification of cDNA ends.

The invention discloses a sugarcane sucrose transport protein SoERD6 (early responsive to dehydration) gene and an encoding protein sequence thereof. On the basis of performing comparative analysis on a full-length sequence of all plant sugarcane sucrose transport protein SoERD6 genes, a primer is designed at a conserved region of the plant sugarcane sucrose transport protein SoERD6 gene, the total RNA (Ribonucleic Acid) is extracted from sugarcane puerile leaves, subjected to inverse transcription, amplified by normal PCR (polymerase chain reaction), and cloned to the full-length cDNA sequence of the sugarcane sucrose transport protein SoERD6 genes by combining an RACE (rapid-amplification of cDNA ends) PCR technique. With the adoption of the gene, not only is the basis provided for researching the transcription and expression mechanism of the sugarcane sucrose transport protein SoERD6 gene and further studying the transportation of sucrose in a sugarcane body and an accumulation mechanism in a sugarcane stem, but also a purified protein with biological activity can be obtained via an amino acid sequence; and the sugarcane sucrose transport protein SoERD6 gene is used for studying the biological function of the sugarcane sucrose transport protein and improving sugarcane high-sugar variety.

The invention relates to a branchiostoma belcheri chitin-binding associated serine protease CASP gene for identifying chitin, a protein coded by the gene and an expression method and application of the protein. The CASP gene is aN MASP (Mbl Associated Serine Protease) similar gene obtained by cloning from a total RNA (Ribose Nucleic Acid) of branchiostoma belcheri by a method of combining primer amplification and RACE (rapid-amplification of cDNA ends) amplification. The protein coded by the CASP gene is expressed in an intracellular soluble form in escherichia coli by a recombinant expression vector PET-32a(+)-CASP. The protein coded by the CASP gene takes an important effect of resisting to foreign pathogen and has the development value of becoming a high-efficiency natural antibacterial medicament.

The invention relates to a tea tree cytoplasm-type glutamine synthetase (GS) gene and an encoding protein thereof. The tea tree cytoplasm-type GS gene is obtained by taking a tea tree (Dragon Well tea 43) total complementary deoxyribonucleic acid (cDNA) as a template through polymerase chain reaction (PCR) amplification reaction. In the tea tree cytoplasm-type GS gene, part of homologous sequence published in national center of biotechnology information (NCBI) is utilized to design degenerate primer amplification to obtain an intermediate conserved sequence, and then, sequences of both ends are obtained through rapid amplification of cDNA Ends reaction; and the sequences of both ends are spliced through DNAman software to finally obtain the full-long sequence of tea tree cytoplasm glutamine synthetase, and the complete sequence analysis on the encoding zone of the gene is completed.

The present invention relates to conventional grape crossbreeding, molecular marker assisted breeding, and gene location and mapping method and technology. By means of resistant geneanalogus (RGAs), the present invention obtains 26 black spot disease resisting candidate gene DNA segments of Chinese V. quinquangularis Read and these segments are cloned and sequenced and have size of 492-550 bp. Black spot disease resisting marker assisted breeding is performed based on these sequences as the probes for detecting black spot disease resisting gene. Black spot disease resisting gene is located and mapped based on the distribution of these segments in the hybrid progeny of Chinese V. quinquangularis Read and European V. Or, specific primer is designed, and the technology of rapid amplification of cDNA ends (RACE) is adopted in cloning the black spot disease resisting gene of Chinese V. quinquangularis Read, so as to improve grape variety in gene engineering technology.

The invention belongs to the technical field of molecular biology, and in particular relates to a Portunus trituberculatus anti-lipopolysaccharide factor PtALF-1 gene, a protein encoded by the gene and an application of the protein. A cDNA (complementary Deoxyribose Nucleic Acid) library and a RACE (Rapid Amplification of cDNA Ends) technology are utilized for amplification to obtain PtALF-1 gene cDNA from the Portunus trituberculatus. The gene performs an important action on the immunologic defense aspect of the Portunus trituberculatus. The recombinant PtALF-1 protein has obvious Gram-negative bacterium and gram-positive bacterium bacteriostatic activity, has the minimum inhibitory concentration on Vibrio alginolyticus, pseudomonas aeruginosa, Staphylococcus aureus and the Micrococcus luteus of respectively 2.33mu M, 18.64mu M, 18.64mu M and 18.64mu M, and does not have obvious inhibition action on fungus Pichia pastoris. The invention lays the foundation on prevention and control of diseases and insect pests, gene-assisted breeding and development of feed additive of Portunus trituberculatus.

The invention discloses a method of the in-vitro expression of a gekko japonicus Hoxc10 (homebox gene c10) protein and the preparation of a polyclonal antibody, comprising the following steps of: obtaining an EST (Expressed Sequence Tag) sequence of gekko japonicus Hoxc10; obtaining an overall length sequence of the gekko japonicus Hoxc10 through an RACE (rapid amplification of cDNA ends) method; predicting an antigenic epitope of the gekko japonicus Hoxc10 protein on line by adopting DNASTAR software and SWISS-PLOT; constructing a prokaryotic expression vector of the gekko japonicus Hoxc10 by selecting superior epitope peptides; inducing the expression of fusion proteins in vitro; purifying the fusion proteins; preparing the polyclonal antibody, and the like. In the invention, an in-vitro gekko japonicus Hoxc10 expression system constructed on the basis of pGEX-4T1 can efficiently express a target protein in BL21 and further purify the target protein; and a large quantity of GST-Hoxc10 fusion proteins can be conveniently obtained; in addition, a rabbit gekko japonicus Hoxc10 antiserum prepared by immunizing a New Zealand rabbit through the GST-Hoxc10 fusion proteins has high titer and good specificity.

The invention discloses a 3'RACE (rapid amplification of cDNA ends) method for acquiring a complete 3' end sequence as a 3' end having a repetitive sequence. The method comprises the following steps: 1) tailing mRNA with terminal transferase, so that a connector sequence is added: tailing the mRNA with dCTP or dGTP, so that a single-base repetitive sequence of 16-22bp is added; 2) synthesizing first chain cDNA: conducting reverse transcription on the tailed mRNA by taking an anchor primer having oligodG or oligodC as a connector sequence; and 3) conducting 3'RACE amplification: with the cDNA having the connector sequence as a template, an internal specific primer GSP2 of the template which is designed in accordance with a RACE primer design principle as well as a corresponding nested primer NGSP2 thereof as forward primers of the amplification, and a complementary sequence (AUAP) of the connector sequence as a reverse amplification primer, conducting 3'RACE on the cDNA sequence having the connector. With the application of the 3'RACE method provided by the invention, the problem that a complete end sequence cannot be acquired in 3'RACE due to a special structure (having the A repetitive sequence) of the sequence can be solved.

The invention discloses a method for promoting gene cloning efficiency, and relates to a gene cloning method. The method comprises the following steps of: designing one pair of specific primers at an intermediate fragment of an object gene; carrying out specific PCR (polymerase chain reaction) amplification on templates of different sources in an RACE (Rapid Amplification of cDNA Ends) process; subjecting an amplification product to the electrophoretic analysis of 1.5% agarose, detecting whether a positive band of the product exists or not and the brightness of the positive band, so as to determine the level of the copy number of the object gene in an RACE to-be-amplified template, and determining the efficiency of each enzymatic reaction step in the RACE process to obtain the most proper template used for 5' RACE amplification, so that the purpose of improving the amplification mission success rate of a target gene is achieved. By using the method, the most proper template for the RACE amplification and a condition are efficiently selected by quickly and sensitively detecting the effective copy numbers of the object genes in the templates of the different sources.

The invention discloses leukocyte chemoattractant genes 2 of a grass carp. By using an EST (Expressed Sequence Tag) sequence of the leukocyte chemoattractant genes 2 in a grass carp intestinal tract cDNA (complementary DNA) library saved in a laboratory as a template design primer and combining with an RACE (Rapid-Amplification of cDNA Ends) technique, the full expression sequence of the leukocyte chemoattractant genes 2 in grass carp intestinal tract cells is obtained by amplification and cloning in a PCR (Polymerase Chain Reaction) method. The invention provides important theoretical basis for the research on the structural composition of the leukocyte chemoattractant genes 2 of the grass carp, the discussion on the action and the functional zone position of the leukocyte chemoattractant genes 2 in the disease resistance of the grass carp and the difference from other species. The invention has a pioneering action on a method for exploring and establishing a link between grass carp genome planning and functional feed industrialization.

The invention relates to a rose skin thorn regulation and control gene RrCPC and application thereof, and the RrCPC gene is cloned by taking purple-branch rose as a material through RACE (Rapid Amplification of cDNA Ends) technology. The expression mode of the RrCPC gene on different levels of lateral branches of roses is detected through a real-time fluorescent quantitative detection technology, meanwhile, arabidopsis thaliana is transformed by constructing an overexpression vector, it is proved that the RrCPC gene is a negative regulatory factor for epidermal hair generation, and the RrCPC gene has important application value for cultivating few-thorn or thorn-free rose new germplasm and improving rose management efficiency. The invention provides a rose skin thorn regulation gene RrCPC and application thereof in few-thorn and thorn-free roses, and the rose skin thorn regulation gene RrCPC has important application value in the field of thorn-free rose breeding.