178 results about "Vector (molecular biology)" patented technology

The invention relates to a cell model obtained after targeted knockout of a rabbit BMP2 gene based on CRISPR/Cas9 and application thereof, belonging to the technical field of molecular biology and biomedicine. According to the invention corresponding oligos are synthesized for three targeting sites of the rabbit BMP2 gene on the design principles of CRISPR/Cas9 and are constructed on px458 vectors; and a CRISPR/Cas9 system is constructed directed at the three targeting sites in rabbit mesenchymal stem cells, and the CRISPR/Cas9 system can effectively knock out the rabbit BMP2 gene, is easy to operate and has high rabbit BMP2 gene knockout efficiency. The cell model disclosed in the invention can greatly promote research related to the functions and signaling pathways of the BMP2 gene.

The present invention relates generally to the field of molecular biology and concerns a method for enhancing various economically important yield-related traits in plants. More specifically, the present invention concerns a method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding an ASPAT (Asparatate AminoTransferase) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding an ASPAT polypeptide, which plants have enhanced yield-related traits relative to control plants. The invention also provides hitherto unknown ASPAT-encoding nucleic acids and constructs comprising the same, useful in performing the methods of the invention. Furthermore, the present invention relates generally to the field of molecular biology and concerns a method for increasing various plant yield-related traits by increasing expression in a plant of a nucleic acid sequence encoding a MYB91 like transcription factor (MYB91 ) polypeptide. The present invention also concerns plants having increased expression of a nucleic acid sequence encoding an MYB91 polypeptide, which plants have increased yield- related traits relative to control plants. The invention additionally relates to nucleic acid sequences, nucleic acid constructs, vectors and plants containing said nucleic acid sequences. Even furthermore, the present invention relates generally to the field of molecular biology and concerns a method for improving various plant growth characteristics by modulating expression in a plant of a nucleic acid encoding a GASA (Gibberellic Acid-Stimulated Arabidopsis). The present invention also concerns plants having modulated expression of a nucleic acid encoding a GASA, which plants have improved growth characteristics relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention. Yet furthermore, the present invention relates generally to the field of molecular biology and concerns a method for enhancing various economically important yield-related traits in plants. More specifically, the present invention concerns a method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding an AUX/IAA (auxin/indoleacetic acid) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding IAA polypeptide, which plants have enhanced yield-related traits relative to control plants. The invention also provides constructs comprising AUX/IAA-encoding nucleic acids, useful in performing the methods of the invention.

The invention relates to a novel gene sequence of a recombinant chicken Alpha interferon, constructs a recombinant expression vector thereof and belongs to a gene engineering biological product obtained by a molecular biology method. The gene sequence of a newly designed chicken Alpha interferon is recombined into a pPICZ Alpha-A vector and then is confirmed on the special position of a microzyme by an electric conversion mode. Besides, a pichia expression system is adopted to express a foreign gene, thus being beneficial to the commercial production of the chicken interferon. The protein expressed by a gene group after being diluted by 4365158.3 times can completely restrain the attraction of vesicular stomatitis virus of 100-1000TCID50. Compared with a natural chicken Alpha interferon, the novel gene sequence of a recombinant chicken Alpha interferon has a higher anti-virus effect; the anti-virus effect thereof is improved by 8 times. Test also detects that the protein expressed by the gene can restrain the proliferation of a newcastle disease virus and an avian influenza virus; besides, the effect of the interferon with high concentration is more remarkable.

The invention discloses a chrysanthemum stress-resistance transcription factor CmMYB2 as well as a plant expression vector, a construction method and an application thereof, belonging to the field of molecular biology. The expression vector pCAMBIA1301-CmMYB2 is constructed with the construction method which comprises the following steps: inserting the CmMYB2 gene into a colon vector pMD19-Tsimplevector (Takara); and after carrying out enzyme digestion on the CmMYB2 gene by BamHI (Takara) and SacI (Takara), connecting to the BamHI locus and SacI locus of an expression vector pCAMBIA1301. The plant expression vector is used for the genetic transformation of plants, the CmMYB2 gene is subjected to over expression under the starting of CaMV35S to synthesize a great quantity of MYB2 proteins, regulate the expression of a downstream target gene and improve plant stress resistance.

The invention discloses lncRNA lnc12 and application thereof in regulation of development of adventitious roots of poplar, and relates to the technical field of plant molecular biology. The lncRNA lnc12 is obtained through cloning by an RACE technology, a cDNA total-length sequence of the lncRNA lnc12 is shown in SEQ ID NO:1, and one small open reading frame with length of 159 bp exists. Construction of 35S::lnc12 sORF-GFP and 35S::lnc12 5'UTR+sORF-GFP vectors and transient expression verification in protoplasts of poplar find that the lnc12 has no encoding capacity. Through lnc12 overexpression vector constructon, stable inheritance conversion of poplar and positive plant screening, lnc12 overexpression transgenic poplar is obtained, the micro-cutting rooting time of the poplar is obviously delayed, the rooting rate is significantly reduced, and the number of adventitious roots and root hair thereof is smaller than that of control plants.

The invention provides a wheat TaARF12 gene and application thereof, and belongs to the field of molecular biology of crops. A designed primer clones a coding region sequence of TaARF12 from cDNA of Chinese spring young ears, and the coding region sequence of the TaARF12 gene in homologous chromosomes A, B and D is represented as SEQ ID NO. 1-3 respectively. A TaARF12-specific RNAi fragment is selected, an RNAi vector is created, and Fierder is transformed. A CRISPR/Cas9 vector is used to create a gene editing vector containing two target sites and transform wild-type Fierder. Obtained RANi transgenic lines and gene editing lines have similar phenotypes, with plants becoming shorter and ears becoming longer. It is indicated that the wheat TaARF12 gene can regulate the plant height and theear length, and the plants become shorter and the ears become longer after the gene is interfered and expressed.

The invention relates to general expression vector pSK-E-P/T construction for plant mitochondria and promoter activity identification. Cucumber mitochondria plasmids pC3 (sequence 1) are discovered for the first time, a gene cob upstream promoter sequence (sequence 2) and a gene atp9 downstream terminator sequence (sequence 3) of the cucumber mitochondria obtained through PCR (polymerase chain reaction) are constructed into a general expression vector pSK-E-P/T (sequence 4) of the plant mitochondria, and the vector also carries replicons copied in escherichia coli and Ampr resistance genes so as to facilitate cloning of exogenous genes and recon screening. Measurement of enzyme activity of promoter -1acZ report gene fusion plasmids in the escherichia coli and fluorescence microscope observation of psk-E-GFP for transforming the green fluorescence of the escherichia coli prove that the promoter on the expression vector has transcriptional activity. The invention provides a novel molecular biology technology tool for researching important life science hotspot problems such as cell apoptosis and the like related with the mitochondria by a mitochondria transformation path.

The present invention provides an E.tenella TERT associated protein gene, and particularly relates to applications of the protein in telomerase activity regulation, wherein the protein has a negative regulation effect on the E.tenella telomerase activity, and develops the new research field for deep research on cell and molecular biology characteristics of coccidian and finding of new chicken coccidian preventing and treating drugs, vaccine acting target sites and the like. According to the present invention, the advantages of high efficiency and practicality are provided, a plurality of positive interaction proteins can be obtained through the yeast two-hybrid screening and verification so as to easily find the new function protein, and the method of the present invention is mature and is easy to operate compared with the comparative analysis, the proteomics, the transcriptomics, and other technologies; and the E.tenella virus transforming vector-mediated ribozyme provides the simple and easy-performing method for the research on the functions of the gene in the insect body.

The invention discloses a porcine reproductive and respiratory syndrome virus with a preservation number of CCTCC NO:V 202020. Accordingly, the inventor constructs virus genome full-length infectiousclone pGXAM and establishes a corresponding construction method and a gene insertion method. According to a vector, Gxnn1396-P96 is used as a female parent, and Gxnn1396-P96 is used as a male parent,a PRRSV full-length infectious clone vector is constructed by means of molecular biology, an independent transcription regulating sequence (TRS) is inserted into ORF4 and ORF5a on the basis of cDNA cloning, and the PRRSV recombinant virus for expressing a green fluorescent protein (GFP) gene is constructed. The successful construction of the fluorescence labeled virus lays a foundation for the development of recombinant porcine reproductive and respiratory syndrome virus genetic engineering vaccines.

The invention relates to an Escherichia coli protein expression vector pLS1128 using a trigger factor originated from Escherichia coli as a fusion tag. The vector is obtained by cloning nucleotide sequences of six coded histidines, a trigger factor gene, a base sequence of a cleavage site of coded tobacco etch virus protease and a filling segment for cloning to an Escherichia coli expression vector pET30a. After a target gene is cloned to the downstream of the trigger factor gene by a gene cloning means, the target gene and the trigger factor gene are inducted to express fusion protein; the trigger factor functions as chaperone; and the target protein is promoted to be folded correctly so as to show a dissoluble state. The vector of the invention can be widely applied to expression on and the dissolubility of the target protein, research of genetics, molecular biology, biochemistry and the like and industrial production of the target protein.

The invention belongs to the technical field of molecular biology and protein engineering, and discloses a recombinant polymerase, an encoding gene, a preparation method, a carrier, a kit and application; wherein the recombinant polymerase is derived from a hybrid DNA polymerase obtained by covalently connecting a protein Cl7 to the N end of a Taq DNA polymerase protein sequence in a Thermous aqueatus YT1 strain through a flexible connection sequence, and the amino acid sequence is shown as SEQ ID No.4; or the sequence is an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function. The expression vector pET30a-Cl7-Taq disclosed by the invention is used for converting escherichia coli and inducing expression to obtain a target protein Cl7-Taq. The continuous synthesis capability of TaqDNA polymerase in PCR nucleic acid amplification is greatly improved, and the TaqDNA polymerase has a wide application prospect.

The invention discloses a specificity overexpression mouse model of a transduction iASPPsv cancer gene in a hemopoietic system and a preparation method and the use thereof. The method comprises the following steps of: establishing an hM HS21/45vav-iASPPsv plasmid, removing a prokaryotic expression vector part after the plasmid is subjected to the Hind III enzyme digestion, and standing the product for low-voltage electrophoresis over night after the linearization followed by glue cutting, recovery, purification and nucleic acid concentration regulation. By the method of microinjection, the transgenic vector, hM HS21/45vav-iASPPsv, is injected to a zygote of a C57 mouse, and the zygote is implanted in a uterus of a pseudopregnancy mouse. 19 to 21 days after the operation, a new mouse is born and subjected to ear cutting, numbering and tail cutting after 3 weeks. DNA is extracted from the tail to detect the integration of the target gene, iASPPsv, by using PCR. The specificity overexpression mouse model of the transduction iASPPsv cancer gene in hemopoietic system is obtained by using molecular biology, experimental zoology and the like to successfully establish an experimental method for detecting the amplification, oriented differentiation, migration, apoptosis and the like of hematopoietic stem/progenitor cells and lay a foundation for the mechanism research and targeted therapy for further explaining the transformation of normal hematopoietic stem/progenitor cells to tumor cells.

The invention belongs to the field of crops, and particularly relates to genetic transformation, gene editing and analysis methods of main soybean varieties. The invention discloses a gene editing andtransforming vector which is based on a CRISPR/Cas9 system and takes an agrobacterium transforming vector pCambia3301 as a skeleton, and a promoter is designed for improving expression of Cas9 and gRNA. According to the invention, rapid and effective genetic transformation of the main soybean varieties is realized for the first time, and a simple and rapid PCR amplification sequencing analysis method for identifying the gene editing effect is developed. Through genetic separation and screening by molecular biology analysis technology, gene editing offspring plants which do not carry exogenousgenes of the soybean varieties for production are successfully obtained, and editing sites are stably inherited.

The present invention provides a method for exogenous gene expression optimization in salmonella. The method comprises the following specific steps: carrying out statistics from a NCBI database to obtain a salmonella codon usage frequency table; analyzing usage frequency of every codon; cloning or synthesizing a target gene, determining a nucleotide sequence of the target gene, adopting a molecular mutation manner or a gene synthesis manner to carry out mutation of the codon having a lowest salmonella codon usage frequency and the codon having a higher salmonella codon usage frequency in the target gene into the codon having a highest usage frequency under a condition of no change of amino acids according to the salmonella codon usage frequency table and an amino acid codon corresponding table to obtain a new nucleotide segment encoding the target gene; adopting a molecular biology method to insert the new nucleotide segment encoding the target gene into an expression vector, transforming the obtained vector to gene engineering escherichia coli, and identifying the obtained clones; and transforming the target gene-containing expression vector into salmonella, wherein the target gene-containing expression vector is identified, and a correct result is obtained.

The invention relates to the technical fields of gene engineering, molecular biology and genetic breeding science, and particularly relates to a wheat salt-tolerant gene AAP3 and application thereof.The invention discloses a genome sequence of an amino acid permease gene TaAAP3 and an expression mode of the gene under the salt stress. An overexpression vector containing the gene TaAAP3 and a wheat transgenic plant for overexpression of the gene are obtained. Experiments prove that according to the invention, the salt tolerance of transgenic wheat is remarkably higher than that of a control (non-transgenic wheat), and the wheat salt-tolerant gene AAP3 can be used for salt-tolerant breeding of wheat and has huge application value.

The invention belongs to the fields of molecular biology and cytobiology, and specifically relates to a design and an application for a lentiviral expression vector. According to the vector designed by the invention, a CMV enhancer and a CMV promoter are used as transcription starting elements of an insertion gene; an EF1a-core promoter is adopted as a transcription starting element for screeningand marking puromycin and green fluorescent protein (GFP), and a puromycin resistance gene and a GFP sequence are connected and expressed in series through T2A; a lentiviral vector is adopted as a vector skeleton, and two above-mentioned elements are inserted into the skeleton vector; thus, the lentiviral expression vector is obtained by reasonable design and optimization. By adoption of the lentiviral vector designed by the invention, the inserted gene can be efficiently expressed, and the expression efficiency of the lentiviral expression vector is superior to the expression efficiency of conventional vectors with the same type of characteristics.

The invention discloses an efficiency and rapid transgenic method for chrysanthemum, belonging to the fields of genetic breeding of the chrysanthemum and molecular biology. The method comprises the following steps: (1) selecting a chrysanthemum stem as an explant, and carrying out pre-culturing on a pre-culturing medium; (2) activating and culturing recombinant expression vector transformed agrobacterium liquid, enriching thalli, and carrying out resuspension by virtue of an MS liquid culture medium, so as to obtain infection liquid; (3) soaking the explant pre-cultured in the step (1) into the infection liquid, and carrying out infection by virtue of a vacuum negative pressure method; and (4) putting the infected explant onto the pre-culturing medium, carrying out co-culturing in a dark condition, carrying out decarboxylation culture on a decarboxylation culture medium, finally, sequentially carrying out selective culture by virtue of a selective culture medium 1 and a selective culture medium 2, when determining that agrobacterium does not outbreak, carrying out subculture on a root culture medium, and carrying out further rooting screening, so as to obtain genetic chrysanthemumplants. Compared with a traditional leaf-disc-method transgenic method for the chrysanthemum, the method has the advantages that the transgenosis time is remarkably shortened, the conversion efficiency of transgenosis of the chrysanthemum is increaed, the workload of science researchers is reduced, a new way is provided for the efficient conversion and gene function analysis of the chrysanthemum,and a new thought is provided for the transgenosis of other plants.

The invention relates to the field of molecular biology, in particular to a drug inducible CRISPR/Cas9 system for gene transcription activation. The drug inducible CRISPR/Cas9 system comprises 16-22ntsgRNA targeting a specific gene site and any one of vector/vector combination as (1)-(3) below: (1) a vector expressing fusion protein dCas9-(ERT2)n-ADs or dCas9-ADs-(ERT2)n; (2) a vector combinationexpressing fusion protein dCas9-(NLS) m or dCas9-(ERT2)n and MCP-(ERT2)n-ADs; (iii) a vector combination expressing fusion protein dCas9-(NLS)m-(GCN4)p and scFv-(ERT2)n-ADs. Compared with the prior art, the system of the invention can more effectively realize the regulation of drug-induced gene transcription activation, and has more flexible and convenient operation and lower background activity.It is helpful to control the dynamic biological process effectively and precisely, and has great value for the research and development of biomedicine.

The invention provides a chrysanthemum anti-retroelement DgZFP1, a plant expression vector thereof, a construction method thereof and application thereof, belonging to the field of molecular biology. The expression vector pCAMBIA1301-DgZFP1 constructed by the invention is a recombinant plasmid which is obtained by the means that a DgZFP1 gene is inserted into a cloning vector pMD19-T simple vector (Takara), is performed by means of double digestion through BamH1 (Takara) and KpnI (Takara), and is connected with the BamH1 site and the KpnI site of the vector pCAMBIA1301 (Invitrogen). The plant expression vector is used for the genetic transformation of the plant, and the DgZFP1 gene is excessively expressed under the promotion of a CaMV35S promoter, so that a large number of the DgZFP1 protein is synthesized, the expression of the downstream gene is regulated and controlled, and the plant salt tolerance of the plant is improved.

The invention discloses siRNA capable of specially inhibiting expression of an LAMB1 gene, a recombinant vector of the siRNA and application of the siRNA in reversing ovarian cancer taxol resistance, and belongs to the technical field of molecular biology and biological medicine. The siRNA comprises a sense strand and an antisense strand, the sense strand is 5'-UGUUUGAAAGCCGAAUCUGCG-3', and the antisense strand is 5'-CAGAUUCGGCUUUCAAACAAA-3'. According to the siRNA, mRNA and protein expression of the LAMB1 gene in a cancer cell can be inhibited specially and efficiently, proliferation of the cancer cell is reduced, cell apoptosis is increased, the invasiveness and migration capability of the cancer cell is reduced, and moreover, the resistance of an ovarian cancer cell to taxol can be reversed effectively. The invention further provides the application of the siRNA and the recombinant vector thereof in preparing medicines treating an ovarian cancer, a hepatocellular cancer, a colorectal cancer, a malignant glioma, a prostatic cancer or a gastric cancer and medicines reversing the ovarian cancer taxol resistance.

The invention discloses siRNA [small interfering RNA(ribonucleic acid)] for specifically suppressing expression of PADI2 (protein-arginine deiminase type-2) genes, a recombinant vector with the siRNA and application of the siRNA to reversing taxol resistance of ovarian cancer, and belongs to the technical field of molecular biology and biomedicines. The siRNA comprises positive-sense strands 5'-AAUGUACAGAACUAUCUCGUA-3' and antisense strands 5'-CGAGAUAGUUCUGUACAUUUC-3'. The siRNA, the recombinant vector and the application have the advantages that expression of mRNA (messenger RNA) and proteins of the PADI2 genes can be specifically and efficiently suppressed by the siRNA, cell proliferation can be reduced, cell apoptosis can be increased, the migration and invasion capacity of cells can be deteriorated, and the taxol resistance of ovarian cancer cells can be effectively reversed; the PADI2 siRNA and the recombinant vector can be applied to preparing medicines for treating ovarian cancer, breast cancer, cervical squamous cell carcinoma, colon cancer, liver cancer and lung cancer or reversing medicine resistance of ovarian cancer.

The invention relates to a T1R3 gene overexpressed lentiviral vector and a T1R3 lentivirus, and construction methods thereof, and belongs to the technical field of molecular biology. By a gene recombination technology, construction is performed by taking a pLVX-Puro lentiviral expression vector as the basis, a T1R3 gene is integrated, and the T1R3 gene overexpressed lentiviral vector pLVX-Puro-T1R3 is obtained. Lentiviral packaging plasmids pRSV-Rev, pMDlg-pRRE and pMD2.G are used for packaging the T1R3 gene overexpressed lentiviral vector pLVX-Puro-T1R3, and HEK293 cells are transfected to obtain the T1R3 lentivirus. The constructed T1R3 gene overexpressed lentiviral vector has high transfection efficiency to host cells, and can specifically, continuously and efficiently promote stable expression of the T1R3 gene in the host cells.

The invention discloses a method for mediating RNAi (RNA interfere) by utilizing slow-virus vectors. The method comprises the following detailed steps of: firstly carrying out homologous analysis on the nucleic acid sequence of goose paramyxovirus NA-1 NP and M genes, finding conserved regions, and designing out siRNA on the conserved regions; cloning the siRNA to slow-virus expression plasmid pLKD-EGFP, then commonly transfecting the slow-virus expression plasmid pLKD-EGFP with pCMV-dR8.2 and pCMV-VSV-G in 293T cells, collecting transfected cell supernatant, forming high-titer slow-virus vectors expressing the siRNA by ultracentrifugation, purification and concentration, then carrying out transduction on the high-titer slow-virus vectors into target cells, and enabling the slow-virus vectors to be stably expressed in the cells and exert the antiviral effect. The method disclosed by the invention has the advantages that the antiviral effect of the siRNA in a body can be really evaluated due to high-efficiency transduction rate and strict control system, the basis of the molecular biology can be laid for disease-resistance breeding of poultry animal molecules, and simultaneously, a new path is provided for preventing and treating the animal-based virus diseases such as newcastle diseases.