95 results about "Single copy" patented technology

The invention relates to the technical field of microorganisms, in particular to recombinant pichia pastoris for expressing plastic degrading enzyme LCCICCG as well as a construction method and application of the recombinant pichia pastoris. According to the invention, pichia pastoris is taken as a host cell, the coding gene lccICCG of the plastic degrading enzyme is heterologously expressed, and single-copy or multi-copy recombinant pichia pastoris is constructed. By optimizing culture conditions, the expression quantity and biomass of the plastic degrading enzyme protein are remarkably improved; by optimizing a fermentation strategy, high-density fermentation production of the LCCICCG protein in a 5L fermentation tank is realized, and a technical basis is provided for realizing large-scale industrial production of the protein. The improvements not only improve the production efficiency and reduce the cost of industrial production, but also provide powerful support for commercial application of the plastic depolymerases.

The invention belongs to the technical field of living cell imaging, and discloses a living cell nucleic acid imaging system and method based on CRISPR (clustered regularly interspaced short palindromic repeats) and a nano antibody and application, the system comprises: a modified dCas protein expression vector containing n antigen polypeptide tags; the modified sgRNA and / or crRNA expression vector comprises a specific guide sequence; the fluorescent nano-antibody expression vector contains m fluorescent nano-antibody coding sequences which are connected in an operable manner, and fluorescent protein serving as an internal tag is inserted between nano-antibody splitting sites, can be combined with a polypeptide tag carried by dCas protein and can stably emit light; and the fluorescent nano antibody in a free state can be self-degraded, so that the fluorescent nano antibody does not emit fluorescence. According to the invention, the single-copy nucleic acid sequence, especially the imaging marker of the single-copy nucleic acid sequence in a living cell, can be visualized with single-molecule resolution and high signal-to-noise ratio.

The invention particularly relates to a saccharomyces cerevisiae genetically engineered bacterium for de novo synthesis of 7-DHC as well as a construction method and application of the saccharomyces cerevisiae genetically engineered bacterium and belongs to the technical field of biology. According to the invention, a saccharomyces cerevisiae engineering bacterium SC9 is taken as an original strain, multiple times of multiple copies of tHMG1, DHCR24 and ERG1 are carried out on TY transposon, multiple copies of ERG2, POS5, ERG7, ERG11, IDI1 and ERG3 are carried out on TY transposon, and POS5, ERG3 and DHCR24 are singly copied on a genome, so that the content of intracellular reducing coenzyme II is increased, and a main path for synthesizing 7-DHC is enhanced. The genome single copy ACL, ACS1, ERG10, ERG19 and the like are used for enhancing the utilization of saccharomyces cerevisiae ethanol and dynamically regulating and controlling the synthesis of 7-DHC. The saccharomyces cerevisiae gene engineering strain constructed by the invention has high product accumulation, and lays a foundation for replacing a traditional multi-step chemical synthesis route and realizing industrial production.

The invention discloses a system for detecting viral nucleic acid on the basis of a CRISPR (clustered regularly spaced short palindromic repeat) technology and an FET (field effect transistor) chip and application of the system, and relates to a method for detecting the viral nucleic acid on the basis of clustered CRISPR (clustered regularly spaced short palindromic repeat) and related proteins (Cas) and a field effect transistor (FET). The CRISPR-FET (clustered regularly interspaced short palindromic repeats-field effect transistor) detection system comprises a non-amplified CRISPR system, a reporter RNA (ribonucleic acid) and gold nanoparticles (AuNPs) connection system, a separation system and a field effect transistor chip detection system. The CRISPR-FET detection method provided by the invention has high sensitivity and high specificity on virus nucleic acid detection, and the lower limit of detection reaches a single copy (100 copies / [mu] L).

The invention belongs to the technical field of gene engineering, and relates to a method for efficiently integrating a target gene into a pichia pastoris genome in a multi-copy manner, which comprises the following steps: S1, constructing a pPIC9K recombinant plasmid containing a single-copy target gene or a pPICZ alpha Amut + recombinant plasmid containing a multi-copy target gene; s2, constructing and integrating a pichia pastoris strain containing the multi-copy target gene by utilizing the pPIC9K recombinant plasmid containing the single-copy target gene or the pPICZ alpha Amut + recombinant plasmid containing the multi-copy target gene; and S3, electrically transforming the recombinant plasmid constructed in the S1 into the pichia pastoris strain integrated with the multi-copy target gene constructed in the S2, and carrying out resistance screening. According to the invention, the contradiction between copy number and screening pressure in the traditional gene integration technology is broken through, and an engineering bacterium construction scheme with higher economical efficiency and stability is provided for industrial production of recombinant protein with high expression quantity.

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to sheep fecundity and application of the SNP molecular marker. The SNP molecular marker comprises an SNP1 (Single Nucleotide Polymorphism) and / or an SNP2; the SNP1 is located at a base at the 115747587 site of a chromosome 6 of a sheep reference genome AR-UIRamV2.0 version, and the polymorphism of the SNP1 is T / G; the SNP2 is located at a basic group at the 41419566th site of a chromosome 22 of a sheep reference genome AR-UIRamV2.0 version, and the polymorphism of the SNP2 is A / G. According to the scheme, the SNP molecular marker related to sheep fecundity is provided, sheep with the high fecundity character can be efficiently and accurately identified and screened out, and the marker is high in typing quality, single in copy and high in polymorphism and can be used for sheep fecundity molecular marker assisted breeding.

The invention belongs to the technical field of molecular biology, and particularly relates to a single-copy nuclear gene marker of elaeagnus mollis, a primer pair and application of the single-copy nuclear gene marker. The elaeagnus mollis single-copy nuclear gene marker comprises any one or more of elaeagnus mollis single-copy nuclear gene markers 1, 2, 3, 4, 5, 6 and 7; the nucleotide sequences of the single-copy nuclear gene marker of the elaeagnus mollis are sequentially as shown in SEQ ID NO.1 to SEQ ID NO.7. The invention further discloses a preparation method of the single-copy nuclear gene marker. The single-copy nuclear gene marker primer pair of elaeagnus mollis provided by the invention has very high universality, amplification sequencing can be performed in selected elaeagnus mollis individuals of different provenances, a genetic diversity index analysis result is good, and all individuals of different provenances can be obviously distinguished according to a genetic structure analysis result; the method is suitable for genetic diversity analysis of large-scale germplasm resources of elaeagnus mollis.

The invention relates to the technical field of biology, and particularly discloses a kit for detecting haemophilus influenzae based on PCR (Polymerase Chain Reaction) / RAA-CRISPR-Cas13a. The invention discloses a kit for detecting a haemophilus influenzae gene on the basis of PCR (Polymerase Chain Reaction) / RAA-CRISPR-Cas13a. The kit comprises a primer pair for specifically amplifying haemophilus influenzae genes and crRNA, the sequence of the crRNA is composed of an anchoring sequence used for being combined with Cas13a protein and a guide sequence of a target sequence of the haemophilus influenzae genes, and the guide sequence is shown as the 39-66th site of SEQ ID No.28. The invention constructs a method for detecting haemophilus influenzae genes by combining a PCR or RAA technology with a CRISPR-Cas13a technology, the method is simple, convenient and rapid, has higher sensitivity and specificity, can reach a single copy level in sensitivity, and can be used for clinically and rapidly identifying whether the haemophilus influenzae is infected or not.

The invention discloses a recombinant saccharomyces cerevisiae engineering bacterium for producing 7-dehydrocholesterol and application, and belongs to the technical field of genetic engineering and biological engineering. On the basis of an original strain, an exogenous gene DHCR24 and key endogenous genes ERG2 and ERG3 are integrated, and a non-path gene ROX1 is knocked out, so that the accumulation of 7-dehydrocholesterol in a saccharomyces cerevisiae body is further improved; the accumulation amount of the 7-dehydrocholesterol is further increased by adding integration of a speed limiting step gene and cell growth promoting genes SET2 and HAP4 and knockout of an RPH1 gene, and the ability of synthesizing the 7-dehydrocholesterol from the beginning in a saccharomyces cerevisiae body is further improved by completing single-copy integration of TPS1, ERG25, ERG26 and ERG27 genes at a saccharomyces cerevisiae GAL80 site. And the yield of 7-dehydrocholesterol reaches 896.2 mg / L after the constructed strain is subjected to shake flask fermentation for 100 hours.

This invention specifically relates to a de novo 7-DHC-synthesizing *Saccharomyces cerevisiae* genetically engineered strain, its construction method, and its applications, belonging to the field of biotechnology. Using *Saccharomyces cerevisiae* engineered strain SC9 as the starting strain, this invention utilizes multiple copies of tHMG1, DHCR24, and ERG1 via the TY transposon, multiple copies of ERG2, POS5, ERG7, ERG11, IDI1, and ERG3 via the TY transposon, and single copies of POS5, ERG3, and DHCR24 in the genome to increase the intracellular content of reducing coenzyme II and enhance the main pathway of 7-DHC synthesis. Single copies of ACL, ACS1, ERG10, and ERG19 in the genome enhance the utilization of ethanol in *Saccharomyces cerevisiae* and dynamically regulate the synthesis of 7-DHC. The *Saccharomyces cerevisiae* genetically engineered strain constructed by this invention exhibits high product accumulation, laying the foundation for replacing traditional multi-step chemical synthesis routes and realizing industrial production.

The invention discloses a mustard SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof. The mustard SNP molecular marker combination is composed of 1694 SNP molecular markers, and the physical positions of the 1694 SNP molecular markers are determined based on sequence alignment of a mustard reference genome Bju.gene. According to the scheme, the mustard SNP molecular marker combination marker is a co-dominant marker, and has the characteristics of high quality, single copy, high polymorphism, high specificity, high sensitivity and high resolution; the marking is not influenced by environmental conditions, seeds or any type of plant tissues can be used, the detection result is accurate, and the repeatability and the stability are good; different detection laboratories and different data results can be compared and verified, and the data has universal comparability, can be accurately used for background detection of mustard breeding materials, and has wide application universality.

The present invention discloses a liquid phase chip of wheat 2K+fusarium head blight resistance loci and its application, wherein the genotyping objects of the liquid phase chip include 2274 single copy SNP loci located in 100 high confidence QTL intervals of fusarium head blight resistance in the wheat reference genome, and the position information of the 2274 SNP loci on the wheat reference genome is shown in Table 2 of the specification. The liquid phase chip of wheat 2K+fusarium head blight resistance loci of the present invention improves the coverage and accuracy of QTL in the study of fusarium head blight resistance, improves the throughput and efficiency of marker-assisted selection, and reduces the cost of genotype analysis.

The invention discloses a method for constructing a transgenic zebrafish model of hypoxia response. The method comprises the following steps: firstly assembling an sv40mp promoter after a hypoxia response element HRE sequence and cloning the promoter into a pT2AL200R150G plasmid carrying green fluorescent protein (GFP), then microinjecting the recombinant plasmid and transposase mRNA into zebrafish embryos, and inserting the recombinant sequence into the genome of F0 generation zebrafish by Tol2 transposon technology; raising the F0 generation fish to adult fish, hybridizing with wild-type zebrafish to produce F1 generation, hybridizing the F1 generation with the wild-type fish again to produce single-copy transgenic zebrafish F2 generation, self-pollinating male and female sibling zebrafish of the F2 generation, and screening out transgenic zebrafish Tg (HRE-sv40mp:GFP) of hypoxia response of F3 generation that can be stably inherited. The transgenic zebrafish Tg(HRE‑sv40mp:GFP) constructed in the present invention can be used as a simple and efficient hypoxia response model for biological research in hypoxia-related fields, as well as for screening molecular targeted drugs for HIF‑1α, providing a living animal screening model for the development of anti-tumor drugs.

The invention discloses a method for identifying pine wood nematode cellulase multi-copy genes, which comprises the following steps: firstly, collecting pine wood nematode strains from different geographic positions, extracting DNA (deoxyribonucleic acid) of the pine wood nematode strains, and performing high-throughput sequencing; the method comprises the following steps: analyzing whole genome information by using bioinformatics software to obtain CNV (Copy Number Variation) information and tandem repeat sequences of the whole genome information, screening out multi-copy genes, namely Glycode hydrolases 45 (GH45) genes related to pine wood nematode pathopoiesis by combining functions of the whole genome information and the tandem repeat sequences, and comparing the GH45 genes of different copies to find that tandem repeat variation exists in a coding region. A proper primer is designed in a TR flanking sequence, different copy numbers of different TR combinations can be identified by a traditional PCR (Polymerase Chain Reaction) method, and most of single-copy insect strains can be found from Guangdong province through PCR and electrophoresis gel, so that a certain theoretical basis and data support are provided for researching bursaphelenchus xylophilus pathogenic related multi-copy genes; and a new research direction is provided for a pine wood nematode molecular marker technology.

The invention discloses a probe, a method for efficiently recognizing licorice chromosomes and application of the probe, and belongs to the field of bioinformatics and molecular cytogenetics. According to the invention, a single copy oligonucleotide sequence of No. 1-8 chromosomes of liquorice is screened by using bioinformatics, and a chromosome specific oligonucleotide probe pool is constructed through an artificially designed universal primer sequence tag splicing strategy. Through multiple rounds of FISH technology, the chromosomes 1-8 of liquorice can be efficiently and accurately identified. The method provided by the invention solves the problem of difficulty in accurate identification caused by tiny morphology and high similarity of the glycyrrhiza chromosome, has the advantages of high resolution, strong specificity, capability of identifying a single chromosome and the like, and provides a powerful tool for follow-up glycyrrhiza chromosome karyotype analysis, evolution research and exploration of a species formation mechanism.

The invention provides a primer combination for identifying a single-copy gene of Australian cyst nematode. The primer combination comprises an upstream outer primer HAUS-F3, a downstream outer primer HAUS-B3, an upstream inner primer HAUS-FIP, a downstream inner primer HAUS-BIP and a downstream loop primer HAUS-LB. The primer combination can specifically distinguish the Australian cyst nematode from other common cyst nematodes such as cereal cyst nematode, Philipse cyst nematode, corn cyst nematode and soybean cyst nematode, the detection limit is low, the detection time is short, and the detection result is identified by means of dye color development or electrophoretogram. The technology is easy to operate, good in sensitivity, high in specificity and high in reaction speed, the result can be directly observed without additional instruments, and the method has important significance in the aspects of rapid detection and early field diagnosis of the Australian cyst nematode.

The invention provides a method and a device for evaluating annotation quality of a genome. The method comprises the following steps: performing single-copy orthologous gene integrity analysis, gene function annotation, expression quantity analysis and gene structure analysis on each gene in a genome to be subjected to quality evaluation to obtain an analysis result file; the analysis result file is subjected to quantitative scoring, the annotation quality of the genome to be subjected to quality evaluation is judged according to the scoring result, multi-dimensional quality evaluation can be carried out on each gene in the genome in four aspects of gene integrity, function annotation, expression quantity and gene structure, the annotation quality of the genome is judged more comprehensively and more accurately, and the quality evaluation efficiency is improved. The method is of great significance to reliability and applicability evaluation of genome data.

The invention provides application of an OG7443 gene of trichophyton in identification of trichophyton, a primer pair and an identification method of trichophyton, and relates to the technical field of biology. Based on comparative analysis of genomes of trichophyton and other fungi, the specific marker gene OG7443 which only exists in trichophyton, has single copy and is free of repetitive sequence interference is screened out, and the gene can be used for identification of trichophyton. The primer provided by the invention is designed aiming at the OG7443 gene of trichophyton, has strong specificity and high sensitivity, can accurately distinguish mixed florae possibly existing in a clinical sample, improves the diagnosis accuracy of fungal infection, not only can accelerate the determination of pathogenic bacterium types, but also can improve the diagnosis accuracy of fungal infection. And important technical support can be provided for targeted antifungal treatment and public health prevention and control.

The invention relates to a plasmid vector capable of easily detecting the copy number of plasmids and a detection kit thereof. According to the plasmid vector, a plasmid replicon ORI gene and a single copy conserved gene of an escherichia coli genome are constructed on the same plasmid. The detection kit contains the plasmid vector or a single-copy quality control product prepared from the plasmid vector. The method has the advantages of high detection result accuracy, high reproducibility and simple detection process, and effectively solves the related problems existing in the traditional escherichia coli plasmid copy number detection method.

The invention belongs to the technical field of molecular biology, and particularly discloses a primer, a primer probe group, a detection kit and a detection method for detecting brucella. The invention provides a novel specific target gene recA for detecting brucella, and primers and probes are designed based on a recA gene conserved sequence, so that the screened primer probe group can accurately detect brucella in a clinical sample; no cross reaction with staphylococcus aureus, acinetobacter baumannii, klebsiella pneumoniae, escherichia coli and pseudomonas aeruginosa exists, and the specificity reaches 100%. The brucella microdroplet digital PCR detection method provided by the invention is simple to operate, wide in linear range, low in detection limit, good in repeatability and high in sensitivity, a single copy in a sample can be detected, and the detection kit prepared from the brucella microdroplet digital PCR detection method has a good clinical application prospect and a relatively high economic value.

The invention relates to the field of genetic engineering and bioengineering, in particular to engineering algae capable of synthesizing alpha-farnesene and a preparation method of the engineering algae. An MEP metabolic network of synechocystis PCC 6803 is modified, through single-copy series recombination and multi-copy combination of dxs, ispA, idi and AFS genes, optimization of a farnesene MEP metabolic pathway is achieved, the carbon flux of oriented synthesis of farnesene is precisely regulated and controlled, under the optimal effect, the yield of alpha-farnesene within 9 days is 83.07 mg / L (25.82 mg / g DCW), and economic application value is achieved.

The application discloses a multiplex method for measuring nucleic acid modification enzyme activity and screening one or more variable elements of enzymatic reaction.The method includes: (i) providing a polynucleotide construct library encoding DNA / RNA target site and variable element to be tested such as nucleic acid modification enzyme variant, (ii) partitioning a single copy of the polynucleotide construct with in vitro transcription and translation (IVVT) reagent, (iii) allowing the IVVT reaction of nucleic acid modification enzyme and DNA / RNA target site to be expressed in each compartment, (iv) depending on the functionality of the nucleic acid modification enzyme encoded, single polynucleotide construct / RNA target is cracked, remains intact or otherwise modified, and (v) by sequencing to cracked, complete or modified polynucleotide construct / RNA target quantification, so as to directly identify the enzymatic activity related to each variable element and to the enzymatic activity quantification.Particularly, nucleic acid modification enzyme is CRISPR associated protein (Cas).

The invention is directed to means and methods for efficient and safe genetic manipulation of a target cell, wherein a knock-out / knock in strategy is performed via HDR in a target gene that is haploinsufficient (and / or in single copy) in the cell.

Proteins, nucleic acids encoding the proteins, compositions comprising the proteins, and methods are provided. The proteins have the ability to be self-targeted to ICAM-1 and, if desired, enzymatically-released at acidic pH. The ICAM-1-targeting peptides are provided as single copies or multiples repeats, and can be separated by linkers from the enzyme segment, from which the ICAM-1 targeting peptides can be released, if desired, at acidic pH. These fusion proteins enhance the activity of the enzyme segment within or liberated from the fusion protein, and provide increased recognition and targeting of diseased organs, transport from the bloodstream across the endothelium into said diseased organ, and intracellular uptake and lysosomal trafficking by cells in them, both in peripheral tissues and the central nervous system. Representative nucleotide and amino acid sequences of these fusion proteins, as well as in vitro, cellular, and in vivo animal data are provided. The described proteins can be used as a protein therapy, a gene therapy, or an implanted cell therapy.

The invention relates to the technical field of microbiological detection, in particular to a detection method based on specificity of PCR (Polymerase Chain Reaction) and CRISPR-Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Cas12a) for bifidobacterium and application. The method comprises the following steps: amplifying a common single copy core gene of bifidobacterium by adopting a polymerase chain reaction (PCR) technology to obtain an amplification product; and an amplification product is detected by adopting a clustered regular interval short palindromic repeat related protein (CRISPR-Cas12a) system. The detection method is simple and convenient to operate, only comprises two parts of PCR amplification reaction and Crispr reaction, and the evaluation method of the detection result is simple, short in overall time consumption, high in efficiency, capable of detecting various bifidobacteria and definitely free of specificity to eight non-bifidobacteria, and wide in application range.

The invention discloses an oligonucleotide-coated fluorescence in-situ hybridization probe for specifically recognizing a tartary buckwheat Chr1-4 chromosome and application, and belongs to the field of cytological markers. The method comprises the following steps: screening a single-copy oligonucleotide sequence of Chr1-4 from a tartary buckwheat whole genome by using Chorus2 software; filtering and removing repetitive sequences to obtain a Chr1-4 chromosome probe sequence library; screening a specific single copy oligonucleotide sequence of the Chr1-4 chromosome; and adding specific primers at the two ends of each single copy oligonucleotide sequence of Chr1-4, and combining the probes into a buckwheat Chr1-4 Oligo-Painting probe mixing pool. By utilizing the constructed Oligo-Painting probe mixing pool and a fluorescence in-situ hybridization technology, accurate identification and karyotype analysis of the buckwheat chromosomes can be realized, the number, morphology, heritable variation and other structural characteristics of the chromosomes can be known, and an important technical support is provided for cytological analysis of buckwheat crossbreeding.

The invention belongs to the technical field of biological detection, and discloses a nucleic acid detection kit, which comprises the following components: a nucleic acid pre-amplification mixed solution, target capture magnetic beads, a multiple PCR reaction solution, a CRISPR-SERS detection solution and an endogenous control system. The nucleic acid detection kit integrates a nucleic acid pre-amplification mixed solution, target capture magnetic beads, a multiple PCR reaction solution, a CRISPR-SERS detection solution and an endogenous control system, and all the components work cooperatively to achieve whole process optimization. The target capture magnetic beads specifically enrich pathogen nucleic acid through a biotin-streptavidin system, so that host background interference is effectively removed; multiple PCR reaction liquid adopts DPO primer design, non-specific amplification is inhibited through a blocking region, and efficient amplification of a target gene is ensured. Finally, the CRISPR-SERS detection liquid activates trans-cleavage of reporter molecules by using cis-cleavage activity of the Cas12a protein, signal amplification is realized by combining with an SERS technology, so that the detection sensitivity reaches a single copy level, and meanwhile, an endogenous control system can monitor the quality of a sample in real time and avoid a false negative result.

The invention discloses a KASP molecular marker for detecting a rice blast resistance gene Pi65 and application of the KASP molecular marker. The KASP molecular marker comprises SNP1 and / or SNP2; the SNP1 is located at the 28376936bp position of the 11th chromosome of a Nipponbare MSUv7 reference genome, and the polymorphism of the SNP1 is T or C; the SNP2 is located at the 28377512bp position of the 11th chromosome of a Nipponbare MSUv7 reference genome, and the polymorphism of the SNP2 is A or C. The KASP molecular marker provided by the invention has the characteristics of high typing quality, single copy and high polymorphism, can quickly realize genotype identification of the rice blast resistance gene Pi65 at high throughput and low cost, is used for marker-assisted breeding for rice blast resistance breeding improvement, and has wide application universality.

The invention discloses a rapid detection method for echinococcus granulosus based on RPA-CRISPR / Cas, and belongs to the technical field of molecular diagnosis. The method comprises the following steps: extracting DNA of a sample to be detected; the specific primers are used for RPA isothermal amplification, a reaction system contains a buffer solution, upstream and downstream primers, RPA enzyme, magnesium acetate and template DNA, and reaction is carried out at 39 DEG C for 20 minutes; and mixing the amplification product with LbCas12a protein, sgRNA and a fluorescence report probe, carrying out CRISPR / Cas reaction at 37 DEG C for 30 minutes, and judging the result through a real-time fluorescence signal. Constant-temperature rapid amplification is achieved through RPA, high-sensitivity and high-specificity detection of the echinococcus granulosus can be achieved under the site condition by combining high-specificity recognition and trans-cleavage activity of CRISPR / Cas12a, the sensitivity reaches the single-site copy number, operation is easy and convenient, complex temperature control equipment is not needed, and the method is suitable for primary medical treatment and site quarantine.

The present invention relates to a method for the relative quantification of GM crops using digital PCR without using certified reference materials (CRMs). More specifically, the present invention relates to a primer and probe set composition for digital PCR for the relative quantification of transgenes in GM crops without using CRMs, which comprises a first primer and probe set capable of specifically detecting genes present in a single copy or in 2-3 copies within the genome of GM (genetically modified) crops; and a second primer and probe set capable of specifically detecting transgenes in GM crops, and uses thereof.