248 results about "Enzyme digestion" patented technology

The present application relates to a virus of reverse genetic operation construction of NADC34-like PRRSV2. Specifically, the full gene sequence of NADC34-like PRRSV2 BJ1805-2 isolated strain is segmented and connected to the vector by using PCR amplification, enzyme digestion and other methods with pACY177 plasmid as the carrier, and the BJ1805-2 full-length infectious clone recombinant plasmid (rBJ1805-2) is obtained. The infectious clone virus rBJ1805-2 is rescued in vitro, and the first NADC34-like PRRSV2 strain infectious clone platform in China is successfully built. The present application also relates to a modification method for constructing NADC34-like PRRSV2 strain which can adapt to Marc-145 cell passage culture. The infectious clone virus constructed by the present application has good in vitro and in vivo proliferation efficiency, and can cause specific viremia after inoculation in pigs; it has good safety, does not cause fever after inoculation in pigs, and will not cause piglet death. The modified strain can be used as a candidate strain for developing the first NADC34-like PRRSV2 specific vaccine in China, and is conducive to the prevention and control of PRRSV epidemic in China.

The invention discloses application of a KASP molecular marker in identification of sweet melon peel color traits, and belongs to the technical field of molecular markers. The invention aims to promote gene localization and molecular-assisted breeding processes by utilizing molecular marker-assisted selective breeding on sweet melon pericarp color characters. The KASP molecular marker is a KASP marker RW2; the KASP marker RW2 is designed according to an SNP (Single Nucleotide Polymorphism) site at the position of Chr04: 526653. According to the application, the KASP molecular marker RW2 is used for performing genetic typing on the red-peel muskmelon and the white-peel muskmelon, the DNA of a target plant can be identified only through one-time PCR amplification, the whole detection process is simple and convenient, and complex steps such as enzyme digestion, electrophoresis and sequencing are not needed. Through the high-throughput genotyping system, a genotyping graph and a genotyping value can be rapidly obtained, so that rapid identification of the genotype of a target plant is realized. The KASP molecular marker RW2 provided by the invention can be more efficiently applied to the breeding work of sweet melon peel color germplasm.

The invention provides a method and system for discovering antioxidant peptides from food and agricultural by-products and outputting enzymolysis recommendation, and relates to the technical field of bioinformatics and artificial intelligence crossing. The method sequentially comprises the steps of protein sequence collection and standardization, candidate peptide fragment interception, prediction of antioxidant activity through fusion of sequence and structural characterization, generation of releasable peptide fragments through enzyme digestion simulation, and construction of comprehensive evaluation indexes based on activity probability and process parameters. And an optimal enzymolysis scheme is searched in an enzyme combination and reaction condition space through multi-objective optimization, so that the method is suitable for high-valued utilization of food industrial residues and agricultural wastes, and has the advantages of high throughput, high accuracy and high implementability.

The invention discloses an EmAGO2 truncated recombinant protein, an echinococcosis multilocularis indirect ELISA (enzyme-linked immuno sorbent assay) diagnostic kit based on the truncated recombinant protein and a use method of the kit. The method comprises the following steps: designing a specific primer by selecting a sequence of 1-738bp at the N end of an Em-AGO2 gene, carrying out PCR (Polymerase Chain Reaction) amplification by taking reverse transcription echinococcosis multilocularis DNA (Deoxyribonucleic Acid) as a template, connecting an amplification product with a pCE2-TA / Blunt vector, and then transforming into a competent cell for culturing, so as to obtain a pCE2-T-EmAGO2 plasmid; respectively carrying out double enzyme digestion on the plasmid and an expression vector pET-28a (+), and connecting enzyme digestion fragments to obtain a recombinant protein expression plasmid pET-28a-EmAGO2; the preparation method comprises the following steps: taking EmAGO2 as a template, transforming the EmAGO2 into competent cells, selecting positive clones for induced expression, and carrying out ultrasonication treatment, separation and purification, concentration and desalination treatment on expressed thalli to obtain the EmAGO2 truncated recombinant protein. Furthermore, a mouse anti-EmAGO2 truncated recombinant protein and goat anti-rabbit IgG are respectively used as an antigen and an antibody to establish an indirect ELISA diagnostic kit for the echinococcosis multilocularis, and the kit has high sensitivity, specificity and good repeatability, and can be used for early diagnosis of the echinococcosis multilocularis.

The invention discloses modified hydroxytyrosol, a cosmetic, a preparation method and application. The provided preparation method of the modified hydroxytyrosol comprises the following steps: in the presence of an acidic solvent, mixing hydroxytyrosol, lipase and fatty acid ester for reaction, centrifugally layering a reaction product, and collecting components of a middle layer to prepare the modified hydroxytyrosol. According to the provided preparation method of the modified hydroxytyrosol, lipase and fatty acid ester are subjected to an enzyme digestion reaction, an active hydroxyl group in a product obtained through the enzyme digestion reaction and hydroxytyrosol are subjected to a dehydration reaction, so that an ester group molecular chain is grafted into hydroxytyrosol, and a modified hydroxytyrosol product with an ester group is prepared. And the obtained modified hydroxytyrosol has relatively high fat solubility and oxidation resistance.

The invention discloses a modular tumor microenvironment response polypeptide and a preparation method and application thereof. The modular tumor microenvironment response polypeptide is composed of the following functional modules: a targeting and chemotactic module used for targeting tumor cell surface p32 protein and tumor lymphatic vessels and promoting tumor tissue penetration and enrichment; the microenvironment double-response module is used for being cut by MMP-2 / 9 enzyme and broken by high-concentration glutathione GSH in a tumor microenvironment to trigger self-assembly; the self-assembly driving module is used for exposing after the microenvironment responds to activation and driving short peptides to be self-assembled to form a nano structure; a killing module: a pro-apoptotic peptide PAD used for targeting a mitochondrial membrane and inducing tumor cell apoptosis; and the stability optimization module is used for enhancing the degradation resistance of the oligopeptide. By integrating targeted delivery, enzyme digestion response, acid-sensitive release and stabilizing structures, the problems of insufficient targeting property, inaccurate release, poor stability, diagnosis-treatment splitting and the like of the anti-tumor drug and the developing agent are solved.

The invention belongs to the technical field of biological drug analysis, and particularly relates to a method and a system for quantitatively detecting antibody drugs by using a dimethyl-labeled modified peptide fragment as an internal standard. Sequentially carrying out dimethyl labeling on the enzymolysis peptide fragments of the antibody drugs to obtain a plurality of dimethyl-labeled modified peptide fragments which are used as internal labels; the method comprises the following steps: enriching antibody drugs in a biological sample, and carrying out enzyme digestion to obtain a plurality of unlabeled modified peptide fragments; and mixing the unlabeled modified peptide fragment with an internal standard, enriching the modified peptide fragment, and then carrying out quantitative detection on the modified peptide fragment by adopting liquid chromatography-tandem mass spectrometry. A one-to-one structural analogue internal standard is provided for the modified peptide fragment, and the matrix effect is eliminated. Meanwhile, the method is combined with a high-pH reversed-phase grading equal peptide fragment enrichment method, so that the detection sensitivity of the modified peptide fragments in the matrix is improved. The technology provides a sensitive and reliable quantitative analysis method for the modified peptide fragment, and can be used for quantitative analysis of antibody drugs in biological samples.

The invention provides a construction method, system and product of a false positive judgment model of a mutation site obtained by sequencing after enzyme digestion fragmentation and related application. Specifically, the false positive judgment model is constructed on the basis of a plurality of characteristic parameters associated with mutation sites obtained by enzyme digestion fragmentation sequencing; the characteristic parameters comprise scores obtained by performing reverse complementary sequence alignment on the front and back extension 30bp of the mutation site, the sequence proportion of 15 basic groups at the tail end of the mutation site, the Bayesian conditional probability of the extracted mutation site, and the KS test statistic of the extracted mutation site. Furthermore, the invention also provides a method for constructing the model by utilizing the crowd detection rate and the corresponding false positive. According to the method, the data characteristics of the false positive mutation sites are introduced by utilizing digestion fragmentation, so that the reliability of the digestion false positive mutation sites is objectively evaluated under the condition that high depth and large data volume are not needed, and the detection accuracy of the mutation sites can be remarkably improved.

The invention belongs to the technical field of biomedical engineering, and particularly relates to a polypeptide preparation method for preparing a GLP-1 analogue through tandem expression. According to the method, a coding gene sequence of the GLP-1 analogue is repeatedly connected in series for a plurality of times, and then expression preparation is carried out on the basis of a genetic engineering technology. The inventor optimizes a series-connected polypeptide structure by adding a section of key peptide sequence. The key peptide serving as a leading peptide can greatly improve the expression quantity of the strain in the fermentation expression process; as a linked peptide, the key peptide sequence can effectively improve the recognition capability of enzyme digestion sites on one hand and can adjust the overall isoelectric point of the sequence on the other hand, so that the solubility, enzyme digestion yield and purity of a target product can be improved. Preliminary experiment results show that the expression quantity of the polypeptide prepared through expression is extremely high, the enzyme digestion efficiency is good, the good enzyme digestion efficiency can be achieved under the condition that the enzyme dosage is low, and the yield and purity of the final GLP-1 analogue are high.

The invention relates to an shRNA (short hairpin Ribonucleic Acid) interference sequence of a targeted silence PCSK9 gene and a construction method and lipid-lowering application of a recombinant adeno-associated virus vector of the shRNA interference sequence. Hyperlipidaemia is a metabolic disease characterized by abnormal rising of cholesterol and triglyceride levels in blood, and the design of lipid-lowering drugs is the focus of attention to improvement of hyperlipidaemia. Proprotein convertase subtilisin / kexin type 9 (PCSK9) can be combined with a low-density lipoprotein receptor (LDL-R) and degrade the LDL-R, so that accumulation of LDL-C in blood is further promoted, and hyperlipidemia is caused. Aiming at the key target PCSK9, a specific shRNA interference sequence is designed, and a recombinant adeno-associated virus vector (rAAV) carrying the sequence is constructed by an enzyme digestion-connection method. In-vitro experiments prove that the vector can remarkably reduce the expression level of PCSK9 protein, so that the cyclic utilization of a low-density lipoprotein receptor (LDL-R) is promoted, and the concentration of low-density lipoprotein cholesterol (LDL-C) in plasma is reduced. The rAAV vector provided by the invention has the characteristics of low production cost, high transfection efficiency, lasting action time and the like, and provides a new thought for gene therapy of hyperlipidemia.

The invention discloses a bubble self-stratification digital detection method and system for miRNA quantification of extracellular vesicles, and belongs to the technical field of biomedical engineering and molecular diagnosis. The technical problem to be solved is to provide a simple and sensitive tumor extracellular vesicle miRNA detection method capable of simultaneously detecting various miRNAs. According to the scheme, a to-be-detected sample and immunocapture bubbles coupled with an anti-EpCAM antibody are incubated to enrich tumor extracellular vesicles, a to-be-detected miRNA sample is obtained through in-situ ultrasonic lysis, then the to-be-detected miRNA sample, a fluorescence / DNA double-coding magnetic bead conjugate and the like are used for constructing an enzyme digestion system for enzyme digestion, multifunctional click glass microbubbles are added to capture unreacted magnetic beads, standing is conducted, self-stratification is conducted, bottom magnetic beads are collected, and the EpCAM antibody is obtained. After fluorescence imaging, an image is input into the AI automatic fluorescence counting and decoding module for counting to obtain a result. The method is used for detecting miR-21 and miR-155 in tumor extracellular vesicles, and is suitable for noninvasive early diagnosis of tumors.

The invention provides a method for improving the expression level of a meat toxin light chain. The method comprises the following steps: connecting a dissolution-promoting tag to an N end or a C end of meat toxin light chain protein to form soluble meat toxin light chain recombinant protein; the dissolution-promoting tag is a wild dissolution-promoting tag NT11 or a mutant protein mut7 of the wild dissolution-promoting tag NT11, enzyme digestion removal is not needed, the activity of a meat toxin light chain is hardly influenced and interfered, and the fusion protein can keep 93% of substrate enzyme digestion activity. The method further comprises overexpressing a molecular chaperone htpG in the genome of the host bacterium. According to the invention, a BoNT / A-LC high-efficiency expression system is constructed through multi-strategy optimization, so that the yield of BoNT / A-LC is increased by nearly 20% compared with that of wild type NT11-Lc, the yield reaches 647 mg / L through overexpression of a molecular chaperone htpG, and after combinatorial optimization, the yield is 8.5 times that of only expressed BoNT / A Lc, 1.79 times that of NT11-Lc and 1.43 times that of Mut7-Lc.

The invention discloses a whole genome methylation detection method based on MeDIP-MSRE, and belongs to the technical field of epigenetics detection. According to the method, methylation immunoprecipitation sequencing and a methylation sensitive restriction enzyme technology are innovatively combined, firstly, a methylation specific antibody is used for conducting immunoprecipitation on sample DNA, and whole genome methylation fragments are enriched; then carrying out enzyme digestion on the enriched product by adopting methylation sensitive restriction enzyme, specifically removing an unmethylated DNA region, and reserving a complete methylation sequence; and finally, constructing a methylation map through high-throughput sequencing. According to the method, traditional hydrosulfite chemical conversion is not needed, DNA damage and base conversion deviation caused by the traditional hydrosulfite chemical conversion are avoided, meanwhile, high sensitivity of MeDIP and high specificity of methylation sensitive restriction endonuclease are fused, and the fidelity, sensitivity and specificity of detection are remarkably improved.

The invention belongs to the technical field of detection, and particularly relates to a nucleic acid detection method based on triple amplification and application of the nucleic acid detection method in ginseng identification. Mixing the liquid to be detected with the hairpin DNA, and assembling to form DNA enzyme; a hairpin substrate is added, enzyme digestion reaction is completed, and a trigger chain is generated; adding a circular DNA probe, carrying out rolling circle amplification, and collecting generated long single-stranded DNA; a G quadruplex structure in the long single-stranded DNA is utilized to catalyze a chromogenic reaction, and the concentration of miRNA in the to-be-detected liquid is analyzed through the RGB value of the color. According to the method disclosed by the invention, the low detection limit of the 651 fM is realized. The analysis of ginseng samples and related products verifies the effectiveness of the triple amplification platform, and a portable and reliable solution is provided for food safety supervision and quality control.

The invention relates to the technical field of biochemistry, and discloses a DNA chimera preparation and application method, which comprises the following steps: synthesizing phosphorylated linear annular L-rTDNA and template DNA, mixing in a 1 * T4 DNA ligase buffer solution according to an equal molar concentration of 10 [mu] M, heating at 95 DEG C for 5 minutes, and then carrying out gradient cooling to room temperature for renaturation for 120 minutes; adding T4DNA ligase to close a tail end notch, and carrying out enzyme digestion treatment to obtain annular rTDNA; the method comprises the following steps: synthesizing hairpin DNAHP (deoxyribonucleic acid), mixing rTDNA and HP according to an equal molar concentration of 2 mu mol / L, denaturating at 95 DEG C for 5 minutes, slowly cooling to 25 DEG C within 2 hours, and self-assembling to obtain the DNA topology limited chimera. According to the invention, a DNA chimera with dynamic response capability is constructed, an integrated detection process of enzyme-free assembly-target triggering-signal cascade amplification is realized, and meanwhile, the problems of anti-interference and ultra-sensitive detection in a complex matrix are solved.

The invention relates to the technical field of crop breeding science, in particular to a molecular marker closely linked with a wheat powdery mildew resistance gene PmNJ3930 and application of the molecular marker. The marker closely linked with the PmNJ3930 is a dCAPS molecular marker, and an upstream primer sequence and a downstream primer sequence for amplifying the molecular marker are provided. A product amplified by the primer is 354 bp after being subjected to NcoI enzyme digestion, which indicates that the wheat to be detected contains the PmNJ3930 gene and is a wheat variety with high powdery mildew resistance. When the primer pair provided by the invention is used for detecting the PmNJ3930 gene, whether the PmNJ3930 gene exists or not and the existence state of the PmNJ3930 gene can be determined, and the powdery mildew resistance of wheat is predicted, so that plants carrying the PmNJ3930 gene are quickly screened and are used for breeding disease-resistant varieties.

The invention discloses construction of a Guangdong small-ear spotted pig spermatogonial stem cell in-vitro culture system and screening of surface markers, and belongs to the technical field of cell culture.The technical scheme includes that testis are broken firstly, then enzyme digestion, density gradient centrifugal purification and differential adherent purification are conducted, and purified spermatogonial stem cells are obtained; inoculating the obtained spermatogonial stem cells to feeder layer cells and culturing in a culture medium; the culture medium is a DMEM culture medium added with FBS and growth factors; the mass fraction of the FBS in the DMEM culture medium is 8-12%, an enzyme digestion method, a density gradient centrifugation method and a differential adhesion method are combined for application, the Guangdong small-ear spotted pig spermatogonia stem cells with certain purity are obtained, cells enriched and purified in vitro can proliferate massively, and an in-vitro culture system of the Guangdong small-ear spotted pig spermatogonia stem cells is established.

This invention discloses a goose double-enzyme digestion simplified genome sequencing library construction kit, library construction method, and application. The kit includes Kit A, Kit B, and Kit C, and magnetic beads. The invention uses two conventional endonucleases to control the location and number of target fragments, and uses improved enrichment primers to significantly increase the number of library pools for GBS sequencing. This leverages the large amount of NGS data and makes goose population genetics research more feasible. In theory, this library construction strategy can exponentially increase the number of individuals to be tested simply by modifying the corresponding adapter and index sequences.

The present invention discloses an application of an insect virus FHV RNA1 replicon in exogenous gene amplification, wherein the application amplifies the exogenous gene at the mRNA level by utilizing the autonomous replication ability of the insect virus FHV RNA1 replicon; the present invention discloses a plasmid system based on the insect virus FHV RNA1 replicon and a method for constructing the plasmid system; the present invention also discloses a method and application of the plasmid system for expressing proteins in cells, wherein the plasmid system is used to obtain a cloning vector after double enzyme digestion, and after homologous recombination with an exogenous gene, the animal or plant protein is expressed in the cell.

Provided in the present application are a cyclic peptide mutant and use and product thereof for the preparation of a coronavirus inhibitor, belonging to the technical field of cyclic peptide drugs. The present application is optimized on the basis of the cyclic peptide 6L3-3P11R. The obtained cyclic peptide mutant can resist the enzyme digestion effect of pancreatic enzymes and exhibits further improved antiviral activity, which is of great significance for expanding antiviral applications of cyclic peptide drugs.

A primer pair, method, and use for rapidly identifying newborn nude mice. On the basis that a deletion mutation occurs in the base G at position 337 of a coding region of the Foxn1 gene in nude mice, a primer pair introducing an enzyme digestion site is designed according to sequence characteristics. Using the genome of a mouse to be tested as a template, the Foxn1 gene is amplified according to a PCR-RFLP method. The PCR product is then digested with a SmaI restriction endonuclease, and the digested product is genotyped by agarose gel electrophoresis. In addition, a reaction system that can be combined with lateral flow technology is screened, thereby achieving instant detection of the newborn nude mice. The method has the characteristics of simple operation, short time consumption, low cost, instant detection, etc.

The present disclosure provides an application method in rice callus differentiation based on an Oryza sativa Leafy Cotyledon 1 (OsLEC1) gene, including the steps of: selecting guide ribonucleic acid (gRNA) target sites; cloning tandem fragments including gRNA; ligating each gRNA fragment; performing polymerase chain reaction (PCR) amplification on a ligation product; performing enzyme digestion on the purified product and a target vector; transforming the ligated vector; performing Agrobacterium-mediated genetic transformation of rice; and screening and identifying transgenetic plants. In the present disclosure, the differentiation of callus directly affects the emergence efficiency of transgenic plants. The knockout of OsLEC1 can promote the differentiation of rice callus, suggesting that OsLEC1 can serve as an important target gene for improving the transformation efficiency of rice and even gramineous crops. OsLEC1 can be used as a starting point to construct various molecular tools to enhance transformation efficiency.

The invention discloses a preparation method of a fibrous protein / bacterial cellulose efficient hemostatic patch. The preparation method comprises the following steps: (1) in-situ preparation of large-aperture bacterial cellulose; (2) infiltration of fibrinogen in the large-aperture bacterial cellulose; and (3) enzyme digestion and freeze-drying treatment of fibrinogen. The problems that fibrinogen is fast in degradation and poor in stability and mechanical property are solved, the prepared fibrous protein / bacterial cellulose efficient hemostatic patch can prevent secondary bleeding and avoid thrombus to a certain extent, the preparation process is simple and easy to implement, and large-scale preparation and storage can be achieved.

The invention provides a construction method of a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model. The construction method comprises the following steps: designing mutation primers H1.3 R80A-F and H1.3 R80A-R; the method comprises the following steps: by taking a pCDH-HA-H1.3-Flag plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification by adopting a mutation primer, digesting a product by DMT enzyme, and converting a competent cell to obtain a mutant plasmid; and co-transfecting the mutant plasmid and a helper plasmid to a packaging cell, collecting a virus solution, filtering, infecting an A549 cell, adding Polybrene to assist infection, culturing, and screening a stably transfected cell strain by using puromycin to obtain the recombinant plasmid. The invention also provides application of the mutant cell model obtained by the construction method. According to the invention, the blank of histone H1.3 methylation research is filled, the 80th arginine is clear as a core modification site, and the established model provides a new tool for lung cancer mechanism research and drug research and development.

This application relates to a method, apparatus, computer device, and storage medium for identifying enzyme substrates. The method for identifying enzyme substrates analyzes the sequence information of substrate peptides before and after enzyme digestion by quantitative detection of control samples and enzyme inhibitor-treated samples, thereby identifying potential enzyme substrates. It effectively identifies enzyme cleavage sites, detects the sequences of substrate peptides before and after enzyme digestion, and promotes enzyme substrate identification and site analysis. This plays an important role in understanding biological processes and identifying biomarkers for diseases.

The invention relates to a CAPS (cleaved amplified polymorphic sequence) molecular marker for identifying the content of rosa chinensis aroma leaf acetate and application of the CAPS molecular marker, and relates to the technical field of molecular breeding, the marker is located at the 27954680 site of the third chromosome of a rosa chinensis genome, and the nucleotide sequence of the CAPS molecular marker for identifying the content of rosa chinensis aroma leaf acetate is shown as SEQ ID No.1. DNA fragments with the length of 447 bp are obtained through amplification of a specific primer pair, and genotypes are distinguished by combining DdeI enzyme digestion analysis: after enzyme digestion, the generated 447 bp and 274 bp fragments are low-content heterozygous types (T / A), and a 447 bp single band is retained as a high-content homozygous type (T / T). The genetic difference of the leaf acetate content of the Chinese rose can be rapidly identified, the flower fragrance character screening efficiency is remarkably improved, the breeding cost is reduced, an efficient molecular tool is provided for high-fragrance Chinese rose variety breeding, and the method has important application value.

The invention relates to the technical field of neutrophile granulocyte separation, in particular to a mouse neutrophile granulocyte separation method which comprises the following steps: (1) anesthetizing and disinfecting a mouse; (2) tissue sampling; (3) tissue digestion; (4) preparing a cell suspension; (5) purifying cells; (6) cell culture; according to the method disclosed by the invention, isoflurane is adopted in the anesthesia disinfection step, so that central nervous activity of mice can be rapidly inhibited, tissue damage caused by stress reaction is avoided, and chemical stimulation can be reduced while body surface microorganisms are cleared through combination of ethanol disinfection and sterile normal saline flushing, and aortic tissues with intact activity are provided for subsequent material taking. In the compound enzyme digestive juice, collagenase I can specifically degrade aortic wall collagenous fibers, trypsin dissociates adhesion connection between cells, neutral protease assists in decomposing elastic fibers, and DNA enzyme I is supplemented to inhibit cell aggregation, so that the contradiction between incomplete digestion and large cell damage of the traditional enzyme juice is effectively solved.

The invention discloses a cell crude sample sex determination primer suitable for chickens, ducks or pigeons and a PCR detection method, and belongs to the technical field of biological detection. A pair of gender-specific universal primers for various poultry such as chicken, duck and pigeon is designed for the first time, a cell crude sample is adopted as a PCR template, stable amplification can be realized without DNA extraction, the operation process is greatly simplified, the detection time is shortened, and the detection cost is reduced. Meanwhile, TrypLE enzyme digestion is very mild to cells, and the method is also suitable for preparation of single-cell suspensions related to single-cell omics. The method is suitable for the fields of early sex identification of poultry, breeding poultry breeding, molecular breeding and the like, and has wide application prospects.

This invention provides a high-throughput method for constructing dual sgRNA libraries and its applications. The method utilizes high-throughput microarray synthesis technology to prepare a set of DNA fragments containing multiple dual sgRNA expression cassettes in a single step. After amplification, these fragments are assembled with a target vector containing a first promoter and a second gRNA backbone sequence in a first round of directed assembly to obtain a preliminary recombinant plasmid set. Then, linearized enzyme digestion and homologous recombination technology are used to insert a fragment containing a transcription termination sequence and a complete second promoter to complete the construction of the dual sgRNA expression unit. Finally, transformation and amplification yield the dual sgRNA plasmid library. This invention avoids the high error rate and high cost of long-chain oligonucleotide synthesis by utilizing microarray synthesis and simplifies the operation process through two rounds of directed assembly, significantly improving the throughput, fidelity, and efficiency of library construction. It is applicable to the construction of genome-wide dual sgRNA libraries, providing an efficient and reliable technical platform for high-throughput gene function screening, drug target discovery, and gene interaction research based on CRISPR.

This invention belongs to the fields of biotechnology and genomics, and relates to a method and kit for capturing plant chromatin conformation. The method includes the following steps: (1) cross-linking and fixing the chromatin of a plant sample; (2) digesting the fixed chromatin using an enzyme digestion combination containing restriction endonuclease AluI and restriction endonuclease HypCh4V; (3) ligating the enzyme fragments generated in step (2) at spatially adjacent locations to form ligation products; (4) enriching the ligation products and constructing sequencing libraries using the enriched ligation products. This invention uses a combination of AluI and HypCh4V enzymes to replace traditional single-enzyme digestion, which significantly improves data resolution while reducing technical bias. It can achieve chromatin interaction mapping at a resolution of 500 bp in plants and has broad application prospects in the study of fine tissue characteristics at the single-gene level in plants.