31 results about "Fungal genome" patented technology

The present disclosure provides expression vectors containing a nucleic acid encoding an RNA polymerase III promoter, a ribozyme, a CRISPR-Cas9 single guide RNA, and an RNA polymerase III terminator, where the ribozyme is 5′ to the CRISPR-Cas9 single guide RNA, as well as ribonucleic acids encoded thereby. Further provided are fungal cells containing an expression vector described herein, as well as methods of fungal genome engineering through use of an expression vector described herein.

The invention relates to a method for detecting fungal diversity in the traditional soybean paste fermentation process. The invention aims to solve the problems that the traditional method for detecting the fungal diversity in the soybean paste fermentation process is high in workload and detection results are inaccurate. The method comprises the following steps of: weighing a soybean paste sample, adding the soybean paste sample into a phosphate buffer solution, suspending, adding glass beads, oscillating, centrifuging, and collecting a supernatant; washing a precipitate, centrifuging, and collecting a supernatant; centrifuging, abandoning a supernatant, washing a precipitate to obtain thalli, adding the phosphate buffer solution into the thalli, blowing and beating until a suspension is obtained, and oscillating to obtain a pretreated sample; extracting deoxyribonucleic acid (DNA) of a fungal genome in the pretreated sample; performing polymerase chain reaction (PCR) amplification to obtain a product A; diluting the product A, and performing PCR amplification to obtain a product B; and loading the product B serving as a sample, and performing electrophoresis by using a denatured gradient gel electrophoresis device to finish the detection. The method is simple, and high in sensitivity, repeatability and reliability, and comprehensively reflects the diversity of flora structures.

The invention belongs to the technical field of gene engineering and discloses a method for knocking out fungus genes. The method includes: respectively introducing nucleotide sequences identified by Vika recombinase into the upstream and downstream of target genes in fungus genomes; identifying protein with Vika recombinase activity, shearing the nucleotide sequences identified by the Vika recombinase, and knocking out the target genes to obtain fungi with the target genes being knocked out. The method has the advantages that a Vika-vox recombinase system is successfully applied to fungus gene knocking out, knocking out of the target genes is achieved, a new method is provided for fungus gene knocking out, and function researches and performance optimization of fungus genes are benefited.

The invention discloses a method for breaking walls for filamentous fungus genomic DNA extraction. The walls are broken in a multi-step centrifugation and glass rod extrusion mode; the method specifically comprises the following steps in sequence: culturing fungi, collecting the thallus, adding an extraction liquid in multiple steps, freezing, crushing the filamentous thallus, collecting a genomic DNA solution, and precipitating the genomic DNA. According to the method, an ordinary refrigerator of -20 DEG C and a self-made glass rod are adopted, the experimental equipment is easily available, the dependence on liquid nitrogen in the conventional method is reduced, living contaminants in grinding by using a mortar are reduced, the method is convenient to operate, economic and practical, filamentous fungus genomic DNA can be conveniently and rapidly obtained, and thus the method is suitable for popularization and application.

The invention discloses a system for multi-round genome editing in fungal cell by a CRISPR system, and a method for multi-round genome editing in fungal cells. The system for multi-round genome editing performs multi-round editing operations on the target fungal genome, wherein the homologous donor DNA in each round of editing operations contains one or more corresponding homologous donor DNA sequences for editing by aiming at different genes, only one donor homologous donor DNA contains a marker gene, and the marker genes of two adjacent rounds are different. The system successfully realizesconvenient, high-efficiency, multi-round (unrestricted) editing of the filamentous fungal genome, and has important significance for genome-directed editing and metabolic engineering of filamentous fungi.

The invention relates to a kit for extracting fungal genome DNA and belongs to the technical field of biology. The kit for extracting the fungal genome DNA comprises the specific steps of crushing fungal cells, dissociating DNA and removing impure proteins, adsorbing DNA by using a DNA adsorbing column, removing impurities and eluting DNA, finally, so as to obtain a liquid which is the fungal genome DNA which is subjected to electrophoresis detection in 1% sepharose gel. The conventional steps for extracting the fungal genome DNA are tedious, consume a long time, and can extract the fungal genome DNA in about 18 hours generally. The method provided by the invention, compared with the conventional fungal genome extraction method, only needs about 7 hours to extract the fungal genome DNA, so that a lot of extraction time is saved, and the fungal genome DNA can be extracted more effectively, quickly and economically. Meanwhile, the kit is suitable for commercial production and has a certain economic value.

The invention relates to a molecular detection method of tea white scab leaves. The method comprises the following steps: designing a primer pair according to the characteristics of the DNA sequence of tea white scab germ; collecting tea leaves and performing separation and purification culture with elsinoe; extracting the tea white scab germ genome DNA in the culture solution by use of a fungal genome DNA kit; performing specific LAMP amplification reaction by adopting the genome DNA as a template; and adding a fluorescent dye into the reaction system to judge whether the collected tea leaves suffer from tea white scab. In the invention, the molecular identification integrates the precise quantification of the spectrum technology and the specificity and sensitivity of DNA hybridization; and in a process of germ inspection, through comparison with morphologic observation and germ separation re-inoculation identification, quick identification of the early no-symptom stage of tea white scab can be realized, and batch sample identification can be accurately finished in 2 days, so that the timely and accurate control of diseases is facilitated, and the quality and efficiency of a tea garden are effectively improved.

The invention relates a method for extracting a fungal genome on the surface of aged tobacco. The method includes the two main steps of aged tobacco surface fungus enrichment and genome DNA extraction. Fungi are enriched through buffering solution soaking and gradient centrifugation, grinding and cell disruption are conducted by means of liquid nitrogen, and the DNA of the genome of fungi is extracted by means of a kit shown in the description. By means of liquid nitrogen grinding, fungal cell walls can be fully split, extraction efficiency of the DNA of the fungi is improved, the DNA isolation kit in the description includes the innovation patent inhibitor removal technology, and the yield of the genome can be effectively increased. The method can be used for efficiently and quickly extracting the DNA of the fungal genome on the surface of aged tobacco.

The invention discloses a method for rapidly extracting fungal genome DNA. The method comprises the following steps: adding lysate into hyphae or sporocarp which is grinded with liquid nitrogen, adding PCA, and centrifuging; adding 3M of sodium acetate into the upper-layer water phase, further adding precooled absolute ethyl alcohol, and leaving to stand for 5-10 minutes at -20 DEG C; centrifuging, abandoning the supernate, bleaching and precipitating with 75% alcohol, centrifuging, and abandoning the supernate; adding ddH2O and RNase to dissolve the components, and preserving at -20 DEG C. Compared with other methods, the method for rapidly extracting fungal genome DNA is simple to operate, high in feasibility, small in DNA extraction material, high in DNA extraction efficiency, good in integrity and stability, relatively low in impurity content, and can be directly applied to further research such as ITS sequencing. DNA of sporocarp can be also extracted, so that the hypha activation and culture time can be shortened, and an efficient and feasible molecular technical method for macro fungus molecular biological research is provided.

Fungal artificial chromosome (FAC) vectors are disclosed. A vector can be replicated in a bacterial or a fungal host, and can comprise an insert of heterologous DNA up to about 500 kilobases. A vector can be used for cloning and expressing a secondary metabolite (SM) gene cluster. An insert sequence can be modified by homologous recombination. A vector can be a plasmid comprising bacterial and fungal origins of replication, as well as bacterial and fungal selection marker genes. Also disclosed are vectors that can be integrated into a fungal genome, and dual function vectors which can be replicated in a bacterial or a fungal host and can also be integrated into a fungal genome. Also disclosed are methods of generating plasmid libraries including vectors comprising intact SM gene clusters.

The invention discloses a reagent and method capable of simply and effectively extracting plant pathogenic fungus genomic DNA. The extracting reagent comprises a solution A and a solution B, wherein the solution A comprises 200mM Tris-HCl (pH 8.0), 250mM NaCl, 25mM EDTA, 0.5wt% of SDS, 1wt% of beta-mercaptoethanol, and 8 microliter of RNA enzyme; the solution B is potassium acetate with the concentration being 5M. When the reagent is used for extraction, high-purity and good-stability genomes can be extracted fast and effectively, the extracted genomes can be directly used for gene cloning andgenome sequencing, and an efficient and feasible molecular detection approach is provided for pathogenic fungus molecular biology study.

The present invention relates to a method of editing a filamentous fungal genome by direct introduction of a genome editing protein molecule or complex. The method includes three modes. In the first mode, a genome editing protein molecule or complex for a target gene is directly introduced into a cell of an Aspergillus fungus, to edit a gene in the Aspergillus fungal genome. In the second mode, a genome editing protein molecule or complex for a target region in a filamentous fungal genome, and a desired DNA fragment, are directly introduced into a filamentous fungal cell, to knock-in the DNA fragment to a desired target site in the filamentous fungal genome. In the third mode, genome editing protein molecules or complexes for plural target genes are directly introduced into a filamentous fungal cell, to carry out simultaneous editing of the plural genes in the filamentous fungal genome.

The invention discloses a novel PyrG screening marker recycling method and application thereof, and belongs to the technical field of biology. According to the method, the pyrG screening marker can beremoved from a fungal genome without depending on homodromous repetitive sequences at the two ends of the pyrG, redundant repetitive sequence fragments cannot be left on the genome, recombinant bacteria without the pyrG can be used as starting strains again for genetic transformation by using the pyrG, and cyclic utilization of the pyrG screening marker is realized. The pyrG cyclic utilization method disclosed by the invention is suitable for fungi taking the whey glycoside-5'-phosphate decarboxylase coding gene URA3/pyrG expression element as a genetic selection marker, is simple and easy tooperate, does not influence the genetic background and character of the strain, and has a wide application prospect.

The invention provides a quantitative fungus detection method based on metagenome sequencing. The method comprises the following steps of: (1) sequentially adding saponin and endonuclease into a sample, and removing a host genome; (2) performing wall breaking treatment on the sample without the host genome, and extracting nucleic acid to obtain a fungus genome; (3) performing library constructionand high-throughput sequencing on the fungus genome to obtain original sequencing data; and (4) comparing the original sequencing data with a fungus database, and performing fungus identification andabundance detection. According to the method, host DNA in the sample is removed by adopting the saponin and the endonuclease before high-throughput sequencing, so that the proportion of a human genomeand the interference to the accuracy of a sequencing result are remarkably reduced; and the wall breaking treatment operation is matched, so that the proportion of the fungus genome is increased, thesequencing cost is reduced, and the method has important significance in the field of fungus detection.

The invention discloses a filamentous fungus replicon. The nucleotide sequence of the replicon is as shown in SEQ ID NO. 2. The replicon is obtained by using 19 fungal genomes as templates to construct a metagenome library and transforming aspergillus niger spores for screening, and is from a rhizopus chlamydosporum endophyte burkholderia HKI454 genome. The replicon provided by the invention can play a replication function in an aspergillus niger expression system, can provide stability higher than that of an AMA1 replicon, is a replicon found in fungi except aspergillus nidulans for the first time, enriches a replicon element library, and expands selection diversity of filamentous fungi utilizing the replicon.