7 results about "Cloning sequencing" patented technology

The application belongs to the field of biotechnology and agricultural pest control, and particularly relates to a membrane protein Sec61 alpha gene and application of dsRNA of the membrane protein Sec61 alpha gene in pest control. After cloning and sequencing of the Sec61 alpha gene of Locusta migratoria, the sequence of the Sec61 alpha gene of SEQ ID NO:1 is obtained; then the gene fragment of SEQ ID NO:2 is selected for synthesis of double-stranded RNA (dsRNA). After the dsRNA of the gene is injected into the body cavity of the Locusta migratoria, specific Sec61 alpha genes can be silenced, and the Locusta migratoria stops feeding and successively dies before molting. The application provides a new specific molecular target for pest control based on RNA interference, and has very important application value.

This invention provides a method for screening highly specific nucleic acid aptamers and specific *Lactobacillus helveticus* aptamers, belonging to the field of nucleic acid aptamers. Specifically, the method for screening highly specific nucleic acid aptamers provided by this invention includes the following steps: S1: A second library is obtained after N rounds of forward screening of a first library; S2: A third library is obtained after M rounds of reverse screening of the second library; S3: The third library is obtained after X rounds of forward screening of the third library to obtain selected random single-stranded oligonucleotides, which are then subjected to PCR amplification, purification, cloning, and sequencing; S4: The sequences cloned and sequenced in step S3 are used for affinity and specificity assays to select sequences with high affinity and specificity, thereby obtaining highly specific nucleic acid aptamers. The aptamers provided by this invention have the advantages of high specificity and strong affinity, and the construction method of the aptamers provided by this invention has the advantage of short screening time.

This invention belongs to the field of biotechnology and discloses a specific primer pair for identifying wheat-specific strains of *Blastomyces oryzae* and its application. Based on the analysis of the genomes of *Blastomyces oryzae* strains specializing in different hosts, this invention identified a conserved fragment in the gene of the wheat-specific strain. Through design, PCR amplification, agarose gel electrophoresis, and cloning sequencing, three pairs of specific primers were ultimately screened to identify the wheat-specific strain of *Blastomyces oryzae*. Using the screened specific primer pairs, the wheat-specific strain of *Blastomyces oryzae* was qualitatively identified by PCR amplification of the test samples. The identification method is simple, efficient, and accurate, and has practical application value. This invention provides molecular markers for screening wheat-specific strains of *Blastomyces oryzae* and offers a convenient and accurate method for observing and studying these strains.

The invention discloses dsRNA of a migratory locust nubbin gene and application of the dsRNA in pest control, and belongs to the technical field of biotechnology and agricultural pest control. The invention belongs to the field of biotechnology and agricultural pest control, and particularly relates to application of dsRNA of a nubbin gene in pest control. The method comprises the following steps: cloning and sequencing a nubbin gene of migratory locust to obtain the nubbin gene with a sequence of SEQ ID NO: 1; and then the gene segment with the sequence of SEQ ID NO: 2 is selected and used for synthesis of double-stranded RNA (dsRNA). After the dsRNA of the gene is injected into the body cavity of the migratory locust, the specific nubbin gene can be silenced, so that wing development is abnormal after 100% of migratory locust molts, and migratory flight cannot be realized. A novel specific molecular target is provided for pest control based on RNA interference, and a novel technical approach is provided for pest control.

The application provides a Marek's disease virus meq gene edited deletion vaccine candidate strain SQ01Delta meq and construction and application. The SQ01Delta meq vaccine candidate strain has a preservation number of CGMCC NO.45864. The parent strain of the SQ01Delta meq vaccine candidate strain is MDV super strong Chinese mutant HNSQ01. After in vitro passage of the parent strain HNSQ01, the CRISPR / Cas9 gene editing technology is used to delete a 925bp fragment on each of the two meq alleles in the viral genome. Through PCR identification, gene cloning sequencing, RT-qPCR analysis, IFA identification and SPF chicken challenge experiment, it is ensured that the SQ01Delta meq strain completely loses the pathogenicity and tumorigenicity to the host chicken, and has no immunosuppressive property, and has good biosafety. Animal immune challenge protection evaluation experiment shows that the SQ01Delta meq has good immune protection, and the immune protection index of the parent strain reaches 80.0%. Therefore, the SQ01Delta meq strain constructed by the application can not only be used as a new MD gene deletion vaccine candidate strain, but also be used for the development and utilization of subsequent MD new type gene engineering vaccine.

This invention discloses a method for sequencing short nucleic acid fragments. The method involves artificially adding a 20-25 bp universal short sequence to both ends of a primer specifically for the target short nucleic acid fragment. After primer synthesis, a first round of PCR amplification is performed to obtain a first-round extended amplification product. Then, a universal long sequence is added to the 5' end of each of the aforementioned 20-25 bp universal short sequences to form universal long primers. After primer synthesis, the first-round extended amplification product is used as a template for a second round of PCR amplification, ultimately obtaining amplification products with 60-70 bp extensions at both ends of the target short nucleic acid fragment. This method eliminates the cumbersome steps of cloning and sequencing, as well as the time required for ligation, transformation, and screening, offering advantages such as high accuracy, shorter time, and lower cost. Furthermore, the two-round PCR amplification method used in this invention to extend the sequencing range is universal and applicable.

The invention provides an sgRNA sequence targeting a Rongchang pig CD163 gene and a gene editing method and application of the sgRNA sequence. The nucleotide sequence of a targeting sequence in the sgRNA sequence is as shown in SEQ ID NO. 1. According to the invention, a part of region of the Rongchang pig CD163 gene exon 7 is accurately knocked out through a CRISPR / Cas9 technology, so that the directional regulation and control of the gene function are realized for the first time, and a key foundation is laid for the cultivation of disease-resistant varieties. According to the invention, the susceptibility of the CD163 gene to PRRSV invasion can be reduced only by knocking out a sequence of 1-18 bp, and the whole gene or most SRCR structural domains do not need to be deleted. According to the gene editing method disclosed by the invention, high editing efficiency and high precision are taken into consideration at the same time, editing, cloning and sequencing are performed on 16 randomly selected genes, the Indel mutation rate is up to 93.75%, the target site precise deletion efficiency is up to 75%, the invalid editing proportion is extremely low, and a reliable guarantee is provided for actual industrial application.