Bombyx Dotaz Zobrazit nápovědu
Bombyx mori is a valuable model organism of high economic importance. Its genome sequence is available, as well as basic genetic and molecular genetic tools and markers. The introduction of genome editing methods based on engineered nucleases enables precise manipulations with genomic DNA, including targeted DNA deletions, insertions, or replacements in the genome allowing gene analysis and various applications. We describe here the use of TALENs which have a simple modular design of their DNA-binding domains, are easy to prepare and proved to be efficient in targeting of a wide range of cleavage sites. Our procedure often allows the production of individuals carrying homozygous mutations as early as in the G1 generation.
- MeSH
- bourec genetika MeSH
- DNA vazebné proteiny genetika MeSH
- endonukleasy chemie genetika MeSH
- geneticky modifikovaná zvířata MeSH
- genom hmyzu MeSH
- genový targeting metody MeSH
- lidé MeSH
- mutageneze MeSH
- trans-aktivátory genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Sericins are soluble silk components encoded in Bombyx mori by three genes, of which Ser1 and Ser3 have been characterized. The Ser1 and Ser3 proteins were shown to appear later in the last larval instar as the major sericins of cocoon silk. These proteins are, however, virtually absent in the highly adhesive silk spun prior to cocoon spinning, when the larvae construct a loose scaffold for cocoon attachment. We show here that the silk-gland lumen of the feeding last instar larvae contains two abundant adhesive proteins of 230 kDa and 120 kDa that were identified as products of the Ser2 gene. We also describe the sequence, exon-intron structure, alternative splicing and deduced translation products of this gene in the Daizo p50 strain of B. mori. Two mRNAs of 5.7 and 3.1 kb are generated by alternative splicing of the largest exon. The predicted mature proteins contain 1740 and 882 amino acid residues. The repetitive amino acid sequence encoded by exons 9a and 9b is apparently responsible for the adhesiveness of Ser2 products. It has a similar periodic arrangement of motifs containing lysine and proline as a highly adhesive protein of the mussel Mytilus edulis.
Engineered nucleases are able to introduce double stranded breaks at desired genomic locations. The breaks can be repaired by an error-prone non-homologous end joining (NHEJ) mechanism, or the repair process can be exploited to introduce precise DNA modifications by homology-directed repair (HDR) when provided with a suitable donor template. We designed a series of DNA donors including long dsDNA plasmids as well as short ssDNA oligonucleotides and compared the effectiveness of their utilization during gene targeting with highly efficient transcription activator-like effector nucleases (TALENs). While the use of long dsDNA donors for the incorporation of larger DNA fragments in Bombyx is still a problem, short single-stranded oligodeoxynucleotides (ssODNs) are incorporated quite efficiently. We show that appropriately designed ssODNs were integrated into germ cells in up to 79% of microinjected individuals and describe in more detail the conditions for the precise genome editing of Bombyx genes. We specify the donor sequence requirements that affected knock-in efficiency, and demonstrate the successful applications of this method of sequence deletion, insertion and replacement in the Bombyx genome.
- MeSH
- bourec genetika MeSH
- DNA genetika metabolismus MeSH
- editace genu metody MeSH
- jednovláknová DNA genetika metabolismus MeSH
- TALENs genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Silkworm diseases caused by fungi infection occur frequently in sericulture and brought huge economic loss to sericulture. However, on the other hand, some fungi such as Beauveria bassiana, as an important entomological fungus, play an important role in biological control of insect pests. Here, two fungal pathogens causing yellow muscardine were isolated from the silkworm and named as SZY1 and SZY2. These two strains showed almost the same conidial morphology which were smooth, near-spherical, spherical, or ovoid and 2.7 ± 0.6 μm × 2.5 ± 0.9 μm in size, and the hyphal growth rate was also similar. However, the conidia production of SZY2 was almost twice as many as that of SZY1. The complete ribosomal RNA gene was sequenced and analyzed. As a result, the gene sequences of internal transcript space 1 (ITS1)-5.8S rRNA-internal transcript space 2 (ITS2) of SZY1 and SZY2 were identical in sequence and size, and for 18S rRNA, 28S rRNA, and intergenic spacer (IGS), the gene identity of SZY1 to SZY2 was 99%, 99%, and 98%, respectively. Results of phylogenetic analysis based on either ITS1-5.8S rRNA-ITS2 or 18S rRNA showed that both SZY1 and SZY2 were closely related to Beauveria bassiana. These results revealed that the pathogens of yellow muscardine SZY1 and SZY2 were identified as two different strains of Beauveria bassiana, which could provide diagnostic evidence for silkworm muscardine and was helpful for the research and development of novel Bombyx batryticatus and fungal biological insecticide.
- MeSH
- Beauveria * genetika MeSH
- bourec * genetika mikrobiologie MeSH
- fylogeneze MeSH
- RNA ribozomální 18S MeSH
- RNA ribozomální 5.8S MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Engineered nucleases are proteins that are able to cleave DNA at specified sites in the genome. These proteins have recently been used for gene targeting in a number of organisms. We showed earlier that zinc finger nucleases (ZFNs) can be used for generating gene-specific mutations in Bombyx mori by an error-prone DNA repair process of non-homologous end joining (NHEJ). Here we test the utility of another type of chimeric nuclease based on bacterial TAL effector proteins in order to induce targeted mutations in silkworm DNA. We designed three TAL effector nucleases (TALENs) against the genomic locus BmBLOS2, previously targeted by ZFNs. All three TALENs were able to induce mutations in silkworm germline cells suggesting a higher success rate of this type of chimeric enzyme. The efficiency of two of the tested TALENs was slightly higher than of the successful ZFN used previously. Simple design, high frequency of candidate targeting sites and comparable efficiency of induction of NHEJ mutations make TALENs an important alternative to ZFNs.
- MeSH
- bourec embryologie genetika MeSH
- deoxyribonukleasy metabolismus MeSH
- genetické vektory MeSH
- genový targeting metody MeSH
- molekulární sekvence - údaje MeSH
- mutační analýza DNA MeSH
- otevřené čtecí rámce MeSH
- Saccharomyces cerevisiae MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
