| Genome Editing is a a new technology to transform biological genome based on gene targeting modified, through Precise gene editing in the genomic level, we can achieve the purpose of gene knockout and knockin structural variation. With gene editing tools, people can be more accurate, in-depth understanding to explore gene function, study disease mechanisms,establish a new strain, find effective treatment of hereditary diseases. So it has an extremely wide range of prospects for the development and application value in research and clinical applications.Silkworm Bombyx mori is not only an important foundation of the silk industry, but also a the Lepidoptera typical model organisms and the development of a new generation of bio-reactor and new insects industrial materials. Silkworm is the first complete genome sequencing Lepidoptera with clear genetic background, accumulated a wealth of basic research, With highly developing of genome research work, especially in functional genomics research, silkworm post-genomic era already has a good foundation. On the basis of whole genome sequencing, research the relationship between the structure and function of genes and their products In different time, space, and the conditions, will greatly promote the development of the whole insect disciplines and the sericulture.Design and construction of TALEN is based on a transcription activator-like effector secreted by Xanthomonas that is plant pathogen. Fusing the DNA-binding domain and the FokI endonuclease enzyme cutting domain of TALE proteins, it can induce double-strand breaks at the specific site and trigger the cell’s own DNA repair mechanisms(Non-homologous end joining and Homologus Recombination), then, it will introduce the gene mutation, gene modification and knock in or out, accordingly, Achieve genome directional edit TALENs achieve great success in many model organisms.We designed a TALENs reagent that target BmBlos2and Bm702gene in silkworm and constructed a few of the TALEN targeting vector successfully several TALEN targeting vector.Combined with ssODN technology, Direct embryo injection of TALEN-ecoding mRNA which is synthesised in vitro into P50and Nistari.therefrom explore the of TALEN technical silkworm genome editing.The main results are as follows.1.Construction of targeting Vector TALENRelies on the standard restriction endonuclease and conventional molecular cloning technology, we try to directly synthesis the DNA fragments according to the amino acid sequence of the silkworm codon and TALEN targeting sites, then load it to backbone vector that contain FOKI. We constructed B2-TALEN, B3-TALEN,702-TALEN expression vector and preparing the corresponding mRNA targeting the second and the third exon of the silkworm BmBlos2gene,Bm702gene.2. knockout of the silkworm genome BmBlos2mediated by TALEN.Synthesized B3-TALEN mRNA expression vector in vitro.The mRNA samples were mi roinjected into the silkworm embryos of silkworm strain Nistari at700ng/m1.Transparent oily skin phenotype was observed at the5th instar. we extracted the genomic DNA of the mutations and analysis the mutant sequences, we observed deletion, insertion mutations, single-base mutations and other types of mutations. This finding suggests that TALENs can be used in silkworm to mediated efficient site-directed mutagenesis and Heritable phenotype.3.Targeted chromosomal deletions mediated by TALENA mixture of B2-TALEN and B3-TALEN mRNA expression vector mRNAs was injected into the silkworm embryos at350ng/ul.7mutant silkworms were randomly chosen from two brood which show mutation phenotype. The genomic DNA of mutants was extracted and the fragments containing two target sites were amplified by PCR. The experiment indicate that TALEN can be used to produce large chromosome deletions by generating two DSBs at two distal sites and Heritable mutations.4.Genomic structure variation in silkworm mediated by TALEN702-TALEN activity was detected by luciferase SSA assay and T7endonuclease I assay:In luciferase SSA assay, the pSSA-Luc vector was transfected into human embryonic kidney (HEK)293T cells, the experiment showed a28fold increase which suggested that TALEN-702has very high activity to promote HR events in HEK293T cells; In T7endonuclease I assay, the PCR product can be digested into two fragments which showed that702-TALEN could induce efficient mutations in the endogenous gene loci through NHEJ repair pathway.in vitro transcribed mRNA of B2-TALEN and B3-TALEN were mixed and microinjected into about100silkworm embryos. PCR was performed using genomic DNA from embryos3day after microinjection and using appreciate primers designed to detect deletions, duplications and inversions could amplify products with expected size. This suggest that targeted GSV could be induced by TALEN.5.Targeted chromosomal deletions using a pair of TALENs and ssODN.An ssODN was synthesized and co-injected with TALEN-B3mRNAs into silkworm embryos. Mosaic mutation and non-mosaic mutation GO moths were subjected to genomic DNA extraction and PCR amplification, we observed smaller amplified DNA segments with the expected size from the mosaic5th silkworms with translucent skin phenotype were obtained from the brood whose male parent was mosaic, the results suggested that ssODN mediated chromosomal deletions was heritable. The results of PCR and sequencing analysis suggested that there are unknown mutants in G1mutants besides the long fragme,... |
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