The Study On Transgenic Silkworm With EGFP-spider [A2S8]₂₈ Protein In The Fiber

In our study, a donor plasmid(piggybac-based vector), p XL-NTD-e GFP-Spider-CTD, constructed with a synthesized Spider gene [A2S8]28 which has a large molecular weight. In order to explore the applications of TALENs(the new genome editing tool) in transgenic silkworm study, a pair of TALEN(9-10) was also successfully constructed and co-transformed into the very fresh silkworm eggs with the donor plasmids by vacuumed electroporation. G0 transgenic silkworm cocoons were screened by green fluorescence. G0 transgenic silkworms, of which the transgenic fibers had high mechanical performance(Max. Stress), hybridized to get G1 transgenic silkworm. e GFP(enhanced green fluorescence protein) gene had been detected in the genome of G0 transgenic moths. 4% e GFP contents(mola r ratio) has been evaluated and then 4% Spider [A2S8]28 contents(molar ratio) has been deduced accordingly in the transgenic silkworm cocoons soluble protein samples. Although the efficiency of TALEN(9-10) was about 13.95% in Bm N cells, TALEN(9-10) did not produce the effect of DNA double-strand break sites of cleavage(DSBs) in the G0 transgenic moth genome(off-target phenomenon). The donor plasmid was investigated to locate on the 23 t h chromosome of transgenic silkworm moth genome. So it was confirmed that the donor was integrated into the silkworm genome by random integration not by specific insertions guided by TALEN(9-10). G0 transgenic silkworm silks have about 40-50% enhanced mechanical performance over that of normal/control silkworm silks. The highest ultimate tensile strength(Max. Stress) of G0 transgenic silkworm silks was about 700 MPa and its average value was 620 MPa. Most importantly, this excellent mechanical performance characteristic was hereditary in G1 transgenic silkworm.