Low-energy Ar~+Ion Beam Induced DNA Transfer In Silkworm Eggs

During the exploration of cosmic space, cosmic radiation has been found to have greatimpact on the living organism. If human beings want to live in the cosmic space for longterm, cosmic radiation is an important life topic which need to be solved. Previous studieshad shown that the radiation can have different effects on life structure, functionappearance and so on. Thus, ion radiation bioeffects will facilitate the development ofefficient countermeasures to radiation damage. Muller discovered the mutagenic effects ofX-ray on fruit flies in1927. Not until the explosive of the first atomic bomb in1945didpeople realize the importance of this discovery. And it motivated the development ofradiation biophysics greatly. However, for a long time, the research of ion radiationbiological effects was restricted in high-energy ion, and low-energy ion radiation has beenignored because of the limitation of condition and depth. In the middle nineteen eighties,Zengliang Yu discovered the mutagenic effects of low energy N+ion implantation on riceseed and founded a new interdisciplinary subject named ion beam bioengineering.Subsequently, this subject has been applied successfully for mutation and improvedbreeding in plants and microbes. But it has not been reported that this technology has beenapplied in transgenic animals. The silkworm, having its clear genetic background, has beenfully utilized as an important economic insect and Lepidoptera model. Efficient and stabletransgenic technology is necessary for model organism research. Using silkworm asreceptor, this thesis studied the technology systerm on transgenic silkworm preparationusing low-energy Ar⁺ion beam bombardment combined with piggyBac transposon. Someresults as follows:1. Preparation of transgenic silkworm induced by low-energy Ar⁺ion beamIn this thesis, a new method to preparate transgenic silkworm named low-energy Ar⁺beam induced gene transfer in silkworm eggs, was proposed. Non-diapause eggs of N4, amultivoltine B. mori variety, were used in this study as radiation receptor. In the optimalcondition for eggs incubation, at25keV to fluences of2.08×10¹⁸ions/cm²in dry stateunder vacuum, Ar⁺beam bombarded the eggs and stopped for ten seconds per1.3×10¹⁵ions/cm²fluences. The ion beam generate etching effects on the eggshell surface andformed micro channels. Transgenic silkworm were prepared after the eggs were immersed in mixture of PiggyBac and helper plasmid.2. Individual test and genetic effects analysis of transgenic silkwormFirstly, the total DNA of G0and G1silkworm was amplified with sequence specificprimers designed from the gfp gene. G0and G1had formed clear aim strip in the gel inPCR experiment having the same size with gfp. Secondly, the flank sequences of piggyBacvector at the insertion site were obtained by iPCR analysis, and the results showed that gfpgene had integrated with B. mori chromosome6of G1individuals. Finally, we detected thenumber of insertion sites on B. mori chromosome by Souther-blotting. The results showedthat gfp gene had been inserted into silkworm genome and the insertion sites were two orthree.3. Analysis on mechanism of low-energy ion beam induced DNA transfer technologyIn this thesis, four control groups compared with experimental group had been used toprove that ion etching channels were the way for foreign gene coming into eggs.In conclusion, etching effects and channels were generated after silkworm eggs werebombarded by low-energy Ar⁺beam. Transposon carrying foreign gene and helper vectormay come into eggs through these micro-holes. Then transpose made the foreign geneintegrate with genome. Several integration sites were found and this kind of integrationwas heritable. The results of this study provided the technical support for low-energy ionbeam study in animal and transgene fields.