| Summary: | 碩士 === 輔仁大學 === 生物學系 === 85 === To develop transgenic plants expressing higher levels of Bacillus thurigiensis δ-endotoxin, we used PCR (polymerase chain reaction) and synthetic complementary oligonucleotides technology to modify several regions of Bacillus thuringiensis var. kurstaki cryIA(b) gene and subsequently cloned them into pBI121, forming three new expression vectors: a.pBT121M1(D) carrying D modified region of cryIA(b) gene, b.pBT121M2(B, D) carrying B, D modified regions of cryIA(b) gene, and c.pBT121M4(A, B, C, D) carrying four modified regions of cryIA(b) gene.
Via direct transformation, we introduc these three new clones and pBT121M(B), a clone we had previously acquirred, into Agrobacterium tumefaciens C58C1/PGV2260 strain. Using Agrobacterium-mediated transformation protocol, we then introduced these four partially modified cryIA(b) genes into tobacco (Nicotiana tabacum xanthi). The transgenic tobacco plant was confirmed by the PCR and the Southern blot analysis after selection by selective regeneration medium (MS 104 medium containing kanamycin 300mg/l). The transgenic plants were homogenized in liquid nitrogen to isolate total RNA and soluble protein. Total RNA was checked by the Northern dot blotting to examine the expressing levels of these four partially modified cryIA(b) genes in transgenic tobacco plants. Soluble protein was checked by the SDS-pAGE and the Western blotting to examine their insectcidal protein expressing level.
Preliminary examination of these three transgenic tobacco strains [pBT121M1(D)(pBT121M1,pBT121M2(B, D)] showed that there were great differences in cryIA(b) m-RNA expressing level among them but no significant differences in protein expressing level. Among these three strains, pBT121M1(D) had the greatest m-RNA expressing level followed by pBT121M2(B, D) and then pBT121M(B).
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