| Summary: | 博士 === 國立陽明大學 === 生物化學研究所 === 92 === Based on the newly revised nomenclature system of cry genes, the PCR amplification method has been adopted to resolve the cry gene combinations of 294 Bacillus thuringiensis isolates from five selected areas of Taiwan. Our results indicate that cry1 (especially cry1A + 1B + 1F) and cry2 were the most abundant cry genes in Taiwan. In contrast, cry3 and cry6 genes were only detected on Yang Ming Mountain, while the cry13 gene was only found on Snow Mountain. In addition, some distinctive combinations of cry genes were detected in distinct areas of Taiwan, such as cry1C, cry1D, cry1C + 1D, cry4, cry1 + 4, cry1 + 11, cry4 + 11 and cry1 + 4 + 11 in Taipei area; cry1A + 1C + 1F in Taichung area; cry1E and cry1A + 1B + 1I on Yang Ming Mountain; cry1 + 13, cry1 + 2 + 11 and cry1 + 2 + 13 on Snow Mountain; and cry1 + 5 and cry1 + 2 + 5 on Jade Mountain. These data clearly indicate that the distribution of cry gene combinations of B. thuringiensis isolates seems to be geographically related.
In addition, five pairs of degenerate universal primers have been designed to identify the general protease gene profiles from some distinct B. thuringiensis strains. Based on the PCR amplification patterns and DNA sequences of the cloned fragments, it was noted that the protease gene profiles of the three distinct strains of B. thuringiensis subsp. kurstaki HD73, tenebrionis and israelensis T14001, are varied. Seven protease genes, neutral protease B (nprB), intracellular serine protease A (ispA), extracellular serine protease (vpr), envelope-associated protease (prtH), neutral protease F (nprF), thermostable alkaline serine protease and alkaline serine protease (aprS), with known functions were identified from three distinct B. thuringiensis strains. Based on the alignment of the derived protein sequences with the protein domain database, it suggested that at least one unknown gene sequence, yunA, might be highly protease-related.
Moreover, an intracellular serine protease A (ispA)-deficient mutant of B. thuringiensis subsp. kurstaki HD73 was created by homologous recombination. It was characterized that sporulation process of the mutant is delayed. In order to reveal the effect of IspA on the process of sporulation, attempt has been made to compare the protein profiles between the wild-type and mutant-type strains using two-dimensional gel electrophoresis (2-DE). In addition, proteins with apparently different expression were characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF MS). Based on database analysis, several sporulation-associated proteins, denoted as pro-σK, SpoVD and SpoVR, were identified. Among them, pro-σK, which is involved in σK-signaling for sporulation, was up regulated, while SpoVD and SpoVR, which are involved in the formation of spore cortex, were down regulated in the ispA-deficient mutant. Therefore, we propose that the delay phenomenon of sporulation process of the mutant might be due to the failure of pro-σK processed to its mature form as well as the deficiency of SpoVD and SpoVR for cortex formation.
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