| Melanin is ubiquitous in nature. It protects protein from degradation, and also has the ability of photon screening or chemical protection, especially resistance to U.V. and radiation. Bacillus thuringiensis, is a ubiquitous gram-positive, spore-forming bacterium that could forms a parasporal crystal during the stationary phase of its growth cycle. As an extensively applied biopesticide, B. thuringiensis has its fatal defaults. For example, the insecticidal crystal protein(ICP), a central insecticidal factor, is easily destroyed by the U.V. of sunlight, and then weakening its potency; melanin can resolve this problem efficiently. On the other hand, B. thuringiensin could produce multi insecticidal crystal protein. Although the diversity of toxin is paralleled by a diversity in pesticidal activity, each class is specific to insect classified to a certain ordo. Therefore, a recombinant with muti-pesticidal activity for a given purpose is desirable.B.thuringiensin has the potency to produce mlanin with high temperature induction. To study the regulation machnism of melanin under high temperature, two strategy were performed. Firstly, a mutant library of B.thuringiensis 4CA1 was constructed with plasmid pIC333 bearing mini-Tn10. Secondly, the genes related to melanin production were interrupted.1. A Bacillus thuringiensis host with high melanin production, BMB181, was obtained after subculturing Bacillus thuringiensis host BMB171 several generations under elevated temperature. The BMB181 possess high yield of melanin reaching 8.55 mg/mL, which is the maximum compared to the reported B. thuringiensis strains with melanin production. Furthermore, the BMB181 could synthesize melanin without additional tyrosine. The electroporation efficiency of BMB181 is 106 cfu/μg. The optical microscope and SDS-PAGE experimental results show that the insecticide crystal protein (ICP) CryIAc10 which is highly toxic to Lepidoptera pest could synthesize efficiently in BMB181. Bioassay results demonstrated that the LC50 value of recombinant BMB32(cry1Ac10/BMB181) was 1.37μg/mL against Helicoverpa armigera while the control recombinant BMB31 (cry1Ac10/BMB171) was 25.85 ug/mL after 4 h ultraviolet radiation (U.V.). It could be concluded that the BMB181 could served as an optimal host for B.thuringiensis preparation with powerful U.V. resistance.2. The mutant library of B. thuringiensis subsp.yosoo 4CA1 was constructed with plasmid pIC333. A strain with weakened melanin production compared to the wide strain 4CA1 at 42℃was obtained on LB plate with 1% tyrosine. Then the flanking sequence of mini-Tn10 was cloned and sequenced. The result showed that the sodA2 gene was interrupted by mini-Tn10. To study the relations between melanin production and sodA2 gene, a double-exchange vector pBMB1252 was constructed. There mutants with Kanamycin resistance were obtained. The mutants showed attenuated melanin production compared to 4CA1 at 42℃.3. Tyrosinase gene was a key gene of melanin synthesis in B. thuringiensis according to the published literature. And this gene was ubiquitous in about 100 strains of B. thuringiensis by PCR technique. The 4CA1 of Bacillus thuringiensis subsp.yosoo produced melanin obviously at 42℃. However, the tyrosinase gene couldn’t be amplified by PCR technique while could be amplified from BMB181 and BMB171. To confirm the function of tyrosinase gene in melanin production, a double-exchange vector pBMB1251 was constructed in this study. Then the tyrosinase gene was interrupted in BMB171 and BMB181. BMB1225 and BMB1226 of tyrosinase deletion mutants(Δtyr) were got respectively. The capacity of melanin production was tested. The result showed that no difference was detected between theΔtyr mutants and the parents. By bioinformatics analysis, an ORF in BMB171 contains conserved dormain of laccase. A hypothesis was proposed that there maybe other melanin synthesis pathway in B. thuringiensis. |
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