To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the co...To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.展开更多
Objective:To investigate the larvicidal and pupicidal activity of spinosad against Anopheles stephensi Listen.Methods:Spinosad from the actinomycete,Saccharopolyspora spinosa was tested against Anopheles stephensi at ...Objective:To investigate the larvicidal and pupicidal activity of spinosad against Anopheles stephensi Listen.Methods:Spinosad from the actinomycete,Saccharopolyspora spinosa was tested against Anopheles stephensi at different concentrations（0.01,0.02,0.04,0.06 and 0.08 ppm.）, and against first to fourth instar larvae and pupae.Results:The larval mortality ranged from 36.1±1.7 in（0.01 ppm） to 79.3±1.8（0.08 ppm） the first instar larva.The LC<sub>50</sub> and LC<sub>90</sub> values of first, second,third and fourth instar larva were 0.001,0.031,0.034,0.036 and 0.0113,0.102,0.111,0.113, respectively.The pupal mortality ranged from 33.0±2.0（0.01 ppm） to 80.0±0.9（0.08 ppm）.The LC<sub>50</sub> and LC<sub>90</sub> values were 0.028 and 0.1020,respectively.The reduction percentage of Anopheles larvae was 82.7%,91.4%and 96.0%after 24,48,72 hours,respectively,while more than 80% reduction was observed after 3 weeks.Conclusions:In the present study spinosad effectively caused mortality of mosquito larvae in both the laboratory and field trial.It is predicted that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.展开更多
基金supported by the National Basic Research Program of China (Grant Nos. 2012CB722301 and 2011CB111605)the National High Technology Research and Development Project of China (Grant No. 2011AA10A203)the National Natural Science Foundation of China (Grant No. 31070006)
文摘To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.
文摘Objective:To investigate the larvicidal and pupicidal activity of spinosad against Anopheles stephensi Listen.Methods:Spinosad from the actinomycete,Saccharopolyspora spinosa was tested against Anopheles stephensi at different concentrations（0.01,0.02,0.04,0.06 and 0.08 ppm.）, and against first to fourth instar larvae and pupae.Results:The larval mortality ranged from 36.1±1.7 in（0.01 ppm） to 79.3±1.8（0.08 ppm） the first instar larva.The LC<sub>50</sub> and LC<sub>90</sub> values of first, second,third and fourth instar larva were 0.001,0.031,0.034,0.036 and 0.0113,0.102,0.111,0.113, respectively.The pupal mortality ranged from 33.0±2.0（0.01 ppm） to 80.0±0.9（0.08 ppm）.The LC<sub>50</sub> and LC<sub>90</sub> values were 0.028 and 0.1020,respectively.The reduction percentage of Anopheles larvae was 82.7%,91.4%and 96.0%after 24,48,72 hours,respectively,while more than 80% reduction was observed after 3 weeks.Conclusions:In the present study spinosad effectively caused mortality of mosquito larvae in both the laboratory and field trial.It is predicted that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.
