Bacterium strain EVA17 was isolated from an oil-contaminated soil, and identified as Sphingomonas sp. based on analysis of 16S rDNA sequence,cellular fatty acid composition and physiological-chemical tests. The salicy...Bacterium strain EVA17 was isolated from an oil-contaminated soil, and identified as Sphingomonas sp. based on analysis of 16S rDNA sequence,cellular fatty acid composition and physiological-chemical tests. The salicylate hydroxylase and catechol 2,3-dioxygenase(C23O) were detected in cell-free lysates, suggesting a pathway for phenanthrene catabolism via salicylate and catechol. Alignment showed that both of the C23O and GST genes of the strain EVA17 had high similarity with homologues of strains from genus Sphingomonas. The phylogenetic analysis based on 16S rDNA and C23O gene sequence indicated that EVA17 should be classified into genus Sphingomonas, although the two phylogenetic trees were slightly different from each other. The results of co-amplification and sequence determination indicated that GST gene should be located upstream of the C23O gene.展开更多
Although CRISPR/Cas9 has been widely used in insect gene editing,the need for the microinjection of preblastoderm embryos can preclude the technique being used in insect species with eggs that are small,have hard shel...Although CRISPR/Cas9 has been widely used in insect gene editing,the need for the microinjection of preblastoderm embryos can preclude the technique being used in insect species with eggs that are small,have hard shells,and/or are difficult to collect and maintain outside of their normal environment.Such is the case with Sogatella furcifera,the white-backed planthopper(WBPH),a significant pest of Oryza sativa(rice)that oviposits inside rice stems.Egg extraction from the stem runs the risk of mechanical damage and hatching is heavily influenced by the micro-environment of the rice stem.To bypass these issues,we targeted embryos prior to oviposition via direct parental(DIPA)-CRISPR,in which Cas9 and single-guide RNAs(sgRNAs)for the WBPH eye pigment gene tryptophan 2,3-dioxygenase were injected into the hemocoel of adult females.Females at varying numbers of days posteclosion were evaluated to determine at what stage their oocyte might be most capable of taking up the gene-editing components.An evaluation of the offspring indicated that the highest G0 gene-edited efficacy(56.7%)occurred in females injected 2 d posteclosion,and that those mutations were heritably transmitted to the G1 generation.This study demonstrates the potential utility of DIPA-CRISPR for future gene-editing studies in non-model insect species and can facilitate the development of novel pest management applications.展开更多
文摘Bacterium strain EVA17 was isolated from an oil-contaminated soil, and identified as Sphingomonas sp. based on analysis of 16S rDNA sequence,cellular fatty acid composition and physiological-chemical tests. The salicylate hydroxylase and catechol 2,3-dioxygenase(C23O) were detected in cell-free lysates, suggesting a pathway for phenanthrene catabolism via salicylate and catechol. Alignment showed that both of the C23O and GST genes of the strain EVA17 had high similarity with homologues of strains from genus Sphingomonas. The phylogenetic analysis based on 16S rDNA and C23O gene sequence indicated that EVA17 should be classified into genus Sphingomonas, although the two phylogenetic trees were slightly different from each other. The results of co-amplification and sequence determination indicated that GST gene should be located upstream of the C23O gene.
基金supported by:the National Natural Science Foundation of China(grants 32370527,for PH,and 32260671,for MH)the Scientific Research Foundation of Guizhou University of China(grant 2017-33,for MH)+3 种基金the Program of Talent Cultivation of Guizhou University(grant(2019)05,for PH)the Science and Technology Support of Guizhou province(grant QKH(2017)2956,for MH)the Program of Introducing Talents of Discipline to Universities of China(111 Program,D20023)the Frontiers Science Center for Asymmetric Synthesis and Medicinal Molecules,Department of Education,Guizhou Province(Qianjiaohe KY number(2020)004).
文摘Although CRISPR/Cas9 has been widely used in insect gene editing,the need for the microinjection of preblastoderm embryos can preclude the technique being used in insect species with eggs that are small,have hard shells,and/or are difficult to collect and maintain outside of their normal environment.Such is the case with Sogatella furcifera,the white-backed planthopper(WBPH),a significant pest of Oryza sativa(rice)that oviposits inside rice stems.Egg extraction from the stem runs the risk of mechanical damage and hatching is heavily influenced by the micro-environment of the rice stem.To bypass these issues,we targeted embryos prior to oviposition via direct parental(DIPA)-CRISPR,in which Cas9 and single-guide RNAs(sgRNAs)for the WBPH eye pigment gene tryptophan 2,3-dioxygenase were injected into the hemocoel of adult females.Females at varying numbers of days posteclosion were evaluated to determine at what stage their oocyte might be most capable of taking up the gene-editing components.An evaluation of the offspring indicated that the highest G0 gene-edited efficacy(56.7%)occurred in females injected 2 d posteclosion,and that those mutations were heritably transmitted to the G1 generation.This study demonstrates the potential utility of DIPA-CRISPR for future gene-editing studies in non-model insect species and can facilitate the development of novel pest management applications.
