Erwinia persicina is a bacterium that has been known to produce secondary metabolites,such as andrimid,pink pigment,and exopolysaccharides,and to infect more than twenty plant species.However,traditional gene manipula...Erwinia persicina is a bacterium that has been known to produce secondary metabolites,such as andrimid,pink pigment,and exopolysaccharides,and to infect more than twenty plant species.However,traditional gene manipulation methods have been hindered by the inefficient of suicide plasmid-mediated genome editing.In this study,we describe the successful application of the CRISPR-Cas9 system in E.persicina.Efficient genome editing was achieved by substituting the native gRNA promoter with J23119 in a single-plasmid system(pRed_Cas9_ΔpoxB)and optimizing the gRNA design.The use of double gRNAs led to the deletion of a 42 kb genomic fragment,and the incorporation of a sacB screening marker facilitated iterative knockouts.Additionally,a 22 kb plasmid containing a self-resistance gene was conjugally transferred into E.persicina,resulting in the insertion of a 6.4 kb fragment with 100%efficiency.Furthermore,we demonstrated the expression of shinorine,an anti-UV compound,within the E.persicina chassis.This study establishes E.persicina as a promising chassis for synthetic biology and provides a model for gene-editing systems in non-model microorganisms.展开更多
Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine...Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep se- quencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phyloge- nomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epi- plasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.展开更多
基金funding from the Hundred Talents Program of the Chinese Academy of Sciences to LZ(E3J56201).
文摘Erwinia persicina is a bacterium that has been known to produce secondary metabolites,such as andrimid,pink pigment,and exopolysaccharides,and to infect more than twenty plant species.However,traditional gene manipulation methods have been hindered by the inefficient of suicide plasmid-mediated genome editing.In this study,we describe the successful application of the CRISPR-Cas9 system in E.persicina.Efficient genome editing was achieved by substituting the native gRNA promoter with J23119 in a single-plasmid system(pRed_Cas9_ΔpoxB)and optimizing the gRNA design.The use of double gRNAs led to the deletion of a 42 kb genomic fragment,and the incorporation of a sacB screening marker facilitated iterative knockouts.Additionally,a 22 kb plasmid containing a self-resistance gene was conjugally transferred into E.persicina,resulting in the insertion of a 6.4 kb fragment with 100%efficiency.Furthermore,we demonstrated the expression of shinorine,an anti-UV compound,within the E.persicina chassis.This study establishes E.persicina as a promising chassis for synthetic biology and provides a model for gene-editing systems in non-model microorganisms.
基金This work was supported by the Natural Science Foundation of China （Grant Nos. 31430077, 91131013, and 31272285）. Many thanks are given to Prof. Weibo Song （Ocean University of China）, for his indispensable advice during the preparation of the manuscript.
文摘Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep se- quencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phyloge- nomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epi- plasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.
