Gene expression quantification at mRNA level is very important for postgenomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expressio...Gene expression quantification at mRNA level is very important for postgenomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction （qRT-PCR）, require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT- PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 （represented by sw22934, sw14876, and sw13956） were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin （sw22671）, the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.展开更多
The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified bas...The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgol and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5＇ end of the small RNA guide strand and of the conserved （aspartate, aspartate and histidine [DDH]） motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.展开更多
文摘Gene expression quantification at mRNA level is very important for postgenomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction （qRT-PCR）, require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT- PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 （represented by sw22934, sw14876, and sw13956） were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin （sw22671）, the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.
文摘The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgol and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5＇ end of the small RNA guide strand and of the conserved （aspartate, aspartate and histidine [DDH]） motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.
