| Argonaute protein are the catalytic core components of the RNAi effector complexes, binding to different small regulatory RNAs such as small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs). In eukaryotes, AGO proteins can be classified into two paralogous subfamilies including AGO subfamily and Piwi subfamily. The AGO subfamily is ubiquitously expressed and involved in cytoplasmic RNAi processes by interacting with miRNAs (miRNA pathway) or siRNAs (siRNA pathway) processed by RNase Ⅲ endonuclease, whereas Piwi subfamily proteins are expressed specifically in the germline cells and function in silencing by interacting with piRNAs. Although the AGO proteins of some insects have been investigated, such as Nilaparvata lugens, Bombyx mori and Anopheles stephensi, the AGOs in most insect species remain unexplored. The small brown planthopper (Laodelphax striatellus; family Delphacidae, order Hemiptera) is one of the most economically important insects mainly because of its ability to efficiently transmit two agriculturally important rice viruses:rice black-streaked dwarf virus (RBSDV) and rice stripe virus (RSV). Using a combination of bio informatics and RACE methods, putative AGO subfamily and Piwi subfamily members were identified, cloned and comprehensively characterized from L. striatellus.1. Genome wide Identification and cloning of AGOs in L. striatellus.EST of two AGO subfamily members (Is-AGO1 and ls-AGO2) and two Piwi subfamily members (ls-AGO3 and ls-Piwi) were identified using local tBLASTn against assembled transcriptome of L. striatellus with AGO sequences of N. lugens, D. melanogaster and B. mori. Complete open reading frame (ORF) of these four genes were further obtained by 5’ and 3’ race technology. ORF of ls-AGO1 is 2,820 bp long, encoding a putative protein of 939 amino acid residues, while ls-AGO2 contains an ORF of 2,490 bp, encoding 829 amino acid residues. ORF length of ls-Piwi is 2,508 bp (encoding 835 amino acid residues) and ls-AGO3 is 2,688 bp (encoding 895 amino acid residues). The expected conserved PAZ and PIWI domains, and the conserved DDH catalytic triad motif in the PIWI domain were observed in both AGO and Piwi subfamilies of L. striatellus. Phylogenetic analysis of the PIWI domain of Is-AGO1, ls-AGO2, ls-Piwi, ls-AGO3 and the AGOs derived from other insects indicated the classification of AGO and Piwi subfamilies of L. striatellus in their respective clades.2. Expression profiles of ls-AGO1 and ls-AGO2 at different developmental stages and in response to various stressesTo obtain a preliminary understanding of the potential roles of AGO and Piwi subfamilies, expression profiles of L. striatellus at different development stages and in response to various stresses were extensively investigated in this study. Both genes were expressed at all the developmental stages. mRNAs of ls-AGO1 and ls-AGO2 were most abundant in eggs, followed by adults, high instar nymphs and low instar nymphs, suggesting a potentially important role for AGOs in the regulation of egg development by targeting maternal mRNAs. High expression of ls-Piwi and ls-AGO3 were observed in male adults compare to female ones, indicating key roles of Piwi subfamily for the regulation of sperm and egg occur in L. striatellus. Surprisingly, significantly decreased expression of both Is-AGO1 and ls-AGO2 was observed in adult L. striatellus infected by RBSDV. We speculate that adult L. striatellus may represent the later stages of RBSDV infection when the efficiency of the anti-viral RNA silencing could be overwhelmed by viral load. Effects of thermal stresses and host shift on the expression of L. striatellus AGOs also suggested that AGO and Piwi subfamilies are actively respond to various stresses. Thus AGO genes may be involved in regulating the stress-response of L. striatellus by controlling related gene expression.3. Polyclonal antibody production for ls-AGO1 and ls-AGO2Peptides corresponding to the amino acid sequence of ls-AGOl and ls-AGO2 gene was designed based on the result of epitope analysis. The peptide was used to immunize rabbits and antibodies were obtained by ammonium sulfate precipitation and protein A preparative chromatography. Highly efficiency of antibodies were confirmed by ELISA and Western blot.In summary, AGO and Piwi subfamilies of L. striatellus were cloned and characterized for the first time in this study. The expression patterns of AGOs in different developmental stages and in response to various response were also explored. These results indicating the involvement of siRNA/miRNA and Piwi pathway involved in these process and providing fundamental clues to further investigate the accurate functions AGOs in L. striatellus. In addition, production of polyclonal antibodies of Is-AGO 1 and ls-AGO2 will facilitate the study on the AGO-binding small RNA which will contribute to a better understanding for the anti-viral mechanism in L. striatellus. |
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