[Objective] MicroRNAs (miRNAs) are small (approximately 22 nt),noncoding RNA molecules that play important roles through post-transcriptional regulation in a wide range of biological process.As an essential component of miRNA silencing complex (RISC),Argonaute1 (AGO1) protein plays a key role in miRNA regulatory pathway.The purpose of this study is to explore the biological function of LmAGO1,and determine its impacts on growth and development in the migratory locust.These results will provide an important theoretical basis for biogenesis mechanism of insect miRNAs and effective pest control.[Method] The cDNA sequence of LmAGO1 was identified from the locust transcriptome database by using bioinformatics approaches,and was translated into protein sequence using online protein translation software (ExPASy).The conserved domains were analyzed by SMART based on the deduced protein sequence.A phylogenetic tree of insect AGO was constructed with the locust AGO1,the homologous amino acid sequences from Bombyx mori,Drosophila melanogaster,Tribolium castaneum and other insects using the Phyml program.The double-strand RNAs (dsRNAs) of AGO1 gene were synthesized in vitro using T7 RiboMAXTM Express RNAi System.The RNA interference (RNAi) experiment was preformed to explore the biological function of LmAGO1 during growth and development in locusts.The dsLmAGO1 was injected into the locust nymphs on day 2 of the 4th-instar and 5th-instar stages,respectively.The injection of dsGFP was used as the control.At the 48 h and 72 h after dsRNA injection,the whole body of locust nymphs was collected for total RNA isolation and cDNA synthesis.The reverse transcript quantitative PCR (RT-qPCR) analyses of the LmAGO1 were performed to determine the gene silencing efficiency.In order to evaluate the influences of the LmAGO1 RNAi on miRNA biogenesis,the five abundantly expressed miRNAs were selected to quantify their expression level by using RT-qPCR after dsLmAGO1 injection.[Result] The amino acid analysis showed that LmAGO1 protein of locust contains 845 amino acids and has the typical AGO protein family conserved domains,including PAZ (Piwi-Argonaute-Zwille) domain which is located in the region from nucleotide 213 to 348 and PIWI (P-element Induced Wimpy Testis) domain which is located in the region from nucleotide 502 to 804.The phylogenetic analysis showed that LmAGO1 was clustered with the AGO 1 s of other insect species.The multiple protein sequence alignments indicated that the amino acid sequence identity is 82.2％ between LmAGOland DmAGO1,and is 86.9％ between LmAGO1 and BmAGO1,respectively;RNAi results showed that the expression of LmAGO1 was significantly reduced at 48 h and 72 h after dsLmAGO1 injection into locust nymphs.The silencing efficiency of LmAGO1 was 88.1％ and 93.0％,respectively.The locust nymphs with dsLmAGO1 treatment did not show any obvious phenotypic defects at the 4th-instar stage.However,compared with dsGFP injected control,the dsLmAGO1 treatment resulted in a high mortality at the 5th-instar stage.A total of 89.3％ locust nymphs were dead in dsLmAGO1 injected group.The RT-qPCR results of LmAGO1 showed that LmAGO1 was significantly silenced at 48 h and 72 h after dsRNA injection.Moreover,the expression analysis of miRNA results indicated that the expression levels of miRNA-252 and miRNA-8 were significantly decreased at 48 h after dsLmAGO1 treatments,and that the expressions of miRNA-7,let-7,miRNA-252 and miRNA-8 were significantly down-regulated at 72 h after dsLmAGO1 treatments.[Conclusion] Besides the role in RSIC formation,LmAGO 1 can enhance the processing of miRNA maturation and regulate the normal development in the migratory locust.
国家科技期刊平台
NSTL
维普
万方数据
