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Bmo-miR-3377-5p down-regulates the Bombyx mori Sericin gene-1
Kandhro Rehana,Tao Jianga,Yanhua Chen,Juan Zhu,Shunming Tang,Xingjia Shen 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.3
MiRNAs are small non-coding molecules, which can regulate a huge number of genes. Based on bioinformatics analysis, we found a target site in the 3′UTR of BmSer-1 for binding bmo-miR-3377-5p. By using semi-quantitative RT-PCR, we detected that miR-3377-5p and BmSer-1 were both more highly expressed in the middle silk gland than in other tissues of 3-day-old fifth-instar Bombyx mori larvae, implying that there is a spatiotemporal condition for miR-3377-5p regulating on BmSer-1. To confirm this prediction, a BmSer-13′UTR recombinant luciferase reporter pGL3.0 [A3-luc-BmSer-1-3′UTR-SV40] and pri-bmo-miR-3377-5p expression pcDNA3.0 [ie1-egfppri-bmo-miR-3377-5p-SV40] were constructed and co-transfected into B. mori ovary cells (BmN cells). The results showed that miR-3377-5p suppressed the expression of BmSer-1 significantly (P < .001). When BmN cells were co-transfected by an artificial inhibitor together with a miR-3377-5p expression vector and a BmSer-1-3′UTR recombinant plasmid, BmSer-1 expression increased significantly (P < .05), indicating that the inhibitor was active against miR-3377-5p, and expression of BmSer-1 was recovered. Moreover, we injected miR-3377-5p expression plasmid and bmo-miR-3377-5p inhibitor into 3-day-old fifth-instar larvae. At 36 h post-injection, silk glands were collected for total RNA extraction. Quantitative RT-PCR analysis showed that miR-3377-5p downregulated the expression of BmSer-1 in vivo, while there was no significant difference inhibitor treatment group compared with NC. Thus, we conclude that miR-3377-5p down-regulated the expression of BmSer-1. Our results provide insight for understanding the function of miRNAs and the regulation network of silk protein genes.
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Ping Qian,Xin Wang,Tao Jianga,Fei Song,Chen Chen,Yangyang Fan,Xing-jia Shen 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.2
MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that serve as important posttranscriptional gene expression regulators and play important roles in the silkworm (Bombyx mori) development, growth, and viral immunity. However, information on the diversity of these regulatory RNAs in the middle silk gland (MSG) of naked pupa (Nd) mutant silkworms is limited. In this study, by using Solexa high-throughput sequencing technology, we identified and compared small RNA libraries from the MSG of wild-type silkworm P50(MSG-P50) and the Nd mutant (MSG-Nd), respectively. A total of 272 conserved and 333 novel miRNAs were identified, in which 141 ones showed significantly different expression patterns between MSG-P50 and MSG-Nd, and 10 ones were randomly selected and validated by stem-loop quantitative reverse-transcription polymerase chain reaction (qRT-PCR). In addition, potential targets were predicted for differentially expressed miRNAs based on sequence complementation between miRNAs and their target genes. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed miRNAs that actively participate in various life processes and three pathways associated with protein synthesis including endoplasmic reticulum pathway, ribosome pathway, and ribosome biogenesis in eukaryotes, were significantly disrupted in MSG-Nd. This is the first comprehensive description of miRNAs in the silkworm MSG. Overall, the results provide useful information for future studies on miRNAs and suggest that the fibroin synthetic deficiency in the posterior silk gland impairs the sericin secretion process in MSG.
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