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The principle of stem-loop RT-PCR in present paper: (a) Stem-loop primer was designed to increase the length of mature miRNAs. PCR forward primer was designed by putative miRNAs, the universal reverse primer was designed by stem-loop primer. The box formed by dotted line represents the bases not included in primers; (b) Six nucleotides of 3′ end of stem-loop RT primer are complementary with 3′ end of mature miRNAs of D. melanogaster. Two bases, three bases and four bases were not included in RT-PCR primers of miR-14, miR-2a and bantam, respectively (colored red), thus miRNAs or non-specific amplification could be distinguished by sequence analysis.
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The indigenous small non-coding RNAs, known as microRNAs (miRNAs), are important regulators of gene expression and many of them are evolutionarily conserved. Whether stem-loop RT-PCR, as a sensitive method, could be utilized to clone conserved miRNAs from non-model insects lacks information. Here, three miRNAs, sli-miR-14, sli-miR-2a and sli-bantam...
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... nucleotides of 3′ end of stem-loop RT primer were complementary with 3′ end of mature miRNAs, other nucleotides of RT primer were partially complementary with themselves to form a stem, and the remaining nucleotides form a loop. The PCR reverse primer was universal, while the forward primer was designed by mature miRNAs (Figure 6a). In order to make a distinction between specific and non-specific amplifications, only partial bases of miRNA were contained in forward primer. ...Context 2
... order to make a distinction between specific and non-specific amplifications, only partial bases of miRNA were contained in forward primer. Two bases, three bases and four bases were not included in primers of miR-14, miR-2a and bantam, respectively (Figure 6b). The designing of stem-loop RT primers often presents learners with difficulties in some way; the primers designed in this assay also provide references for later research on other miRNAs. ...Similar publications
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... Briefly, total RNA was extracted from mixed tissues including gill, hepatopancreas, stomach, and muscle of L. vannamei using Trizol reagent (Ambion, Austin, TX, USA), and cDNA was then synthesized with stem-loop primer of miR-10c-RT (Table 1) using PrimeScript RT reagent kit (Takara, Japan). To verify the sequence of miR-10c, the mature miRNA sequence was cloned and sequenced with primers of miR-10c-IF and miR-10c-IR as previously described (24). ...
Toll-like receptors (TLRs) are canonical cell membrane receptors functioning to recognize pathogens and transduce signals to activate immune responses. It has been known that Toll3 in Pacific white shrimp Litopenaeus vannamei (LvToll3) plays a critical role in antiviral immunity by inducing the transcription of interferon regulatory factor (IRF), which mediates a signaling axis that is similar to the interferon system of vertebrates. However, the regulatory mechanism of the Toll3-IRF signaling is still unclear. In this study, a novel microRNA (miRNA) of miR-10 family, temporarily named as miR-10c, was identified from L. vannamei. miR-10c may play a nonnegligible regulatory role in shrimp immune responses since it was constitutively expressed in all detected tissues and transcriptionally induced by immune stimulation. Functional analysis validated that miR-10c could target LvToll3 to inhibit its expression, through which miR-10c blocked the nuclear translocation of IRF and facilitated white spot syndrome virus (WSSV) infection. To our knowledge, the present study revealed the first report of a Toll targeted by miRNA in crustaceans and provided a solid evidence base for supporting the role of LvToll3 in antiviral defense by activating IRF signaling in L. vannamei. Identification of the miR-10c/Toll3/IRF regulatory axis in shrimp provides new insights into the participation of miRNA in the regulation of immune responses and contributes to in-depth understanding of the mechanisms of Toll-induced immune responses in L. vannamei.
... Published work [14] and our early data showed that microRNA bantam was among the few most abundant microRNAs in Sf9 cells. bantam is an insect-specific microRNA identified in Drosophila, B. mori [32], and some other insects [33,34]. It has also been reported in blood fluke Schistosoma japonica [35,36]. ...
The role of microRNA bantam, one of the most abundant microRNAs in Sf9 cells, was studied for its role in baculovirus infection in vitro and in vivo. The expression level of bantam was increased after AcMNPV infection in Sf9 cells and in Spodoptera litura larvae. In Sf9 cells, application of bantam inhibitor or mimic altered the expression of many virus genes, the most affected gene being lef8, gp41 and p10, the expression level of which was increased by 8, 10 and 40 times, respectively, in the presence of bantam inhibitor. Virus DNA replication was decreased in the presence of bantam mimic and increased in the presence of bantam inhibitor in a dose dependent manner. However, the production of budded virus did not change significantly. Feeding the larvae of S. litura and Spodoptera exigua with bantam antagomiR, a more stable form of the inhibitor, resulted in an abnormal larval growth and a decreased pupation rate. In S. litura, larvae died 3.5 days sooner than the control when bantam antagomiR was applied, together with AcMNPV. In infected S. exigua, larval mortality increased from 47% without antagomiR to 80% with it. The results suggest that microRNA bantam plays an important role in insect growth, as well as in baculovirus-insect interaction.
... Because miRNAs are involved in regulating development and metamorphosis in insects [4,[12][13][14][15][16][17][18], the identification of miRNAs in these two pests will help to uncover their physiological roles and provide potential targets for pest control. In S. litura, many miRNAs have been identified by computational prediction or stem-loop polymerase chain reaction (PCR) [19,20]. However, deficient genome sequences have restricted the further identification of miRNAs. ...
... The precursors of the miRNA homologs from species in the same genus were more analogous. These results are in accordance with a previously published study on highly conserved miRNAs in S. litura [19]. Thus, when B. mori pre-miRNAs and mature miRNAs are used as references for lepidopteran miRNA identification, conserved miRNAs in H. armigera and S. litura can be obtained. ...
The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.
... S. exigua larvae were reared at 26 °C with an L14:D10 photoperiod using an artificial diet [31][32][33]. The developmental stages were synchronized at each molt by collecting new larvae or pupae. ...
Elongation factor (EF) is a key regulation factor for translation in many organisms, including plants, bacteria, fungi, animals and insects. To investigate the nature and function of elongation factor 1β' from Spodoptera exigua (SeEF-1β'), its cDNA was cloned. This contained an open reading frame of 672 nucleotides encoding a protein of 223 amino acids with a predicted molecular weight of 24.04 kDa and pI of 4.53. Northern blotting revealed that SeEF-1β' mRNA is expressed in brain, epidermis, fat body, midgut, Malpighian tubules, ovary and tracheae. RT-PCR revealed that SeEF-1β' mRNA is expressed at different levels in fat body and whole body during different developmental stages. In RNAi experiments, the survival rate of insects injected with SeEF-1β' dsRNA was 58.7% at 36 h after injection, which was significantly lower than three control groups. Other elongation factors and transcription factors were also influenced when EF-1β' was suppressed. The results demonstrate that SeEF-1β' is a key gene in transcription in S. exigua.
Spodoptera litura is a destructive agricultural pest in tropical and subtropical areas. Understanding the molecular mechanisms of S. litura adaptation to its preferred host plants may help identify target genes useful for pest control. We used high‐throughput sequencing to characterize the expression patterns of mRNAs and microRNAs (miRNAs) in the midgut of S. litura fed on Brassica juncea for 6 h and 48 h. A total of 108 known and 134 novel miRNAs were identified, 29 miRNAs and 237 mRNAs were differentially expressed at 6 h of B. juncea feeding, 26 miRNAs and 433 mRNAs were differentially expressed at 48 h. For the mRNAs, the up‐regulated genes were mostly enriched in detoxification enzymes (cytochrome P450, esterase, glutathione S‐transferase, UDP‐glucuronosyl transferase), while the down‐regulated genes were mostly enriched in proteinases and immune related genes. Furthermore, most detoxification enzymes begin to up‐regulated at 6 h, while most digestion and immune related gene begin to up or down‐regulated at 48 h. 18 and 37 differently expressed transcription factors were identified at 6 h and 48 h, which may regulate the functional genes. We acquired 136 and 41 miRNA vs mRNA pairs at 6 h and 48 h, respectively. Some down‐regulated and up‐regulated miRNAs were predicted to targeting detoxification enzymes and proteinases, respectively. RT‐qPCR of 9 randomly selected miRNAs and 28 genes confirmed the results of RNA‐seq. This analyses of miRNA and mRNA transcriptomes provided useful information about the molecular mechanisms of S. litura response to B. juncea.
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MicroRNAs (miRNAs) constitute some of the most significant regulatory factors involved at the post-transcriptional level after gene expression, contributing to the modulation of a large number of physiological processes such as development, metabolism, and disease occurrence. This review comprehensively and retrospectively explores the literature investigating silkworm, Bombyx mori L. (Lepidoptera: Bombicidae), miRNAs published to date, including discovery, identification, expression profiling analysis, target gene prediction, and the functional analysis of both miRNAs and their targets. It may provide experimental considerations and approaches for future study of miRNAs and benefit elucidation of the mechanisms of miRNAs involved in silkworm developmental processes and intracellular activities of other unknown non-coding RNAs.
