RNA interference of Argonaute-1 disrupts ovarian development and enhances insecticide susceptibility in Tribolium castaneum.

With the development of rapid resistance, new modes of action for pesticides are needed for insect control, such as RNAi-based biopesticides targeting essential genes. To explore the function of Argonaute-1 (Ago-1) and potential miRNAs in ovarian development of Bactrocera dorsalis, an important agricultural pest, and to develop a novel control strategy for the pest, BdAgo-1 was first identified in B. dorsalis. Spatiotemporal expression analysis indicated that BdAgo-1 had a relatively high transcriptional level in the ovarian tissues of adult female B. dorsalis during the sexual maturation period. RNA interference (RNAi) experiment showed that BdAgo-1 knockdown significantly decreased the expression levels of ovarian development-related genes and delayed ovarian development. Although RNAi-mediated silencing of Ago-1 led to a reduced ovary surface area, a subsequent oviposition assay revealed that the influence was minimal over a longer time period. Small RNA libraries were constructed and sequenced from different ovarian developmental stages of B. dorsalis adults. Among 161 identified miRNAs, 84 miRNAs were differentially expressed during the three developmental stages of the B. dorsalis ovary. BdAgo-1 silencing caused significant down-regulation of seven differentially expressed miRNAs (DEMs) showing relatively high expression levels (>1000 TPM (Transcripts per kilobase of exon model per million mapped reads)). The expression patterns of these seven core DEMs and their putative target genes were analyzed in the ovaries of B. dorsalis. The results indicate that Ago-1 and Ago-1-dependent miRNAs are indispensable for normal ovarian development in B. dorsalis and help identify miRNA targets useful for control of this pest.

PDF HTML阅读 XML下载 导出引用 引用提醒 青虾Ran基因的克隆、表达及其在卵巢发育中的功能 DOI: 作者: 作者单位: 1. 南京农业大学 无锡渔业学院, 江苏 无锡 214081;2. 中国水产科学研究院 淡水渔业研究中心, 农业部淡水渔业和种质资源利用重点实验室, 江苏 无锡 214081 作者简介: 卜宗元(1991-),男,硕士研究生,研究方向为水产动物遗传育种.E-mail:375334258@qq.com 通讯作者: 中图分类号: S917 基金项目: 江苏省重点研发计划（现代农业）重点项目（BE2016308）；中国水产科学研究院基本科研业务费专项项目（2016HY-ZD0402）；国家自然科学基金面上项目（31572617）；江苏省农业科技自主创新基金项目[CX（15）10124]；江苏省水产三新工程（D2015-16）；无锡科学科技发展基金项目（CLE02N1514） Molecular characterization and developmental expression of Ras-related nuclear protein in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference on ovarian maturation Author: Affiliation: 1. Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China;2. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture; Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:本研究应用RACE技术克隆了青虾（（Ras related nuclear protein，Ras 相关核蛋白）基因全长cDNA序列，该基因cDNA全长1191 bp，包括218 bp的5'UTR，648 bp的开放阅读框（ORF），405 bp的3'UTR，编码215个氨基酸。青虾基因属于P-loop-NTPase超级家族，拥有PTZ00132跨结构域，多肽分子量约为24.57 kDa，理论等电点7.13。系统进化树分析表明，在动物界进化中非常保守的青虾Ran多肽与罗氏沼虾（基因在成体青虾不同组织和卵巢不同发育期的表达差异，结果显示，基因的表达水平呈现上升趋势，在卵巢消退期又恢复到较低水平。RNA干扰后，实验组（vitellogenin）在卵巢中的表达量也显著低于对照组（基因的表达起到调控作用。 Abstract:Ras-related nuclear protein (Ran) is a small GTPase with many functions, such as hydrolysis of GTP, control of cell development, replication of DNA, and RNA transcription. In this study, the cDNA-encoding ) was cloned using expressed sequence tag (EST) analysis and a rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of was 1191 bp, comprising a 5' untranslated region of 218 bp, a 3' untranslated region of 405 bp, and an open reading frame of 648 bp. The deduced protein had 215 amino acid residues with a molecular mass of 24.6 kD, and 7.13 point of theoretical isoelectric. Ran belongs to the P-loop NTPase super family. Ran has a PTZ00132 model that crosses multiple domains. The members of P-loop NTPase super family have extremely conservative nucleotide sequences. Phylogenetic analyses indicate evolution of Ran proteins within the animal kingdom is very conservative, with that of . Quantitative real-time RT-PCR analysis revealed the gene was expressed in testis, ovary, brain, muscle, eyestalk, abdominal nerve, heart and gill tissues. The ovary has the highest level of expression and the eyestalk has the lowest level of expression ( gene expression of ovary is seven-eight times higher than that of testis, and the expression level of gene increased with the development of ovary. After ovulation in ovarian regression period, the expression level of gene was at a low level. After RNA interference (RNAi), expression of gene in an experimental group of adult females was significantly lower than in the control group (<0.05). After RNA interference (RNAi) in the mature female prawns, the expression of gene in experimental group (injected dsRNA solution into the shrimp's pericardial cavity) was significantly lower than in the control group (injected equal amount of DEPC water into the pranw's pericardial cavity) ( gene changes with the development of ovary. The expression of gene increased from the early stage of ovarian development to the mature stage and decreased rapidly after ovulation in the ovary. Expression of in ovarian tissues in the experimental group was also significantly lower than that of control group (<0.05), indicating RNA interference was effective. Expression of vitellogenin was significantly affected by RNA interference, with expression in the experimental group significantly lower than in the control group ( gene plays a regulatory role in expression of the gene is involved in female ovary development. 参考文献 相似文献 引证文献

Latrophilin participates in insecticide susceptibility through positively regulating CSP10 and partially compensated by OBPC01 in Tribolium castaneum

Many insect cytochrome P450 proteins (CYPs) are involved in the metabolic detoxification of exogenous compounds such as plant toxins and insecticides. Tribolium castaneum, the red flour beetle, is a major agricultural pest that damages stored grains and cereal products. With the completion of the sequencing of its genome, two T. castaneum species-specific CYP genes, CYP4BN6, and CYP6BQ11, were identified. However, it is unknown whether the functions of most CYPs are shared by TcCYP4BN6 and TcCYP6BQ11, and the upstream regulatory mechanism of these two CYPs remains elusive. QRT-PCR analysis indicated that TcCYP4BN6 and TcCYP6BQ11 were both most highly expressed at the late pupal stage and were mainly observed in the head and gut, respectively, of adults. Moreover, the transcripts of these two CYPs were significantly induced by dichlorvos and carbofuran, and RNA interference (RNAi) targeting of each of them enhanced the susceptibility of beetles to these two insecticides. Intriguingly, knockdown of the latrophilin (lph) gene, which has been reported to be related to the insecticide susceptibility, reduced the expression of TcCYP4BN6 and TcCYP6BQ11 after insecticide treatment, suggesting that these two CYP genes are regulated by lph to participate in insecticide susceptibility in T. castaneum. These results shed new light on the function and mechanism of CYP genes associated with insecticide susceptibility and could facilitate research on appropriate and sustainable pest control management. © 2019 Society of Chemical Industry.

BackgroundThe ovary is an important reproductive organ in mammals, and its development directly affects the sexual maturity and reproductive capacity of individuals. MicroRNAs (miRNAs) are recognized as regulators of reproductive physiological processes in various animals and have been shown to regulate ovarian development through typical targeting and translational repression. However, little is known about the regulatory role of miRNAs in ovarian tissue development during sexual maturity in goats. To comprehensively profile the different physiological stages of sexual maturation in goats, we performed small-RNA sequencing of ovarian tissue samples collected at four specific time points (1 day after birth (D1), 2 months old (M2), 4 months old (M4), and 6 months old (M6)). In addition, we used ELISAs to measure serum levels of reproductive hormones to study their temporal changes.ResultsThe results showed that serum levels of gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, oestradiol, progesterone, oxytocin, and prolactin were lower in goats at the D1 stage than at the other three developmental stages (P < 0.05). The secretion patterns of these seven hormones show a similar trend, with hormone levels reaching their peaks at 4 months of age. A total of 667 miRNAs were detected in 20 libraries, and 254 differentially expressed miRNAs and 3 groups of miRNA clusters that had unique expression patterns were identified (|log2-fold change|> 1, FDR < 0.05) in the 6 comparison groups. RT‒qPCR was employed to confirm that the expression pattern of the 15 selected miRNAs was consistent with the Illumina sequencing results. Gene ontology analyses revealed significant enrichment of GO terms such as cell proliferation regulation, epithelial cell development, and amino acid transport, as well as important signaling pathways including the MAPK signaling pathway, the PI3K-Akt signaling pathway, and the oestrogen signaling pathway. Further miRNA‒mRNA regulation network analysis revealed that 8 differentially expressed miRNAs (chi-miR-1343, chi-miR-328-3p, chi-miR-877-3p, chi-miR-296-3p, chi-miR-128-5p, chi-miR-331-3p, chi-miR-342-5p and chi-miR-34a) have important regulatory roles in ovarian cell proliferation, hormone secretion and metabolism-related biological processes.ConclusionsOverall, our study investigated the changes in serum hormone and miRNA levels in the ovaries. These data provide a valuable resource for understanding the molecular regulatory mechanisms of miRNAs in ovarian tissue during the sexual maturity period in goats.

Identification and ovarian developmental regulation of ribosomal protein S6 kinase in Macrobrachium nipponense.

Dynamic and Temporal Transcriptomic Analysis Reveals Ferroptosis-Mediated Antileukemia Activity of S-Dimethylarsino-Glutathione: Insights into Novel Therapeutic Strategy

Argonaute family genes encode a highly conserved group of proteins that have been associated with RNA silencing in both animals and plants. This study investigates the importance of microRNA biogenesis key regulators Argonaute1 (Ago1) and Gawky genes in the post-embryonic and ovarian development of the melon fly, Zeugodacus cucurbitae. The expression levels of these genes were mapped in all developmental stages and different adult tissues. Their roles in development were investigated using RNA interference (RNAi) via two different dsRNA delivery techniques. Embryo microinjection and oral feeding of third instar larvae successfully knocked down and greatly reduced the expression level of the target genes. Additionally, ex vivo essays revealed the stability of dsRNA in food was sufficient for gene silencing, although its integrity was affected in midgut. A wide range of phenotypes were observed on pupation, segmentation, pigmentation, and ovarian development. RNAi-mediated silencing of Gawky caused high mortality and loss of body segmentation, while Ago1 knockdown affected ovarian development and pigmentation. Developmental abnormalities and ovarian malformations caused by silencing these genes suggest that these genes are crucial for viability and reproductive capacity of Z. cucurbitae, and may be used as potential target genes in pest management.

In captivity, the Japanese eels can not undergo further gonadal development, and the population size of wild elvers had gradually decreased year by year. In the silver Japanese eel, artificial induction of gonadal development and maturation by injection of salmon pituitary homogenate (SPH) has been applied for 40 years. One of the major problems on the eel artificial maturation is that, the development of ovarian follicles are rather asynchronous during artificial maturation by SPH injection. But according to the previous in vivo induction data in our laboratory, although both of 17α-methyltestosterone (MT) combined with SPH (SPH+MT treatment) and SPH alone (SPH treatment) induction could promote ovarian development, SPH+MT enhanced the survival and synchronized ovarian follicles development. Previous results reveal that SPH is essential, and MT may directly regulate ovarian development during induction of sexual maturation. Current research focus on growth differentiation factor 9 (GDF-9) which is one of the oocyte-secreted factors, and it is essential for early follicle development. We have already obtained the cDNA encoding for GDF-9 from female Japanese eel. The overall sequence of GDF-9 cDNA isolated from Japanese eel contained 2135 bp, and this GDF-9 cDNA encodes 430 amino acids of peptide sequence. The deduced amino acid sequence shares 38 %-62 % identity with other teleost GDF-9. We propose that MT would interact with GDF-9 to regulate the SPH action in ovarian development during SPH and MT induction in Japanese eel, and GDF-9 might be regulated by androgen receptor. Cultivated female Japanese eels obtained from local supplier were acclimated to seawater. After acclimation, eels were intraperitoneal injected respectively with different exogenous hormone for 2 times and 6 times at 1-week interval. Another treatment, eels were intraperitoneal injected with exogenous hormone above combined with anti-androgen flutamide. Real-time RT-PCR (RT-qPCR) was employed to investigate the ovarian gene expression of GDF-9 and follicle stimulating hormone receptor (FSHR). Our data showed that the ovarian GDF-9 expression in SPH alone treatment declined significantly after two injections. By contrast, the supplement of MT (SPH+MT) could maintain the expression level after two injections. MT alone treatment could significantly increase the expression level compared with control group (SW control). The gene expression of GDF-9 and FSHR in hormone induction showed the significantly positive correlation, and it implied the interaction between GDF-9 and FSHR. Flutamide could significantly inhibit the SPH-induced GDF-9 expression. In SPH+MT group after six injections, most of larger follicles with larger diameter (280~300μm) were still at the oil droplet stage, on the other hand, those of follicles by SPH tretment with similar oocyte diameter (280μm) had entered the period of yolk accumulation. In conclusion, MT might induce GDF-9 expression and then increase FSHR expression level, and thus promoted folliculogenesis in smaller follicles within the ovary, and the steroidogenesis in larger follicles would be inhibited by the amount of GDF-9 expression. As result, pre-vitellogenic stage could be prolonged, so that oil droplets could be fully cumulated, egg quality could also be improved, and the development of ovarian follicles could be more synchronized. These results imply that the interaction of SPH and MT might be mediated through GDF-9 action to affect ovarian development in Japanese eel. However, when is the initiation of GDF-9 expression in the ovary of Japanese eel, and how does GDF-9 regulate the development of ovarain follicles in Japanese eel needs further elucidation.

Identification of ribosomal protein L24 (RPL24) from the oriental river prawn, Macrobrachium nipponense, and its roles in ovarian development

Anautogeny is a reproductive strategy by which females do not reproduce until they feed. Therefore, nutritional signals must inform the reproductive tissues, and cells that the organism has reached a nutritional status suitable for triggering reproductive processes. One of the possible pathways involved in anautogeny is the "target of rapamycin" (TOR) pathway, which has been described as connecting the nutritional status with growth, proliferation, and cancer. The German cockroach, Blattella germanica, is an anautogenous species whose vitellogenesis is governed by juvenile hormone. In the present report, we describe the cloning of TOR cDNA from B. germanica (BgTOR). Expression studies showed that BgTOR is expressed in adult female corpora allata and fat body. BgTOR knockdown using systemic RNAi in vivo produced a severe inhibition of juvenile hormone synthesis in adult female corpora allata, together with a reduction of mRNA levels corresponding to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, and HMG-CoA reductase. In addition, there was a reduction of vitellogenin mRNA in the fat body, and ovaries did not grow. Analysis of TOR expression in corpora allata of fed and starved females suggested that TOR is not regulated at the transcriptional level. Nevertheless, there was a reduction in HMG-CoA synthases and reductase mRNA in corpora allata (but not in the fat body) of starved females, together with a dramatic reduction of juvenile hormone production and ovary development. Taken together, our results indicate that TOR knockdown mimics starvation in terms of corpora allata activity, and suggest that nutritional signals that activate juvenile hormone biosynthesis and vitellogenin production are mediated by the TOR pathway.

Identification of Foxl2 in freshwater mussel Hyriopsis cumingii and its involvement in sex differentiation.

Objective To study the effects of tumor-associated calcium signal transducer-2 (TROP-2) gene small interfering RNA (siRNA) on adhesion, migration, and invasion of human gastric cancer cells.Methods Real time polymerase chain reaction (PCR) was used to detect the TROP-2 mRNA expression of human gastric cancer cell lines MGC-803, HC, C-27 and BGC-823. The cells with highest expression of TROP-2 were transfected with different doses of TROP-2 siRNA. The expression of TROP-2 mRNA and protein was detected by real-time quantitative PCR and immumoflureseence method. Cell adhesion, migration,and invasion were exmined by hoyden chamber, respectively. Results Cell line BGC-823 showed the highest elevation of TROP-2 mRNA among three gastric cancer cell lines. Real-time quantitative PCR and immumoflurescence method revealed that the expression of TROP-2 mRNA and protein was reduced in a time- and dose-dependent manner ( r = 0. 935 ; r = 0. 922). The ability of adhesion, migration and invasion of BGC-823 cells treated with TROP-2 siRNA was decreased as compared with control group (P <0. 01 ). Conclusion TROP-2 gene might play an important role in adhesion, migration, and invasion of human gastric cancer cells, siRNA targeted TROP-2 could effectively inhibit adhesion, migration, and invasion of human gastric cancer cells. Key words: Gastric carcinoma; TROP-2; RNA interference; Invasion

Clathrin-dependent endocytosis is a vesicular transport process by which cells take macromolecules from the extracellular space to the intracellular space. It plays important roles in various cellular functions, but its biological significance in insect development and reproduction has not been well studied. We characterized and functionally analyzed four major clathrin-dependent endocytic pathway genes (TcChc, TcAP50, TcVhaSFD, TcRab7) in Tribolium castaneum. RNA interference (RNAi) by injecting double-stranded RNA (dsRNA) targeting each gene at three doses (50, 100, or 200 ng per insect) in 20-day-old larvae led to 100% larval mortality. When the expressions of TcChc, TcVhaSFD, and TcRab7 were suppressed by injecting their respective dsRNAs at each dose in 1-day-old pupae, the adults that emerged from the dsRNA-injected pupae were deformed, with the absence of wing development. The deformed adults died within 2 days after eclosion. When the expression of TcAP50 was suppressed by injecting its dsRNA into 1-day-old pupae, although no apparent deformed adults were observed, all the adults died within 35 days after eclosion. In addition, when the expressions of TcChc and TcVhaSFD were suppressed by injecting their respective dsRNAs at a reduced dose (10ng per insect) in 5-day-old pupae, the ovarian development and oocyte production in the resultant females were completely inhibited. Our results indicate that clathrin-dependent endocytosis is essential for insect development and reproduction. The results from this study can help researchers identify potential molecular targets for developing novel strategies for insect pest management. © 2023 Society of Chemical Industry.

Choline acetyltransferase (Tcchat) is essential for metamorphosis, reproduction, and susceptibility to insecticide in Tribolium castaneum. However, the mechanisms through which Tcchat contributes to these biological processes remain poorly understood. Here we report the findings of RNA sequencing‐based transcriptome profiling of T. castaneum following RNA interference against Tcchat. In all, 173 differentially expressed genes were identified: 120 upregulated genes and 53 downregulated genes. Gene Ontology analysis classified the genes into 30 functional groups. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the genes were distributed over 22 different pathways. Silencing Tcchat reduced the activity of endopeptidase and inhibited regulation of protein decomposition in the beetles. Hence, knockdown of Tcchat inhibited the synthesis of chitinase and serine protease, which disturbed embryonic development in T. castaneum, thus affecting its development and reproduction. RNA interference of Tcchat also suppressed the synthesis of cytochrome P450 in the extracellular region and adversely affected normal functioning of the lysosomal pathway, which might have hindered the insects' ability to recognize, phagocytose, and degrade exogenous toxic substances, resulting in increased mortality. Furthermore, knockdown of Tcchat increased the susceptibility of larvae to carbamate and organophosphate insecticides, which supports the notion that Tcchat is indeed involved in susceptibility to insecticide in T. castaneum. Finally, Tcchat knockdown adults showed progressively reduced locomotor activity. These results provide novel insights into biological processes affected by Tcchat and provide a potential theoretical basis for the biological control of T. castaneum.