Genome-wide analysis of microRNAs induced and regulated by colchicine in mulberry

A variety of plants were colchicine treated to double their chromosome number.Chromosomes are genetic carriers that determine the individual traits of organisms.The doubling of chromosomes will lead to modifications in plant morphology,physiology and genetics.To determine the response of mulberry trees induced by colchicine,using mulberry variety Yu-711 leaves as research materials,two small RNA libraries(control and experimental groups)were constructed.It was found that 45 known miRNA genes and 78 predicted novel miRNA genes in the sequence results.A comparison of data between the control group and the experimental group revealed 37 differentially expressed miRNA genes,19 genes of which were up-regulated and 18 genes of which were down-regulated.Eight miRNAs were selected from 37 differentially expressed miRNA genes.These miRNAs were verified by quantitative real-time PCR,and the expression levels of these miRNAs were found to be consistent with those obtained by sequencing,which proved the accuracy of sequencing results.The PsRNATarget software was used to predict the target genes of 8 miRNAs.The Gene Ontology and Kyoto Encycopedia of Genes and Genomes analysis were used to collect the functions of target genes and confirm that target genes are mainly involved in biological processes,cell components and molecular functions.novel-77 miRNA was selected from 37 differentially expressed miRNAs.What will serve as a fundamental data in understanding mulberry miRNAs function in molecular biology research and further provides the molecular mechanism of mulberry gene regulation as induced by colchicine is that the prediction of novel_77 precursor sequence and the target gene(XM_010104258.2),as well as the secondary structure analysis.

Expression and Activity Analysis of Non-Structural Protein 2 (NS2) of Bombyx mori Densovirus Zhenjiang Strain

The gene of the non-structure protein 2 （NS2） was cloned by PCR from the genome ofBombyx mori densovirus Zhenjiang strain （BmDNV-Z）, inserted into prokaryotic expression vector pET28a to construct recombinant plasmid pET28a-NS2 and then expressed in bacteria Escherichia coli BL21 （DE3）. The expressed recombinant protein was identified by SDS-PAGE and Western blot analysis. Then, the recombinant protein was purified by Ni-NTA column, renatured and tested for enzyme activities. The purified NS2 protein exhibited a helicase activity unwinding double-stranded DNA substrates into single-strand primers, and higher unwinding activity to polarity substrate. Similarly, the purified NS2 protein possessed an ATPase activity and its enzyme activity was 0.276 μmol gg^-1 h^-1 in this study. The results indicated that the non- structure protein which encoded by the gene of BmDNV-Z NS2 possesses the biological activities of helicase and ATPase, and the helicase prefers to polarity substrates. Based on these results, it is speculated that the gene of BmDNV-Z NS2 plays an important role in the viral DNA replication.

Single-cell transcriptomes provide insights into expansion of glial cells in Bombyx mori

The diversity of cell types in the brain and how these change during different developmental stages,remains largely unknown.The life cycle of insects is short and goes through 4 distinct stages including embryonic,larval,pupal,and adult stages.During postembryonic life,the larval brain transforms into a mature adult version after metamorphosis.The silkworm,Bombyx mori,is a lepidopteran model insect.Here,we characterized the brain cell repertoire of larval and adult B.mori by obtaining 50708 single-cell transcriptomes.Seventeen and 12 cell clusters from larval and adult brains were assigned based on marker genes,respectively.Identified cell types include Kenyon cells,optic lobe cells,monoaminergic neurons,surface glia,and astrocyte glia.We further assessed the cell type compositions of larval and adult brains.We found that the transition from larva to adult resulted in great expansion of glial cells.The glial cell accounted for 49.8%of adult midbrain cells.Compared to flies and ants,the mushroom body kenyon cell is insufficient in B.mori,which accounts for 5.4%and 3.6%in larval and adult brains,respectively.Analysis of neuropeptide expression showed that the abundance and specificity of expression varied among individual neuropeptides.Intriguingly,we found that ion transport peptide was specifically expressed in glial cells of larval and adult brains.The cell atlas dataset provides an important resource to explore cell diversity,neural circuits and genetic profiles.

A study of miRNAs targets prediction and experimental validation

microRNAs(miRNAs)are 20–24 nucleotide(nt)RNAs that regulate eukaryotic gene expression post-transcriptionally by the degradation or translational inhibition of their target messenger RNAs(mRNAs).To identify miRNA target genes will help a lot by understanding their biological functions.Sophisticated computational approaches for miRNA target prediction,and effective biological techniques for validating these targets now play a central role in elucidating their functions.Owing to the imperfect complementarity of animal miRNAs with their targets,it is difficult to judge the accuracy of the prediction.Complexity of regulation by miRNA-mediated targets at protein and mRNAs levels has made it more challenging to identify the targets.To date,only a few miRNAs targets are confirmed.In this article,we review the methods of miRNA target prediction and the experimental validation for their corresponding mRNA targets in animals.

Construction and detection of expression vectors of microRNA-9a in BmN cells

MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.

Fumarate mitigates disruption induced by fenpropathrin in the silkworm Bombyx mori(Lepidoptera):A metabolomics study

The silkworm Bombyx mori L.is a model organism of the order Lepidoptera.Understanding the mechanism of pesticide resistance in silkworms is valuable for Lepidopteran pest control.In this study,comparative metabolomics was used to analyze the metabolites of 2 silkworm strains with different pesticide resistance levels at 6,12,and 24 h after feeding with fenpropathrin.Twenty-six of 27 metabolites showed significant differences after fenpropathrin treatment and were classified into 6 metabolic pathways:glycerophospholipid metabolism,sulfur metabolism,glycolysis,amino acid metabolism,the urea cycle,and the tricarboxylic acid(TCA)cycle.After analyzing the percentage changes in the metabolic pathways at the 3 time points,sulfur metabolism,glycolysis,and the TCA cycle showed significant responses to fenpropathrin.Confirmatory experiments were performed by feeding silkworms with key metabolites of the 3 pathways.The combination of iron(II)fumarate+folic acid(IF-FA)enhanced fenpropathrin resistance in silkworms 6.38 fold,indicating that the TCA cycle is the core pathway associated with resistance.Furthermore,the disruption of several energy-related metabolic pathways caused by fenpropathrin was shown to be recovered by IF-FA in vitro.Therefore,IF-FA may have a role in boosting silkworm pesticide resistance by modulating the equilibrium between the TCA cycle and its related metabolic pathways.

BmSd gene regulates the silkworm wing size by affecting the Hippo pathway

Insect wings are developed from the wing disc during metamorphosis.Bombyx mori,a model lepidopteran insect,loses flight ability after long-term domestication from the wild silkworm,Bombyx mandarina.The mw mutant(ul 1 strain)shows minute wings compared to wild type(e.g.,p50 strain)wings.RNA sequencing analysis previously revealed differential Hippo-pathway-related gene expression between the ull and p50strains.The Hippo pathway is an evolutionarily conserved signaling cascade that controls organ size during development in animals.In this study,the function of BmSd which has been characterized as one of the Hippo-pathway-related genes was analyzed for silkworm wing development.We found that mats,warts,and hippo expression levels were higher in u11 compared to p50 wing discs.BmSd(scalloped)expression,which encodes a prominent transcriptional partner to Yorkie(Yki),gradually decreased during the wandering stage in ull,but exhibited the opposite expression pattern in p50.When BmSd was knocked down by small interfering RNA during the wandering stage in the p50 strain,57.9%of the individuals showed minute wings.Additionally,ex,kibras and wingless expression levels decreased in the BmSd knockdown mutant.Further,BmSd deletion mediated by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 induced 50%of individuals with minute wings,a phenotype similar to the mw mutant.This result demonstrates that BmSd plays pivotal roles in silkworm wing development.Our results show that the Hippo signaling pathway participates and plays crucial roles in the regulation of silkworm wing development,and our findings provide a basis for further research on B.mori wing development.

Functional characterization of a low-density lipoprotein receptor in the lepidopteran model,Bombyx mori

The growth and development of metabolous insects are mainly regulated by ecdysone and juvenile hormone.As a member of the low-density lipoprotein receptor(LDLR)family,megalin(mgl)is involved in the lipoprotein transport of cholesterol which is an essential precursor for the synthesis of ecdysone.Despite extensive studies in mammals,the function of mgl is still largely unknown in insects.In this study,we characterize the function of mgl in the silkworm Bombyx mori,the model species of Lepidoptera.We find that mgl is broadly present in the genomes of lepidopteran species and evolved with divergence between lepidopterans and Drosophila.The expression pattern suggests a ubiquitous role of mgl in the growth and development in the silkworm.We further perform clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9-based mutagenesis of Bmmgl and find that both the development and the silk production of the silkworm are seriously affected by the disruption of Bmmgl.Our results not only explore the function of mgl in Lepidoptera but also add to our understanding of how cholesterol metabolism is involved in the development of insects.

家蚕miR-0001和miR-0015体外下调BmFib-L基因的表达（英文）

目的:探究新发现的两个miRNA对家蚕丝素轻链基因BmFib-L的调控作用。创新点:在家蚕后部丝腺中发现两个新的miRNA,并首次证明它们对家蚕丝素蛋白轻链基因BmFib-L有负调控作用。方法:本实验通过生物信息学分析,从家蚕后部丝腺miRNA高通量测序获得的新miRNA中,筛选出两个可能对家蚕丝素蛋白轻链基因BmFib-L有调控作用的miRNA,即bmo-miR-0001和bmo-miR-0015。设计茎环引物,采用反转录聚合酶链反应(RT-PCR)方法对家蚕5龄3 d头部、表皮、脂肪体、马氏管、精巢/卵巢、丝腺(前、中、后)、气管、中肠和血淋巴细胞等12个不同组织的bmo-miR-0001和bmo-miR-00015进行半定量表达分析。并采用双荧光报告基因检测系统进一步在细胞水平上验证bmo-miR-0001和bmo-miR-0015对BmFib-L表达的调控作用。结论:本实验中RT-PCR结果显示,这两个新miRNA在家蚕后部丝腺中表达量最高(图4)。双荧光报告基因检测结果显示,报告基因荧光素酶活性明显低于阳性对照组(图7),转染bmo-miR-0001和bmo-miR-0015表达载体的细胞,报告基因和荧光素酶活性分别只及对照组60%和69%。t检验分析结果显示两个实验组与对照组之间差异都达到显著水平(P<0.05)。由此可见,bmo-miR-0001和bmo-miR-0015在体外对BmFib-L的表达具有显著的抑制作用。

PLA_(2) mediates the innate immune response in Asian corn borer, Ostrinia furnacalis

The eicosanoid signaling pathway mediates insect immune reactions to a wide range of stimuli. This pathway begins with the biosynthesis of arachidonic acid (AA) from the hydrolysis of phospholipids catalyzed by phospholipase A_(2) (PLA_(2)). We report here that the PLA_(2) inhibitor, dexamethasone (DEX), impaired the innate immune response including nodulation, encapsulation, and melanization in Ostrinia furnacalis larvae, while AA partially reversed these effects of DEX. We cloned a full-length complementary DNA encoding a PLA_(2), designated as OfsPLA_(2), from O. furnacalis. The open reading frame of OfsPLA_(2) encodes a 195-amino acid residue protein with a 22-residue signal peptide. Sequence alignment analyses indicated that O. furnacalis PLA_(2) might be a Group III secretory PLA_(2). The highest transcript levels of OfsPLA_(2) were detected in the fat body, and its transcript levels increased dramatically after infection with Escherichia coli, Micrococcus luteus, or Beauveria bassiana. Recombinant OfsPLA_(2) significantly induced prophenoloxidase (PPO) activation in larval hemolymph in the presence of Ca^(2+) and encapsulation of agarose beads. Injection of recombinant OfsPLA_(2) into larvae resulted in increased transcript levels of attacin, defencin, and moricin-3 genes. Our results demonstrate the involvement of the eicosanoid signaling pathway in the innate immune response of O. furnacalis larvae and provide new information about the roles of O. furnacalis secretory PLA_(2) in activating PPO and antimicrobial peptide production.

CRISPR/Cas9-based knockout reveals that the clock gene timeless is indispensable for regulating circadian behavioral rhythms in Bombyx mori

Circadian rhythms,which are ubiquitous and adaptive,occur across all species,from microbes to humans,in which they organize and modify behavior and physiology.timeless(im)is a canonical clock gene.The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated;however,in lepidopteran insects,including Bombyr mori,the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities.To arrive at a comprehensive understanding of the role of tim in the B.mori endogenous circadian clock,we exploited the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 gene editing system.We attermpted to elucidate the functions of tim in the circadian clock of B.mori using Bmtim mutants.The knockouts affected two circadian behavioral activities:adult emergence and embryo hatching rhythms.Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels,and thereby the oscillation amplitudes,of Bmper and Bmclk messenger RNAs during both the photophase and scotophase.Additionally,the daily rhythmic expression of Bmndbt was up-regulated in the photophase and downregulated in the scotophase in a tim-knockout.Our study reveals that tim is integral to the B.mori circadian clock and may be involved in regulating eclosion and hatching rhythms.

Two dehydroecdysone reductases act as fat body-specific 20E catalyzers in Bombyx mori

Periodic post-embryonic changes in insects, including growth, development and metamorphosis, are strictly controlled by many compounds, including steroid hormones. The biosynthesis and clearance of 20-hydroxyecdysone (20E), the major active form of the insect steroid hormone ecdysone, result in titer fluctuations that help control insect development. The inactivation of 20E in the silkworm Bombyx mori is highly tissue-specific, with CYP18A1 and ecdysone oxidase controlling 20E inactivation specifically in the mid-silk gland and midgut, respectively. Here, we characterized silkworm 3-dehydroecdysone 3α reductase (Bm3DE3α) and 3-dehydroecdysone 3β reductase (Bm3DE3β), two enzymes involved predominantly in the C-3-mediated catalysis of 20E in fat bodies. The ubiquitous and silk gland-specific overexpression of Bm3DE3α decreased the 20E titer, resulting in larval lethality and larval–pupal transition failure, respectively. In contrast, the ubiquitous and mid-silk gland-specific overexpression of Bm3DE3β increased the 20E titer, resulting in larval growth delays and lethality at the mid-fifth larval stage, respectively. Thus, Bm3DE3α and Bm3DE3β mediate fat body-specific steroid hormone metabolism in B. mori, indicating that highly diversified 20E metabolism-related mechanisms exist in different insect species.