Molecular characterization and expression analysis of BmNOX in two strains of Bombyx mori with contrasting viral resistance phenotype

We recently documented the identification of a 26.5 kDa protein named BmNox in the gut fluid of Nistari strain of Bombyx mori, which possessed antiviral activity against BmNPV in vitro. In this report, we report the characterization of the full‐length gene encoding BmNOX and the levels of expression of this gene in select tissues of silkworm larvae from a BmNPV‐susceptible and a BmNPV‐resistant strain to the defense capability in Bombyx mori larvae challenged with BmNPV. We also evaluated the BmNox expression in various stages of larval life of a resistant and a susceptible strain of Bombyx mori selected from among a panel of strains of silkworm. Nistari, a multivoltine strain of silkworm, expressed BmNOX during all five larval stages, and were highly resistant to BmNPV infection. In sharp contrast, CSR₂, a bivoltine strain, showed weaker expression of BmNOX in the anterior midgut in larval life and was highly susceptible to BmNPV infection. BmNOX is a secretory protein with dual expression in gut fluid and mid gut tissue. BmNOX is expressed heavily in the posterior mid gut, with weaker expression in the fore‐ and mid‐gut regions. © 2010 Wiley Periodicals, Inc.     

“两广二号”家蚕抗病毒基因Bmlipase-1和BmSP-2克隆与原核表达

本研究以优良杂交品种"两广二号"家蚕为试材,克隆了该杂交品种家蚕两个抗家蚕核型多角体病毒(BmNPV)基因:脂肪酶基因Bmlipase-1和丝氨酸蛋白酶基因BmSP-2,测序并分别与不同品种蚕的同源基因序列进行比较。结果显示,"两广二号"家蚕Bmlipase-1基因ORF长度为885bp,编码294个氨基酸,BmSP-2扩增长度为855bp,编码284个氨基酸;它们的核苷酸和推导氨基酸序列同源性皆达92%以上,Bmlipase-1更保守,同源性大于99%";两广二号"家蚕的Bmlipase-1基因脂肪酶活化部位和BmSP-2基因酶催化三联体位点的氨基酸残基与不同品种蚕的完全相同。以上结果说明这两个抗病毒基因在蚕的遗传进化过程中高度保守,提示其可能在机体消化或者免疫防御方面起着重要生理作用。将这两个抗病毒基因在大肠杆菌BL21中进行融合表达,获得的融合Bmlipase-1和BmSP-2蛋白分子量分别为47kD和42kD左右。     

IN VITRO ANTIVIRAL ACTIVITY OF AN ALKALINE TRYPSIN FROM THE DIGESTIVE JUICE OF Bombyx mori LARVAE AGAINST NUCLEOPOLYHEDROVIRUS

Alkaline trypsin protein of molecular mass 25,436 Da purified from the digestive juice of Bombyx mori larvae indicated strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) under in vitro conditions. Partial N-terminal amino acid sequence of the protein was determined and the cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 55% identity with Helicoverpa armigera trypsin and the active site of this protein was completely conserved. Hence, the protein was designated B. mori trypsin (Bmtryp). The results suggest that Bmtryp, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection.     

A lipase isolated from the silkworm Bombyx mori shows antiviral activity against nucleopolyhedrovirus

A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. A homology search of the deduced amino acid sequence of the protein cDNA revealed 56% homology with Drosophila melanogaster lipase and 21% homology with human lipase. As lipase activity of the protein was confirmed in vitro, this protein was designated Bmlipase-1. Northern blot analysis showed that the Bmlipase-1 gene is expressed in the midgut but not in other tissues, nor is it activated by BmNPV infection. In addition, the Bmlipase-1 gene was shown not to be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that an insect digestive enzyme has potential as a physiological barrier against BmNPV at the initial site of viral infection.     

苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达

【目的】本研究旨在从苜蓿夜蛾Heliothis viriplaca中肠克隆出丝氨酸蛋白酶（serine protease, SP）基因的cDNA序列,测定原核表达后的蛋白经纯化及复性后的活性。【方法】运用RT-PCR和cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆苜蓿夜蛾幼虫中肠丝氨酸蛋白酶cDNA全序列,用大肠杆菌Escherichia coli表达系统进行表达。重组蛋白经纯化后,利用梯度透析法进行复性,以BApNA为底物,进行活性测定。【结果】克隆获得的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为HvSP（GenBank登录号：JX866720）,该基因全长880 bp,开放阅读框长762 bp,编码254个氨基酸,推测分子量和pI值分别为26.9 kDa和9.49。由HvSP推导的氨基酸与鳞翅目昆虫SP氨基酸序列的一致性在52%~95%之间,其中与棉铃虫Helicoverpa armigera SP（GenBank登录号：CAA72962）的氨基酸序列一致性最高,达95%。成功构建重组载体pET21b-HvSP进行原核表达,Western-blot鉴定确定为目的蛋白。蛋白可溶性分析发现重组蛋白为包涵体。在Glycine-NaOH缓冲液中,当pH为10.0时,复性的重组蛋白活性达到最高,为35.74 U/mL。【结论】本研究在苜蓿夜蛾体内获得了一个新的丝氨酸蛋白酶基因,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性。该结果为进一步研究丝氨酸蛋白酶在鳞翅目昆虫体内的生理功能奠定了基础。     

Low METTL3 level in midgut of the Bombyx mori inhibit the proliferation of nucleopolyhedrovirus

N6-methyladeosine (m⁶A) plays an important role in virus infection and replication. Bombyx mori nuclear polyhedrosis is caused by Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Expression levels of m⁶A-modification-related genes after the infection of BmNPV were detected at first. Then, expression levels of BmNPV nucleocapsid protein gene VP39 and envelope fusion protein gene GP64 after knockdown of METTL3in vitro were quantified to identify the effect of m⁶A modification on BmNPV. BmNPV firstly infects the larval midgut in case of oral infection. Subsequently, to clarify the relationship between m⁶A modification and resistance of the silkworm to BmNPV, we detected the expression levels of m⁶A-modification-related genes invivo before and after infection of BmNPV. The results indicated that low METTL3 level hindered the proliferation of BmNPV to some extent, and silkworm strain with low METTL3 level showed stronger resistance against BmNPV. This study will accumulate new experimental data for elucidating the resistance mechanism of silkworm against BmNPV.     

A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity

Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins has been proposed to be the regulation of protein turnover and defense against pathogens as well as the balance of the host-parasite immune relationship. Genes encoding cystatins have been found in several species of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from tick H. longicornis and designated it as Hlcyst-2 (H. longicornis cystatin-2). Its full-length cDNA is 569 bp, and it encodes a putative 133 amino acid protein with an obvious signal peptide. Sequence analysis demonstrated that it has significant homology with the known cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain, cathepsin L, and cathepsin B was identified by fluorogenic substrate analysis. Cystatin was mostly expressed in the tick midgut and hemocyte. Blood feeding induced significantly increased expression in the midgut. Real-time PCR confirmed that LPS-injected adult ticks expressed Hlcyst-2 1.6 more times than the PBS-injected control; Babesia gibsoni-infected larvae ticks expressed Hlcyst-2 1.8 more times than normal larvae ticks. The recombinant protein also showed a significant growth-inhibitory effect on Babesia bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.     

Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV.     

甜菜夜蛾泛素延伸蛋白基因cDNA的3′RACE-PCR扩增及其克隆和表达

根据已知的草地夜蛾Spodoptera frugiperda的泛素延伸基因 5'端核苷酸序列设计引物,应用3'RACE-PCR技术,从甜菜夜蛾S. exigua脂肪体组织总RNA中反转录扩增泛素基因的cDNA片段。扩增得到的片段全长513 bp,3'末端有123 bp的非翻译区,翻译区编码一个长为129个氨基酸残基的蛋白质,预测分子量为14.8 kD。同源分析表明,此cDNA序列为ubiquitin-53aa extension protein(ubi-53) 基因,在泛素蛋白后融合了一个核糖体L40蛋白（ribosomal L40 protein）。用MagAlign和Genedoc软件对cDNA编码的氨基酸序列进行了同源性分析,结果表明： 甜菜夜蛾的ubi-53基因与真核生物家蚕Bombyx mori、草地夜蛾、果蝇Drosophila melanogaster和人Homo sapienes泛素的同源性分别为96.9%、98.5%、95.3%和93.0%,与甜菜夜蛾核型多角体病毒(SeNPV)泛素的同源性为78.8%,说明真核生物的泛素基因与核型多角体病毒的泛素基因可能存在不同的分子进化途径。将甜菜夜蛾的ubI-53基因克隆到原核表达载体pET-28a上,转化至BL21（DE3）中,用IPTG进行诱导表达,用异源泛素单克隆抗体进行Western blot检测,证明原核表达蛋白是目的蛋白。     

Molecular and biochemical characterization of juvenile hormone epoxide hydrolase from the silkworm, Bombyx mori

One major route of insect juvenile hormone (JH) degradation is epoxide hydration by JH epoxide hydrolase (JHEH). A full-length cDNA (1536 bp) encoding a microsomal JHEH was isolated from the silkworm, Bombyx mori. Bommo-JHEH cDNA contains an open reading frame encoding a 461-amino acid protein (52 kDa), which reveals a high degree of similarity to the previously reported insect JHEHs. The residues Tyr298, Tyr373, and the HGWP motif corresponding to the oxyanion hole of JHEHs and the residues Asp227, His430, and Glu403 in the catalytic triad are well conserved in Bommo-JHEH. Bommo-JHEH was highly expressed in the fat body, where its mRNA expression pattern was in contrast to the pattern of hemolymph levels of JH during the larval development, suggesting that Bommo-JHEH plays an important role in JH degradation. Recombinant Bommo-JHEH (52 kDa) expressed in Sf9 insect cells was membrane-bound and had a high level of enzyme activity (300-fold over the control activity). This Bommo-JHEH study provides a better understanding of how JH levels are regulated in the domesticated silkworm.     

家蚕中肠组织抗核型多角体病毒病的相关蛋白分析

家蚕中肠上皮是病毒经口侵入遇到的第一个组织。昆虫幼虫抵御杆状病毒的感染,可通过选择性的使感染的中肠上皮细胞发生调亡并在释放病毒粒子进入血淋巴之前使感染的细胞从中肠脱落。为研究家蚕抗核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)病的机制,通过对BmNPV高度抗性和高度敏感性的家蚕品系杂交和回交构建了近等基因系。本文对家蚕高抗,敏感及近等基因系5龄起蚕中肠组织的蛋白质表达谱进行了二维电泳 (two-dimensional gel electrophoresis,2-DE) 分析,并利用基质辅助激光解吸电离飞行时间 (matrix-assisted laser desorption/ionization-time of flight, MALDI-TOF) 质谱对差异蛋白进行鉴定。结果发现了5个差异表达的蛋白。推测这些蛋白可能与家蚕中肠对BmNPV的抗性或感性有关。     

hsc70-4基因促进家蚕核型多角体病毒复制增殖

【目的】热休克应答(heatshockresponse,HSR)是机体细胞应对环境压力的一种重要防御策略,鉴定热休克蛋白在杆状病毒侵染宿主过程中的功能,并揭示其作用的分子机制,为探明宿主与病毒相互作用的分子基础提供理论依据。【方法】通过分子克隆技术对Bmhsc70-4基因进行克隆,并利用BioEdit及GeneDoc对其进行多序列比对分析;分别通过真核表达和基于CRISPR/Cas9的基因编辑系统对Bmhsc70-4基因进行过表达和敲除;利用荧光定量PCR技术检测相应基因的表达量;通过对Caspase-9和Caspase-3/7活性的检测确定Bmhsc70-4基因对细胞凋亡的影响;通过免疫荧光验证BmHSC70-4和BmIAP的共定位情况,并进一步通过免疫共沉淀验证它们的相互作用。【结果】Bmhsc70-4基因开放阅读框为1950bp,编码649个氨基酸,在昆虫间具有较高的保守性;BmNPV能够诱导Bmhsc70-4基因上调表达,过表达Bmhsc70-4基因能够促进BmNPV的增殖,敲除Bmhsc70-4基因能够抑制BmNPV的增殖,表明Bmhsc70-4基因的表达利于BmNPV的增殖;Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能;荧光共定位显示BmHSC70-4和BmIAP共定位于细胞质中,免疫共沉淀结果表明两者可以相互作用;BmNPV侵染过程中Bmhsc70-4基因能够促进Bmiap基因的表达。【结论】Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能,在BmNPV侵染家蚕细胞过程中,能够与BmIAP相互作用,并促进BmNPV复制增殖。     

Molecular cloning and expression characterization of translationally controlled tumor protein in silkworm pupae

A Bombyx mori (B. mori) cDNA was isolated from silkworm pupae cDNA library encoding a homologue of translationally controlled tumor protein (BmTCTPk). BmTCTPk was expressed in E. coli; SDS–PAGE and Western blot showed the molecular weight of recombinant and native BmTCTPk is approximately 28 and 25 kDa, respectively; they are larger than the theoretical molecular weight. Immunohistochemical studies showed that BmTCTPk is uniformly distributed throughout the cytoplasm of BmN cells. In silkworm pupae, BmTCTPk is expressed in the midgut wall, the midgut cavity, and some fat body tissues lying between the midgut wall and body wall. Western blot and ELISAs performed on total protein extracts isolated from silkworm pupae at different development stages showed that, although BmTCTPk is expressed during all pupae stages, its expression level increases dramatically during late pupae stages, suggesting that BmTCTPk may play an important role during the developmental transition from pupa to imago.     

Molecular characterization, tissue distribution, subcellular localization and actin-sequestering function of a thymosin protein from silkworm

We identified a novel gene encoding a Bombyx mori thymosin (BmTHY) protein from a cDNA library of silkworm pupae, which has an open reading frame (ORF) of 399 bp encoding 132 amino acids. It was found by bioinformatics that BmTHY gene consisted of three exons and two introns and BmTHY was highly homologous to thymosin betas (Tβ). BmTHY has a conserved motif LKHTET with only one amino acid difference from LKKTET, which is involved in Tβ binding to actin. A His-tagged BmTHY fusion protein (rBmTHY) with a molecular weight of approximately 18.4 kDa was expressed and purified to homogeneity. The purified fusion protein was used to produce anti-rBmTHY polyclonal antibodies in a New Zealand rabbit. Subcellular localization revealed that BmTHY can be found in both Bm5 cell (a silkworm ovary cell line) nucleus and cytoplasm but is primarily located in the nucleus. Western blotting and real-time RT-PCR showed that during silkworm developmental stages, BmTHY expression levels are highest in moth, followed by instar larvae, and are lowest in pupa and egg. BmTHY mRNA was universally distributed in most of fifth-instar larvae tissues (except testis). However, BmTHY was expressed in the head, ovary and epidermis during the larvae stage. BmTHY formed complexes with actin monomer, inhibited actin polymerization and cross-linked to actin. All the results indicated BmTHY might be an actin-sequestering protein and participate in silkworm development.