家蚕卵黄原蛋白及其受体基因

家蚕小卵突变体 (Smalleggmutant ,sm) ,其卵体积仅及正常卵的 2 / 3,不能受精而致死。因其卵母细胞不能正常吸收卵黄原蛋白 (Vg) ,人们认为其原因可能是卵黄原蛋白受体基因 (VgR)突变所致。本研究首先通过克隆筛选和基因组序列分析 ,获得了 2 5 6 4bp含有 ployA的家蚕卵黄原蛋白受体基因 (BmVgR)片段。将该基因片段的预测蛋白与其它物种的VgR/YPR和低密度脂蛋白受体 (LDLR)家族比较 ,发现该基因具有LDLR家族的基本结构特征。其次 ,经RT PCR检测 ,结果表明BmVgR在sm的不同时期的卵母细胞中都能正常转录。最后 ,分别对sm不同发育时期的体液和卵的总蛋白进行SDS PAGE分析 ,发现该突变体的卵母细胞不能正常摄取体液蛋白 (包括Vg)。综合分析 ,sm不能正常摄取Vg ,可能并不是VgR的功能异常导致 ,而是与滤泡细胞异常有关     

Molecular characterization and functional analysis of the vitellogenin receptor in the rice stem borer,Chilo suppressalis

As a member of the low-density lipoprotein receptor (LDLR) superfamily, vitellogenin (Vg) receptor (VgR) is responsible for the uptake of Vg into developing oocytes and is a potential target for pest control. Here, a full-length VgR complementary DNA (named as CsVgR) was isolated and characterized in the rice stem borer, Chilo suppressalis. The composite CsVgR gene contained an open reading frame of 5,484 bp encoding a protein of 1,827 amino acid residues. Structural analysis revealed that CsVgR contained two ligand-binding domains (LBDs) with four Class A (LDLR_A) repeats in LBD1 and seven in LBD2, which was structurally different from most non-Lepidopteran insect VgRs having five repeats in LBD1 and eight in LBD2. The developmental expression analysis showed that CsVgR messenger RNA expression was first detectable in 3-day-old pupae, sharply increased in newly emerged female adults, and reached a peak in 2-day-old female adults. Consistent with most other insects VgRs, CsVgR was exclusively expressed in the ovary. Notably, injection of dsCsVgR into late pupae resulted in fewer follicles in the ovarioles as well as reduced fecundity, suggesting a critical role of CsVgR in female reproduction. These results may contribute to the development of RNA interference-mediated disruption of reproduction as a control strategy of C. suppressalis.     

Identification and characterization of the vitellogenin receptor in Macrobrachium rosenbergii and its expression during vitellogenesis

In oviparous organisms, oocyte maturation depends on massive production of the egg yolk-precursor protein, vitellogenin (Vg). Vg is taken up by the developing oocytes through receptor-mediated endocytosis (RME), a process essential to successful reproduction. The aims of this study were to identify and characterize the yet-unknown vitellogenin receptor (VgR) from the pleocyamate crustacean Macrobrachium rosenbergii, and to investigate its expression levels during vitellogenesis and its interaction with Vg. The VgR gene was cloned, and its translated protein was specifically located at the oocyte membrane. Moreover, for the first time, a VgR protein was identified and sequenced by mass spectrometry. The putative MrVgR displayed high sequence similarity to VgRs from crustaceans, insects, and vertebrates, and its structure includes typical elements, such as an extracellular, lipoprotein-binding domain (LBD), EGF-like, and O-glycosylation domains, a transmembrane domain, and a short, C-terminal, cytosolic tail. In this article, we identify the first crustacean VgR protein, and present data demonstrating its high affinity for a Vg column followed by elution with suramin and EDTA. Additionally we demonstrate that VgR expression in the oocyte is elevated during vitellogenesis. Our results contribute to the fundamental understanding of oocyte maturation in crustaceans, and particularly elucidate Vg uptake through RME via the VgR.     

Insect vitellogenin/lipophorin receptors: Molecular structures, role in oogenesis, and regulatory mechanisms

Insect vitellogenin and lipophorin receptors (VgRs/LpRs) belong to the low-density lipoprotein receptor (LDLR) gene superfamily and play a critical role in oocyte development by mediating endocytosis of the major yolk protein precursors Vg and Lp, respectively. Precursor Vg and Lp are synthesized, in the majority of insects, extraovarially in the fat body and are internalized by competent oocytes through membrane-bound receptors (i.e., VgRs and LpRs, respectively). Structural analysis reveals that insect VgRs/LpRs and all other LDLR family receptors share a group of five structural domains: clusters of cysteine-rich repeats constituting the ligand-binding domain (LBD), epidermal growth factor (EGF)-precursor homology domain that mediates the acid-dependent dissociation of ligands, an O-linked sugar domain of unknown function, a transmembrane domain anchoring the receptor in the plasma membrane, and a cytoplasmic domain that mediates the clustering of the receptor into the coated pits. The sequence analysis indicates that insect VgRs harbor two LBDs with five repeats in the first and eight repeats in the second domain as compared to LpRs which have a single 8-repeat LBD. Moreover, the cytoplasmic domain of all insect VgRs contains a LI internalization signal instead of the NPXY motif found in LpRs and in the majority of other LDLR family receptors. The exception is that of Solenopsis invicta VgR, which also contains an NPXY motif in addition to LI signal. Cockroach VgRs still harbor another motif, NPTF, which is also believed to be a functional internalization signal. The expression studies clearly demonstrate that insect VgRs are ovary-bound receptors of the LDLR family as compared to LpRs, which are transcribed in a wide range of tissues including ovary, fat body, midgut, brain, testis, Malpighian tubules, and muscles. VgR/LpR mRNA and the protein were detected in the germarium, suggesting that the genes involved in receptor-endocytotic machinery are specifically expressed long before they are functionally required.     

From hepatopancreas to ovary: molecular characterization of a shrimp vitellogenin receptor involved in the processing of vitellogenin

We report the first cloning and characterization of cDNA encoding a putative vitellogenin (Vg) receptor (VgR) from the shrimp, Penaeus monodon. The shrimp VgR cDNA is 6.8 kb; the deduced protein has 1943 amino acids with a molecular weight of 211 kDa. VgR is ovary specific and consists of conserved cysteine-rich domains, epidermal growth factor-like domains, and YWTD motifs similar to the low-density lipoprotein, very low-density lipoprotein, and VgR of insects and vertebrates. VgR expression level in the ovary is low during early vitellogenesis and increases to maximum levels in females with a gonadosomatic index of 3-4, presumably when needed for receptor-mediated endocytosis during the rapid phase of extraovarian Vg production by the hepatopancreas. A peptide from the C-terminal end of VgR was synthesized for antibody production. Anti-VgR antibody recognized an ovarian membrane protein, and the level of this protein was high when extraovarian production of Vg reached peak levels. By immunohistochemical analysis, VgR was detected strongly in the membranes of larger oocytes. VgR expression was knocked down after the shrimp were injected with VgR double-stranded RNA, leading to a decrease in VgR protein content in the ovary, but an increase in the hemolymph level of Vg. This study represents the first report of the functional analysis of a putative VgR in a crustacean.     

In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say)

Injection of the hormone 20-hydroxyecdysone (20-E) into partially fed (virgin) female adults of the American dog tick, Dermacentor variabilis, while they are attached and feeding on the rabbit host, initiated the expression of the vitellogenin (Vg) gene, and Vg protein secretion and uptake by the ovary. The induction of egg production by 20-E in this bioassay was dose dependent in the range of 1-50 times the concentration normally found in a replete, vitellogenic female. Ticks examined 4 d after the 50 x treatment were still attached to the host, had numerous enlarged vitellin-filled (brown) oocytes in their ovaries, but had not engorged to repletion. The ovaries reached weights similar to those found in untreated, replete (mated) females (pre-oviposition) while solvent-injected controls demonstrated no increase in oocyte size or increase in ovary weight. An increase in the levels of a putative Vg protein was observed in hemolymph samples collected 1, 2 and 3d post-20-E injection but was not observed in the corresponding solvent controls as determined by native PAGE. Analysis of the ecdysteroid-induced protein by tryptic digestion-mass fingerprinting and BLASTP found that the putative Vg had the strongest match to GP80 (U49934), the partial sequence for the vitellogenin protein from Boophilus microplus. A partial Vg cDNA was cloned and sequenced from replete females of D. variabilis with a high similarity to GP80. Using this message as a probe, Northern blots conducted with RNA collected from partially fed, virgin females 1, 2 and 3d post-20-E injection showed upregulation of the Vg mRNA on all 3 days. Controls injected with solvent only showed no Vg mRNA. Injections with juvenile hormone III did not stimulate Vg expression, oocyte growth or full engorgement. These studies indicate that ecdysteroids and not JH can initiate expression of the Vg gene, Vg protein synthesis and release into hemolymph, and Vg uptake into developing oocytes under bioassay conditions mimicking normal feeding on the host.     

昆虫卵黄原蛋白受体的研究进展

昆虫卵黄发生的一个重要过程是卵黄蛋白的摄取,已有的研究表明脂肪体合成的卵黄原蛋白(vitellogenin,Vg)是通过受体介导的内吞作用(receptor mediated endocytosis,RME)被正在发育的卵母细胞所摄取。昆虫卵黄原蛋白受体(vitellogenin receptor,VgR)是介导昆虫卵黄原蛋白胞吞作用主要受体,它属于低密度脂蛋白家族,在结构与特性上具低密度脂蛋白家族的共性。卵黄原蛋白及其受体在昆虫生殖过程中起着重要的作用,本文综述了昆虫VgR的基本特性、分子结构及表达调控等方面的研究进展。     

Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins

The receptor-mediated uptake of major yolk protein precursor, vitellogenin (Vg) is crucial for oocyte growth in egg laying animals. In the present study plasma membrane receptor for Vg was isolated from the oocyte of the red mud crab, Scylla serrata. Vitellogenin receptor (VgR) protein was visualized by ligand blotting using labeled crab Vg ((125)I-Vg) as well as labeled low density lipoprotein ((125)I -LDL) and very low density lipoprotein ((125)I-VLDL) isolated from rat. The endocytosis of Vg was visualized in the crab oocyte by ultrastructural immunolocalization of Vg. The Vg receptor was purified by gel filtration high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 230 kDa. In direct binding studies, the receptor exhibited high affinity (dissociation constant K(d) 0.8x10(minus sign6) M) for crab Vg. Vitellogenin receptor was observed to have an increased affinity to crab Vg in the presence of Ca(2+) and the binding was inhibited by suramin, suggesting similarities between crab VgR and low density lipoprotein receptor (LDLR) superfamily of receptor protein. Furthermore, the crab VgR showed significant binding ability to mammalian atherogenic lipoproteins such as LDL and VLDL. This suggests that there is a tight conservation of receptor binding sites between invertebrate (crab) Vg and vertebrate (rat) LDL and VLDL.     

Vitellogenin transcytosis in follicular cells of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis> and the wasp <Emphasis Type="Italic">Polistes simillimus</Emphasis>

Vitellogenin receptor (VgR) is a low-density lipoprotein receptor responsible for the mediated endocytosis of vitellogenin (Vg) during egg formation in insects. The maturing oocyte is enveloped by a follicular epithelium, which has large intercellular spaces during Vg accumulation (patency). However, Vg has been reported in the cytoplasm of follicular cells, indicating that there may be a transcellular route for its transport. This study verified the presence of VgR in the follicular cells of the ovaries of the honeybee Apis mellifera and the wasp Polistes simillimus in order to evaluate if Vg is transported via transcytosis in these insects. Antibodies specific for vitellogenin receptor (anti-VgR), vitellogenin (anti-Vg), and clathrin (anti-Clt) were used for immunolocalization. The results showed the presence of VgR on the apical and basal plasma membranes of follicular cells of the vitellogenic follicles in both species, indicating that VgR may have been transported from the basal to the apical cell domain, followed by its release into the perivitelline space, evidenced by the presence of apical plasma membrane projections containing VgR. Co-localization proved that Vg bind to VgR and that the transport of this protein is mediated by clathrin. These data suggest that, in these social insects, Vg is transported via clathrin-mediated VgR transcytosis in follicular cells.     

Multiple vitellogenins from the Haemaphysalis longicornis tick are crucial for ovarian development

Ovarian development and egg maturation are crucial processes for the success of reproduction in ticks. Three full-length cDNAs encoding the precursor of major yolk protein, vitellogenin, were obtained from cDNA libraries of the Haemaphysalis longicornis tick and designated as HlVg-1, HlVg-2 and HlVg-3. The HlVg mRNAs were found in fed females with major expression sites in the midgut, fat body and ovary. Native PAGE and Western blot demonstrated that HlVgs in the hemolymph, fat body and ovary of fed females consisted of four major polypeptides. RNAi results showed that HlVg dsRNA-injected ticks obtained lower body weight, egg weight and showed higher mortality of engorged females after blood sucking than control groups. Our results indicate that all HlVgs are essential for egg development and oviposition.     

Full-length sequence, regulation and developmental studies of a second vitellogenin gene from the American dog tick, Dermacentor variabilis

Vitellogenin (Vg) is the precursor of vitellin (Vn) which is the major yolk protein in eggs. In a previous report, we isolated and characterized the first Vg message from the American dog tick Dermacentor variabilis. In the current study, we describe a second Vg gene from the same tick. The Vg2 cDNA is 5956 nucleotides with a 5775 nt open reading frame coding for 1925 amino acids. The conceptual amino acid translation contains a 16-residues putative signal peptide, N-terminal lipid binding domain and C-terminal von Willebrand factor type D domain present in all known Vgs. Moreover, the amino acid sequence shows a typical GLCG domain and several RXXR cleavage sites present in most isolated Vgs. Tryptic digest-mass fingerprinting of Vg and Vn recognized 11 fragments that exist in the amino acid translation of DvVg2 cDNA. Injection of virgin females with 20 hydroxyecdysone induced DvVg2 expression, vitellogenesis and oviposition. Using RT-PCR, DvVg2 expression was detected only in tick females after mating and feeding to repletion. Northern blot analysis showed that DvVg2 is expressed in fat body and gut cells of vitellogenic females but not in the ovary. DvVg2 expression was not detected in adult fed or unfed males. The characteristics that distinguish Vg from other similar tick storage proteins like the carrier protein, CP (another hemelipoglycoprotein) are discussed.     

Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick,Amblyomma hebraeum

Ovaries of the ixodid tick, Amblyomma hebraeum Koch, grew rapidly after engorgment as a result of yolk uptake. At 26 °C, oviposition began by day 10 post-engorgement, plateaued on days 16-18, and ended by day 38. Vitellin (Vt) was partially purified from ovaries of day 10 engorged ticks by gel filtration and ion exchange chromatography. This Vt comprises seven major and several minor polypeptides. Two polypeptides (211 and 148 kD) from haemolymph of engorged female ticks corresponded to minor polypeptides of similar molecular weight in the ovary. The haemolymph titre of the 211 and 148 kD polypeptides increased up to the onset of oviposition. These polypeptides were absent in males and non-vitellogenic females (day 0 engorged or day 10 partially-fed females), and were thus designated as vitellogenin (Vg). Antibodies raised against haemolymph Vg211 and 148 recognized these polypeptides in partially purified Vt, as well as six of the seven major polypeptides. Using these antibodies we developed an indirect, competitive ELISA to quantify Vg. Rise in haemolymph Vg-concentration lagged slightly behind the rise in haemolymph ecdysteroid (ES)-concentration, and Vg-synthesis was stimulated by injections of 20E into non-vitellogenic females. These observations indicate that an ES is the vitellogenic hormone in A. hebraeum.     

烟粉虱MEAM1隐种卵黄原蛋白受体基因cDNA的克隆、 序列分析及在不同发育时期的表达

卵黄原蛋白受体（vitellogenin receptor, VgR）是卵黄原蛋白被卵母细胞摄取的关键因子, 在卵黄发生和卵母细胞发育等生理过程中发挥着重要作用。为探讨烟粉虱Bemisia tabaci VgR的功能, 我们采用RT-PCR和RACE等技术扩增了烟粉虱MEAM1隐种B. tabaci Middle East-Asia Minor 1 (MAEM1) 的VgR基因cDNA 全长序列。生物信息学分析表明, 烟粉虱MEAM1隐种的VgR基因cDNA全长5 774 bp, 编码1 919个氨基酸, 推测分子量约201 kDa, N-端前31个氨基酸为信号肽。烟粉虱MEAM1隐种的VgR属于低密度脂蛋白受体（low density lipoprotein receptor, LDLR）家族, 蛋白质三维结构预测分析表明, 该受体具有LDLR家族基因典型的保守功能结构域。通过实时荧光定量PCR技术研究了烟粉虱MEAM1隐种VgR基因不同发育时期的表达, 结果表明VgR基因在伪蛹期开始表达, 并在羽化后1 d达到高峰, 此后逐渐降低, 3 d后又逐渐升高, 直至羽化后7 d达到峰值。研究结果丰富了卵黄原蛋白受体家族基因的数据库, 为今后深入研究并揭示烟粉虱卵黄发生的调控机制奠定了基础。