三疣梭子蟹Spook基因克隆及其表达分析

甲壳动物蜕皮激素(Ecdysteroids)主要在Y器官(YO)中合成与分泌,参与调控蜕皮、繁殖等多项生理活动。Spook基因编码的细胞色素P450(CYP)307a1是蜕皮激素合成通路早期的关键酶。为了研究其在三疣梭子蟹蜕皮过程中的调控作用,采用反转录PCR(RT-PCR)和c DNA末端快速扩增(RACE)技术,克隆得到了三疣梭子蟹Spook基因的全长c DNA序列(GenBank登录号:KM030021)。该序列全长为2 200 bp,包含一个长度为1 563 bp的开放阅读框,编码520个氨基酸;比对分析显示该氨基酸序列含helix-C、helix-K、helix-I、PERF、heme-binding 5个P450特征保守区域;系统进化树分析发现推导的三疣梭子蟹Spook蛋白与其他物种Spook聚为一支,而其他Halloween基因则分别聚为一支,表明推导的氨基酸确实是三疣梭子蟹Spook的蛋白序列。采用实时荧光定量PCR(qPCR)技术分析了其在不同组织中的表达情况,结果显示Spook基因主要在三疣梭子蟹的YO中表达。在三疣梭子蟹的蜕皮周期中,Spook基因的表达水平自蜕皮后期(A、B期)逐渐上升,并在蜕皮间期(C期)上升至最大,随后在蜕皮前期逐渐下降至D_3、D_4亚期最低。研究结果表明Spook基因可能参与调控三疣梭子蟹的蜕皮过程。     

中国对虾蜕皮抑制激素全长cDNA的克隆及序列分析

对虾的蜕皮活动由蜕皮抑制激素和蜕皮激素调控,蜕皮抑制激素是甲壳动物CHH家族神经肽的成员之一,通过抑制Y器官蜕皮激素的合成而调节蜕皮,以中国对虾（Fennropenaeus chinensis）眼柄总RNA为材料,采用cDNA末端快速扩增（RACE）方法。首次得到蜕皮抑制激素的全长cDNA（GenBank登录号：AF469187）。该全长cDNA大小为697bp,是由320bp的3′RACE产物和468bp的5′RACE产物拼接而成,Blast搜索结果显示,该全长cDNA与甲壳动物的MIH基因序列具有较高的相似性,用Clustal X进行多序列比较结果表明,由该全长cDNA推导的氨基酸序列与对虾类的MIH的氨基酸序列同源性最高,其中与日本对虾,斑节对虾,刀额新对虾MIH的同源性分别为95.1%,83.1%,79.1%,根据以上数据,推断该697bp的全长cDNA为编码中国对虾MIH前体的cDNA。进一步序列分析表明,编码中国对虾MIH前体cDNA包括312bp的开放阅读框,81bp的3′UTR和302bp的5′UTR;编码103个氨基酸的MIH前体分子包括信号肽和成熟肽,信号肽由28个氨基酸组成,成熟肽由75个氨基酸组成,成熟肽中6个半胱氨酸非常保守。     

中华绒螯蟹蜕皮抑制激素基因全长cDNA克隆和重组表达

根据实验室分离自中华绒螯蟹(Eriocheir sinensis)的一种蜕皮抑制激素(Molting-inhibiting hormone,MIH)N端氨基酸测序结果设计简并引物,采用RACE方法,首次从中华绒螯蟹眼柄中克隆到蜕皮抑制激素基因全长cDNA(Es-MIH,GenBank登录号:DQ341280),该基因全长为1457 bp,开放阅读框为330 bp,编码110个氨基酸(含有35个氨基酸的信号肽);其成熟肽包含C7-C44、C24-C40和C27-C53三个二硫键,有典型的CHH家族结构域。该cDNA编码的氨基酸序列与地蟹(Gecarcinus lateralis)MIH同源性最高,达到了85%。Northern杂交和半定量RT-PCR显示蜕皮间期成体蟹仅在眼柄中有MIH基因表达,提示该基因的表达具有一定组织特异性。利用pCR T7/NT TOPO TA系统重组表达MIH成熟肽,纯化的重组蛋白得率为0.3 g/L,纯化产物经质谱鉴定为中华绒螯蟹MIH。研究解决了CHH家族神经肽在机体中的表达量少,直接纯化较难的问题,为深入研究MIH的作用机制和在生产上控制中华绒螯蟹蜕皮和生长奠定了基础。     

三疣梭子蟹aqp2基因克隆及其在蜕皮中的功能

为研究水通道蛋白AQP的生理功能, 克隆了三疣梭子蟹 (Portunus trituberculatus) 水通道蛋白2基因(ptaqp2), 该基因全长4126 bp, 编码522个氨基酸, 具有水通道蛋白基因家族的保守结构域及功能结构域; 进化树聚类分析结果显示, ptaqp2基因属于水通道蛋白基因家族C-AQP类基因; 组织表达结果显示, ptaqp2呈泛组织表达特征, 在眼柄中的表达量最高, 在肠和肌肉中也有较高表达, 在血淋巴中的表达最低; ptaqp2基因在不同蜕皮时期呈显著差异表达(P<0.05), 且在蜕皮前期表达量最高; 在蜕皮激素(Molting Hormone简称MH)刺激下, 该基因在肌肉、肠及胃中均呈不同程度的上调表达趋势; 通过RNAi技术在蜕皮前期敲降ptaqp2的表达, 发现能够显著推迟蜕皮过程。研究初步证明了ptaqp2基因在三疣梭子蟹蜕皮中发挥重要作用。     

甲壳动物蜕皮抑制激素调控机制的研究进展

甲壳动物的蜕皮过程主要是由Y器(Y-organ)分泌的蜕皮类固醇激素与X器-窦腺复合体(X-organ-sinus gland,XO-SG)分泌的蜕皮抑制激素MIH相互拮抗而进行调控的。而MIH调控机制较为复杂,且存在争议。本文就MIH调控机制的研究进展,包括研究方法,以及目前调控机制中争议最大的3个问题:MIH受体、cAMP与cGMP功能以及Ca2+功能作一综述。     

罗氏沼虾蜕皮周期中内分泌调控和蜕皮信号通路相关基因的表达分析

蜕皮是罗氏沼虾重要的生理过程,为了探究罗氏沼虾蜕皮周期中内分泌调控与蜕皮通路中相关基因在蜕皮周期中的表达模式,阐明罗氏沼虾蜕皮的分子调节通路。本研究测定了罗氏沼虾肝胰腺和血淋巴组织中蜕皮周期内蜕皮相关酶活性（谷氨酰胺合成酶,β-N乙酰氨基葡萄糖苷酶和几丁质酶）与蜕皮激素含量,并通过RT-qPCR分析了蜕皮信号通路中Mr-ETHR、MrFTZ-F1以及RXR、ECR和MIH基因在罗氏沼虾不同蜕皮周期内的表达模式。酶活测定结果表明,谷氨酰胺合成酶在血淋巴组织中活力高于肝胰腺组织（P<0.05）;β-N乙酰氨基葡萄糖苷酶在肝胰腺和血淋巴组织中蜕皮前期的活力远高于蜕皮后期（P<0.05）。在肝胰腺中几丁质酶在蜕皮后期活力显著高于其他时期（P<0.05）。肝胰腺和血淋巴组织中蜕皮激素含量在蜕皮间期最低,蜕皮后期达到最高,呈上升趋势。通过PCR扩增与测序验证获得了Mr-ETHR、Mr-FTZ-F1基因ORF全长序列,Mr-ETHR基因ORF全长1 173 bp,编码390个氨基酸;Mr-FTZ-F1基因ORF全长为1 206 bp,编码401个氨基酸。荧光定量结果表明Mr-ETH...     

喷射自吸管式生化反应器研究

本文研究了喷射自吸管式生化反应器的吸气及所液传质特性,提出了吸气量(Qs)和容积氧传递系数(kLa)的数学表达式：Qg=5.2×10^-2W^0.144_cLR^0.079_cD(L-D)^0.328D²_nPoπ——P^o_gTo[(D-Dn)²-1](Pn-Pl)-1)(Kla)₁=0.999(W-V)^0.38V^0.90_sg(L-D)^-0.16(Kla)2=1.003(W-V)^0.71V^0.28_sg(L-D)^-0.32(加C圈)反应器的具优工况为：L/D=320-400,D/D=2.7—3。8,Pn=5—13×10⁴N/m²。Kla最高达4280h^-1,比能耗为0.72—2.16×10³kJ/kgO₂用于培养饲料酵母,酵母浓度达40.04kg/m³.最大生长速率为6.24kg/m³·h,比能耗为1.66—2.52×10³KJ/kg DBM.空气利用率为10—20%.是一般生化反应器的2—4倍。     

三疣梭子蟹腺苷酸转移酶(ANT)基因的克隆与分析

腺苷酸转移酶(Adenine nucleotide translocase,ANT)是线粒体内膜上负责能量分子传导的转运蛋白,在能量代谢中起着关键作用。为了研究ANT基因在甲壳动物蜕皮中的作用,采用RT-PCR和c DNA末端快速扩增技术(RACE技术)克隆得到三疣梭子蟹ANT基因的序列全长(Gen Bank登录号:KM921660),该序列全长1 414 bp,包括132 bp的5'端非编码区,352 bp的3'段非编码区,具有930 bp的完整开放阅读框(ORF),编码309个氨基酸。将该ANT基因序列推导的氨基酸序列与已公布的其他物种ANT序列进行系统进化树分析发现,三疣梭子蟹ANT基因与其他甲壳动物ANT基因聚为一支,其中与拟穴青蟹ANT一致性高达96%。通过氨基酸序列比对发现,三疣梭子蟹ANT序列具有3个保守的线粒体穿膜功能结构域,是形成能量分子传导的转运通道,催化细胞质中ADP和线粒体内ATP间进行跨膜交换。采用实时荧光定量PCR技术,分析三疣梭子蟹ANT基因的组织差异表达,结果表明ANT基因在三疣梭子蟹肌肉(Ms)中的表达量最高,在其他组织中表达量均较低,具显著差异(P0.05);在三疣梭子蟹蜕皮周期中,肌肉中ANT基因的表达量A期最高(P0.05),然后下降,至C期最低,随后又逐渐上升至D1期,在D1期出现第2个峰值后再逐渐下降。研究结果说明ANT基因与三疣梭子蟹肌肉活动密切相关,可能在蜕皮调控中发挥重要作用。     

苄基异喹啉类化合物 D20抑制钙调素激活磷酸二酯酶的研究

苄基异喹啉化合物是一类钙调素拮抗剂.对新合成的双苄基异喹啉化合物 D₂₀对钙调素依赖的磷酸二酯酶的抑制作用进行了研究,IC₅₀=5μmol/L,表明其拮抗作用大于三氟啦嗪,是强的拮抗剂.荧光分析表明,钙调素与化合物 D₂₀的结合常数为2.64(μmol/L)^-1,一个化合物 D₂₀分子与两个钙调素分子结合,并显示了结合方向性及空间位阻影响.     

棉铃虫蛹期血淋巴的蜕皮甾类

目前为止仅在少数几种昆虫中研究过蛹期的蜕皮激素。关于蜕皮甾类的性质分析,结果也颇不一致。本文采用放射免疫分析、薄层层析、高压液相色谱及质谱对棉铃虫Heliothis armigera蛹血淋巴内的蜕皮激素进行了研究。结果如下:1.物理-化学方法证明蛹血淋巴内存在二种蜕皮甾类:蜕皮酮和20-羟基蜕皮酮。2.蛹期蜕皮甾类滴度呈一宽峰,高峰出现在化蛹后的第5天(3435ng/ml)。3.在高峰时,蜕皮酮与20-羟基蜕皮酮的比例为1:3.57,说明20-羟基蜕皮酮是主要的蜕皮甾类。4.比较雌雄两性蛹的蜕皮甾类滴度,未见明显差异。研究表明在棉铃虫中影响成虫发育的主要激素是20-羟基蜕皮酮而不是蜕皮酮。     

Hemolymph ecdysteroids during the last three molt cycles of the blue crab,Callinectes sapidus: quantitative and qualitative analyses and regulation

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210–330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20‐OH ecdysone (20‐OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt‐inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20‐OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity. © 2009 Wiley Periodicals, Inc.     

Stress reduces hemolymph ecdysteroid levels in the crab: mediation by the eyestalks

In decapod crustaceans, molt hormone (ecdysone) production by Y-organs is suppressed by an eyestalk neurosecretory product, molt-inhibiting hormone (MIH). Environmental stressors are known to delay or prevent molting in crabs. The present study assesses the function of the MIH-Y-organ neuroendocrine system in the crab Cancer antennarius under conditions of daily handling stress. After three days, stressed crabs showed significant suppression of hemolymph ecdysteroid levels, which continued to fall to 20% of controls by day 14. Ecdysteroid titers of stressed crabs returned to prestress levels seven days after stress termination. Ecdysteroid levels in de-eyestalked (DES) crabs rose 160% within 48 hr post-DES. Stressing DES crabs over 16 subsequent days did not significantly alter ecdysteroid levels compared with unstressed DES controls. Handling stress thus depresses hemolymph ecdysteroid levels in the crab, a response that is mediated by eyestalks and appears to result from stress-induced MIH release.     

In vivo effects of a recombinant molt-inhibiting hormone on molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus

In order to determine the function of molt-inhibiting hormone (MIH) in vivo, we examined the effects of injecting of a recombinant MIH on the molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus. The injection of recombinant MIH significantly prolonged the molt interval (9.0 +/-0.4 days in the control group, 9.5+/-0.5 days in the 2500 ng/g-body weight/injection-group, mean+/-SD), and significantly decreased the hemolymph ecdysteroid level (ratio of levels between after and before injection: 1.94+/-1.09 in the control and 1.28+/-0.39 in the 3000 ng/g-body weight/injection-group, mean+/-SD). These results conclusively show the inhibitory effects of MIH on molting in vivo.     

Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement

The finding that molt-inhibiting hormone (MIH) regulates vitellogenesis in the hepatopancreas of mature Callinectes sapidus females, raised the need for the characterization of its mode of action. Using classical radioligand binding assays, we located specific, saturable, and non-cooperative binding sites for MIH in the Y-organs of juveniles (J-YO) and in the hepatopancreas of vitellogenic adult females. MIH binding to the hepatopancreas membranes had an affinity 77 times lower than that of juvenile YO membranes (K_D values: 3.22 × 10^-8 and 4.19 × 10^-10 M/mg protein, respectively). The number of maximum binding sites (B_MAX) was approximately two times higher in the hepatopancreas than in the YO (B_MAX values: 9.24 × 10^-9 and 4.8 × 10^-9 M/mg protein, respectively). Furthermore, MIH binding site number in the hepatopancreas was dependent on ovarian stage and was twice as high at stage 3 than at stages 2 and 1. SDS-PAGE separation of [¹²⁵I] MIH or [¹²⁵I] crustacean hyperglycemic hormone (CHH) crosslinked to the specific binding sites in the membranes of the J-YO and hepatopancreas suggests a molecular weight of ~51 kDa for a MIH receptor in both tissues and a molecular weight of ~61 kDa for a CHH receptor in the hepatopancreas. The use of an in vitro incubation of hepatopancreas fragments suggests that MIH probably utilizes cAMP as a second messenger in this tissue, as cAMP levels increased in response to MIH. Additionally, 8-Bromo-cAMP mimicked the effects of MIH on vitellogenin (VtG) mRNA and heterogeneous nuclear (hn) VtG RNA levels. The results imply that the functions of MIH in the regulation of molt and vitellogenesis are mediated through tissue specific receptors with different kinetics and signal transduction. MIH ability to regulate vitellogenesis is associated with the appearance of MIH specific membrane binding sites in the hepatopancreas upon pubertal/final molt.     

Hemolymph concentrations of host ecdysteroids are strongly suppressed in precocious prepupae of Trichoplusia ni parasitized and pseudoparasitized by Chelonus near curvimaculatus.

Regulation of ecdysteroid production in lepidopteran prepupae was studied using a parasitic wasp (C. near curvimaculatus) which specifically suppresses host prepupal ecdysteroid production after the induction of precocious host metamorphosis. At the developmental stage at which the hemolymph of the unparasitized metamorphosing host has its maximum titer of prepupal ecdysteroids, the hemolymph of 4th instar "truly parasitized" hosts (hosts with a surviving endoparasite) had a strongly reduced ecdysteroid titer. However, during the photophase about 12 h later, just prior to emergence of the parasite larva, an ecdysteroid peak was observed in the host hemolymph. Fourth instar pseudoparasitized prepupal hosts (in which the endoparasite was not present or died early in development) exhibited a sustained suppression in the hemolymph ecdysteroid titer. Small 5th instar pseudoparasitized hosts, which normally would molt to a 6th instar prior to metamorphosis, but which precociously attained the prepupal stage, also had a strongly reduced ecdysteroid titer. The late increase observed in truly parasitized hosts could be completely prevented by surgical removal of the parasite 24 h earlier, resulting in a titer similar to that in pseudoparasitized hosts. HPLC analysis of ecdysteroids in normal, truly parasitized, and 4th or 5th instar pseudoparasitized prepupae showed that both ecdysone and 20-OH ecdysone* were suppressed in truly and pseudoparasitized prepupae, with ecdysteroid levels being lowest in pseudoparasitized hosts. These data, and those of Brown and Reed-Larsen (Biol Contr 1, 136 [1992]), showing endoparasite secretion of ecdysteroids just prior to its emergence from the host, strongly indicate that: (1) the prepupal peak in truly parasitized hosts originates from the endoparasite, and (2) the low level of ecdysteroids in pseudoparasitized hosts results from the host's intrinsic inability to express a normal level of prepupal ecdysteroid titer. While precocious 4th or 5th instar prepupae of similar size had similarly suppressed ecdysteroid titers, smaller 4th instar prepupae had a lower ecdysteroid titer than larger, precocious 5th instar prepupae. Rare 5th instar pseudoparasitized prepupae that were of nearly normal size showed a prepupal ecdysteroid titer distinctly greater than those of the usual smaller, precocious 5th instar prepupae. The data suggest that the competence of the host to express a normal hemolymph titer of prepupal ecdysteroids is more closely correlated with the size of the prepupae than with the instar attained.     

Changes in intracellular calcium concentration in crustacean (Callinectes sapidus) Y-organs: relation to the hemolymphatic ecdysteroid titer

Secretion of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated (inhibited) by molt-inhibiting hormone (MIH), a neuropeptide produced by neurosecretory cells in eyestalk ganglia. The inhibitory effect of MIH is mediated by one or more cyclic nucleotide second messengers. In addition, available data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular calcium. However, despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca(2+) in Y-organs cells has not been previously determined. In studies reported here, eyestalks were ablated from blue crabs (Callinectes sapidus) to remove the endogenous source of MIH and activate Y-organs. At 0, 3, 6, and 9 days after eyestalk ablation (D0, D3, D6, and D9, respectively), the level of Ca(2+) in Y-organ cells was determined using a fluorescent calcium indicator (Fluo-4), and the hemolymphatic ecdysteroid titer was determined by radioimmunoassay. Calcium fluorescence in D6 Y-organs was 3.5-fold higher than that in D0 controls; calcium fluorescence in D9 Y-organs was 3.9-fold higher than in D0 controls (P<0.05). Measurement of fluorescence along a transect drawn through representative cells indicated that the calcium fluorescence was localized to cytoplasm and not to nuclei. Associated with the increase in intracellular Ca(2+) was a significant increase in the hemolymphatic ecdysteroid titer: The level of ecdysteroids in hemolymph rose from 5.5?ng/mL on D0 to 49.6?ng/mL on D6 and 87.2?ng/mL on D9 (P<0.05). The results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca(2+).     

Crustacean molt-inhibiting hormone: Structure,function, and cellular mode of action

In Crustacea, secretion of ecdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks. This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a) structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis. Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.