红螯光壳螯虾卵黄磷蛋白的分离纯化和鉴定

采用凝胶层析、聚丙烯酰胺凝胶电泳及SDS-聚丙烯酰胺凝胶电泳法对红螯光壳螯虾(Cherax quadricarinatus)卵巢中的卵黄磷蛋白进行了分离、纯化和鉴定。结果显示该虾的卵黄磷蛋白是一个分子量为369ku的多聚体,由分子量为85．4、80．6、76．6、73．7ku的四个亚基组成。染色分析表明卵黄磷蛋白为糖-磷-类胡萝卜素结合的复合蛋白。氨基酸组成分析显示天冬氨酸(Asp)和谷氨酸(Glu)为主要氨基酸。     

红螯螯虾胚胎发育研究Ⅰ.胚胎外部结构的形态发生

详细研究了红螯螯虾（Ｃｈｅｒａｘ ｑｕａｄｒｉｃａｒｉｎａｔｕｓ）胚胎的外部形态发育过程。在水温２８℃的条件下,整个发育过程约需３９ｄ,顺次经历卵裂期、囊胚期、原肠期、前无节幼体期、后无节幼体期、复眼色素形成期及孵化准备期。刚孵化出的幼体在形态结构上与成体相似。     

红螯螯虾感光器中Gq蛋白的鉴定及光波长对其含量的影响

用蛋白质常规提取方法分别提取红螯螯虾复眼视网膜的可溶性和膜结合蛋白质,用SDS-PAGE和免疫印迹法进行G蛋白的分离和鉴定;在经光、暗适应及红、绿、蓝、黄4种波长光照处理后,对其感光器中G蛋白分别进行SDS-PAGE分析.暗适应、自然光及4种波长光适应的感光器可溶性蛋白、膜结合蛋白条带数量和分子量大小基本相同,在42?kD处都出现一条清晰的蛋白条带.兔抗Gαq-11多克隆抗体能识别这一蛋白条带.表明红螯螯虾感光器中存在可溶性和膜结合Gq蛋白α亚基,分子量为42?kD.在不同光照条件下感光器中可溶性和膜结合Gqα亚基的含量有差异.可溶性Gqα蛋白占总蛋白的百分含量依次为:日光(7.71%)>黄光(7.32%)>红光(7.06%)>暗适应(6.94%)>蓝光(6.46%)>绿光(5.74%);除暗适应与红光之间差异不显著外,红光与黄光差异显著,其余差异极显著.而膜结合Gqα亚基的百分含量则相反,依次为:绿光(13.94%)>蓝光(10.56%)>暗适应(10.25%)>红光(10.14%)>黄光(9.76%)>日光(9.44%);除暗适应与红光差异不显著外,红光与黄光差异显著,其余差异极显著.推测红螯螯虾感光器中Gq蛋白的激活与光照刺激的波长有关,其感光器对不同波长光刺激的敏感度有差别.     

红螯螯虾胚胎发育的研究:Ⅱ.消化系统的发生

应用组织切片技术 ,研究了红螯螯虾胚胎发育过程中消化系统的发生。红螯螯虾的消化系统由前肠、中肠和后肠 3部分组成 ,前肠和后肠由外胚层形成 ,而中肠源自原肠期由胚胎表面向囊胚内迁移的中内胚层细胞团。前无节幼体期前肠开始发生 ,至后无节幼体期先后形成口道、食道和胃等结构 ;中肠起始于后无节幼体期的次级卵黄锥 ,包括管状中肠和 1对囊状消化腺 -中肠腺 ;后肠端部是前无节幼体期形成的肛道 ,肛道不断向胚胎前端延伸逐渐形成后肠     

红螯螯虾胚胎发育期主要消化酶和同工酶的活性变化

采用生物化学方法测定了红螯螯虾(Cherax quadricarinatus)胚胎发育各期主要消化酶(胃蛋白酶、胰蛋白酶、淀粉酶、纤维素酶和脂肪酶)的比活力及主要同工酶(乳酸脱氢酶、苹果酸脱氢酶、葡萄糖-6-磷酸脱氢酶和酯酶)的活力。结果显示,5种消化酶各自表现出不同的变化模式,胃蛋白酶和胰蛋白酶的比活力早期均逐渐上升,到发育后期胃蛋白酶出现快速下降,而胰蛋白酶却仍保持较高水平;淀粉酶比活力呈“V”字型变化趋势,晚期活性较高;纤维素酶和脂肪酶的比活力则均较低。4种同工酶酶谱随胚胎的发育渐趋复杂,酶活性也随之增强。结果表明,消化酶和同工酶活力的高低均受其基因的调控,并随胚胎发育适时表达,为胚胎组织、器官和系统的形成以及未来仔虾的开口摄食提供物质保证。     

红螯蟹虾胚胎发育的研究：Ⅱ，消化系统的发生

应用组织切片技术,研究了红螯螯虾胚胎发育过程中消化系统的发生,红螯螯虾的消化系统由前肠,中肠和后肠3部分组成,前肠和后肠由外胚层形成,而中肠源自原肠期由胚胎表面向囊胚内迁移的中内胚层细胞团,前无节幼体期前肠开始发生,至后无节幼体期先后形成口道,食道和胃等结构,中肠起始于后无节幼体期的次级卵黄锥,包括管状中肠和1对囊状消化腺－中肠腺;后肠端部是无节幼体期形成的肛道,肛道不断向胚胎前端延伸逐渐形成后肠。     

切除眼柄对加速克氏原螯虾卵巢发育进程的组织学研究

应用组织切片和透射电镜技术研究眼柄切除对加速克氏原螯虾(Procambarus clarkii)卵巢发育进程的影响。实验分成4组,选取眼柄切除15 d和30 d两个时间点进行。切除眼柄30 d后,雌虾体长增长显著,从(11.50±2.30)cm增长至(18.20±3.40)cm(P0.05),性腺指数(GSI)从4.56%±2.81%增长至8.05%±2.51%(P0.05),体重和螯肢长也有所增长(P0.05)。电镜观察发现卵母细胞从发育Ⅱ期迅速度过Ⅲ期进入到Ⅵ期阶段,卵母细胞体积变大,变圆;液泡压缩变小;卵黄颗粒加速积累,逐渐占满整个胞内空间;滤泡细胞胞质从黏稠变稀薄。这些结果表明,切除眼柄在短期内显著加快卵母细胞内卵黄颗粒的积累,并且加快个体生长。     

红螯光壳螯虾白斑综合症血液病理学研究

采用Wright-Geimsa染色法和电镜技术对人工感染的红螯光壳螯虾(Cherax quadricarinatus)白斑综合症(White spot syndrome,WSS)血液病理学进行了研究。结果显示:患病螯虾血细胞总数、透明细胞(AH)数量极显著减少(P<0.01),大颗粒细胞(LGH)极显著增加(P<0.01);病毒感染后3种血细胞大小均有增加趋势,透明细胞和大颗粒细胞的核质比(NP)较感染病毒前极显著下降(P<0.01)。显微病理学变化主要表现为血涂片中血细胞明显减少,病变、破损或解体的细胞增多,至濒死期螯虾血液呈典型的溶血状态。超微病理学变化表现为血细胞受到了损伤。高尔基体变形、线粒体结构模糊破损;核膜变形核固缩、细胞核高度异染色质化;濒临死亡的螯虾血细胞细胞器和染色质溶解,胞浆水肿,细胞溶解坏死。在患病螯虾的血细胞核中清晰可见WSSV粒子。     

不同水温对中华绒螯蟹胚胎发育的影响

本文研讨了各种人控恒温和自然水温对中华绒螯蟹胚胎发育周期,各发育阶段以及卵裂同步率等的影响,在水温9．6±3．6度到23度之间,中华绒螯蟹的胚胎均能发育;最适发育水温为18－23度,29度以上,胚胎不能发育,变温对胚胎发育有益,胚胎在卵裂阶段对水温特别敏感,水温越高,卵裂虽然越快,但卵裂期同步率越低;胚胎处在自然水温中,经过低温,卵裂期同步率最高。     

氨氮和亚硝酸盐对红螯螯虾幼虾和亚成虾的急性毒力

【目的】近年来,红螯螯虾养殖面积越来越广泛,明确不同规格的红螯螯虾对氨氮和亚硝酸盐的耐受力,有利于提高其养成率,促进其养殖业的健康发展。【方法】在水温24~25℃、p H 7.9~8.0的条件下,研究了氨氮和亚硝酸盐对红螯螯虾幼虾和亚成虾的急性毒性,分析半致死浓度(LC50)和安全浓度(SC)。【结果】总氨氮对红螯螯虾幼虾的24、48、72和96 h LC50分别为188.0、136.15、104.67和88.00 mg·L~(-1),SC为8.80 mg·L~(-1);总氨氮对亚成虾的24、48、72和96 h LC50分别为344.01、270.46、205.15和167.68 mg·L~(-1),SC为16.77 mg·L~(-1);非离子氨对幼虾的24、48、72和96 h LC50分别为10.16、7.35、5.65和4.75 mg·L~(-1),SC为0.48 mg·L~(-1);非离子氨对亚成虾的24、48、72和96 h LC50分别为18.58、14.60、11.08和9.05 mg·L~(-1),SC为0.91 mg·L~(-1);亚硝酸盐对幼虾的24、48、72和96 h LC50分别为46.76、33.88、27.97和22.81 mg·L~(-1),SC为2.28 mg·L~(-1);亚硝酸盐对亚成虾的24、48、72和96 h LC50分别为77.56、59.33、45.41和37.48 mg·L~(-1),SC为3.75 mg·L~(-1)。【结论】红螯螯虾对氨氮的耐受力高于亚硝酸盐,亚成虾对氨氮和亚硝酸盐的耐受力高于幼虾。     

Changes in vitellin and other yolk proteins during embryonic development in the silkworm, Bombyx mori

Changes in the amounts of vitellin and other yolk proteins of the eggs of the silkworm, Bombyx mori were investigated during embryonic development using polyacrylamide gel electrophoresis and immunotitration techniques. In the newly laid eggs, soluble proteins were separated into at least nine bands after electrophoresis. The major band was identified as vitellin, accounting for about 40% of the total proteins. The four predominant bands including vitellin exhibited the same mobility as the proteins of haemolymph, but one other major band was specific to the eggs, accounting for about 20% of the proteins.During embryonic differentiation 6–7 days after oviposition, the total protein content did not decrease and the banding patterns and their relative concentrations remained unchanged as a whole. However the concentration of the egg specific protein steadily decreased. During subsequent larval differentiation until hatching, the total proteins were utilized to about 50% of the initial levels: the rapid degradation was observed in almost every species of proteins.An immunotitration experiment further demonstrated that vitellin was not utlilized during embryonic differentiation but was consumed markedly during larval differentiation. However, about 30% of initial level was reserved in the newly hatched larvae. Such a prolonged persistence of vitellin is discussed in relation to protein metabolism during embryonic development in silkworms.     

Purification and characterization of the common yolk protein,vitellin, from the estuarine amphipod Leptocheirus plumulosus

Vitellin is a major yolk protein that plays a significant role in the embryonic development of crustacean embryos. This protein was rapidly purified from embryos of the estuarine amphipod, Leptocheirus plumulosus, by subjecting the crude protein homogenate to high affinity column chromatography. SDS-PAGE revealed a single band with an approximate molecular weight of 200,000 daltons. Vitellin was characterized by SDS-PAGE techniques and amino acid composition analysis. L. plumulosus vitellin is a lipoglycophosphoprotein with serine, glutamic acid/glutamine, alanine, and aspartic acid/asparagine accounting for almost 66% of all amino acid residues. Polyclonal antibodies were raised against L. plumulosus vitellin and antibody reactivity was verified by dot-blotting and immuno-fluorescence confocal microscopy. These antibodies are specific for purified vitellin and show little cross-reactivity with other embryonic proteins.     

龟纹瓢虫卵黄蛋白的分子特性及发生动态

研究了龟纹瓢虫Propylea japonica (Thunberg) 卵黄蛋白的基本特性以及卵黄发生过程中卵黄蛋白的动态变化。PAGE和SDS-PAGE实验表明,龟纹瓢虫卵黄蛋白分子量为294.81±40.70 kD,并由分子量分别为144.68±0.03 kD和51.23±0.27 kD的两种亚基组成。对卵黄蛋白的氨基酸组成和含量分析发现,其必需氨基酸总量占57.48％,略高于非必需氨基酸,其中谷氨酸（Glu）含量最高,为15.26％;色氨酸（Trp）和蛋氨酸（Met）含量较低,分别为0.50％和0.11％。采用间接竞争ELISA法,系统测定了龟纹瓢虫成虫期脂肪体、血淋巴和卵巢中卵黄蛋白的动态变化,结果表明：脂肪体是卵黄原蛋白合成的场所,卵黄原蛋白的合成始于羽化后第2天;脂肪体、血淋巴和卵巢中卵黄原蛋白的滴度在羽化后第4天开始迅速上升,至成虫期的第8天左右达到高峰期。     

Identification,purification, and partial characterization of vitellin from the eggs of Eurygaster integriceps putton (heteroptera,pentatomidae)

A single vitellin was identified in the eggs of the wheat bug, Eurygaster integriceps, and purified by a two-step procedure including ion exchange and gel permeation chromatography. Its nondenatured molecular mass is estimated to be 385 kDa. Under reducing conditions—SDS-PAGE—the wheat bug vitellin gives five polypeptides at 110 kDa, 80 kDa, 69 kDa, 53 kDa, and 38 kDa. Its amino acid composition is characterized by high content of aspartic acid/asparagine and glutamic acid/glutamine and low content of methionine and histidine. The lipid moiety (5.65% by weight) includes diacylglycerols, cholesterol, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin. Carbohydrate content amounts to 4.54% by weight. A part of the wheat bug vitellin is dimerized during the course of deposition into the yolk granules. © 1995 Wiley-Liss, Inc.     

Postendocytic Provitellin Processing in the Growing Oocyte of the Short Horned Grasshopper, Oxyajaponicajaponica (Orthoptera: Acrididae)

Polyclonal antibodies made against 86 kDa (86 k), 80 kDa (80 k) and 54 kDa (54 k) vitellins of Oxya japonica japonica are used for Western blotting. Anti‐80k vitellin antibody is cross‐reacted with a 95 kDa (95 k) vitellin. While 95 k vitellin is present both in the female hemolymph and in the oocyte, 80 k vitellin is detected only in the oocyte and the laid egg. In the growing oocytes, as 95 k vitellin is faded out gradually, 80 k vitellin is accumulated increasingly, indicating postendocytic processing of 95 k vitellin brings 80 k vitellin. Further conforming the hypothesis, partial digestion of 95 k vitellin with pepsin and α‐chymotrypsin makes several protein bands of molecular weight around 80 kDa. Thus, the 95k vitellin may have a cleavage site (s) to produce 80 k vitellin which forms fairly stable tertiary structure. In the reduced condition (20 mM glutathion), both 95 k and 80 k vitellins were digested throughly by endogenous proteinase at pH 4. Both 86 k and 54 k vitellins, respectively, show no apparent molecular weight changes in the growing oocyte and in the hemolymph.     

Embryo lipoproteins and yolk lipovitellin consumption during embryogenesis in Macrobrachium borellii (Crustacea: Palaemonidae)

The prawn Macrobrachium borellii has lecithotrophic eggs with highly-abbreviated development. The major yolk component is lipovitellin (LV), a lipoprotein with 30% lipids (by weight). LV consumption during embryogenesis was followed by ELISA and Western blot analysis using an anti-LV polyclonal antibody. No cross-reacting proteins were observed and LV-like lipoproteins were strongly recognized by the antibody in hemolymph (vitellogenin), yolk (LV) and embryos (LVe), as determined by Western Blot analysis. LV decreased significantly along development from 9.4 to 1.1 microg/mg egg. Consumption rate of LV was slow in early embryogenesis, followed by a rapid utilization in late embryonic stages. Significant LVe amounts were still present at hatching. LV apolipoproteins were selectively degraded during embryo development, being the highest molecular weight subunit the most affected. Comparison among in vitro, in vivo and theoretical proteolysis suggested that trypsin may be involved in LV degradation during late embryogenesis. Embryo lipoprotein (HDLe) synthesis was first detected at stage 6. HDLe shared the same density, MW and subunit composition as adult hemolymph HDL(1) and did not cross-react with LV-like lipoproteins. Though expressed at low concentration, it fulfilled embryo needs for lipid transport among organs.     

Changes in embryonic protein profile and economic characters of Bombyx mori (Lepidoptera:Bombycidae) following UV irradiation

Eggs of B. mori were irradiated with UV (254.4 nm wavelength) for different durations. Increase in the time of exposure to UV decreased the percentage hatchability of the eggs, cocoon and pupal weights. The shell weight remained unaltered proving the stability of silk gland DNA. Irradiation of eggs also delayed the degradation/utilization of the embryonic proteins, viz. vitellin (heavy and light subunits), egg-specific protein and 30K protein.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori,during early developmental stages

Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.     

褐飞虱卵黄蛋白的分离及其生化特性

电泳结合不同染色方法证实, 褐飞虱Nilaparvata lugens (Stål)卵黄蛋白为一种糖脂结合蛋白,其分子量约为314 kD,由148 kD、124.5 kD和39.6 kD 3个亚基组成。免疫反应证明,卵黄蛋白只存在于生育期的雌性褐飞虱成虫体内。褐飞虱卵黄蛋白具有种的特异性,其免疫血清与白背飞虱的卵黄蛋白无交叉反应。