反相高效液相色谱法分离中华绒螯蟹性腺抑制激素的初步研究

采用反相高效液相色谱层析法(RP-HPLC)分离中华绒螯蟹(Eriocheir sinensis)眼柄视上神经节中的性腺抑制激素,收集在高血糖激素家族区域内出峰的洗脱液组分,得到7组单一的样品组分。通过活体注射的方法,观察和统计每一组分对去眼柄中华绒螯蟹卵母细胞直径大小的影响,以检验每个分离组分的性腺抑制活性。结果发现,其中的第7组分对中华绒螯蟹的卵巢发育具有最强的抑制作用,推测这一组分为中华绒螯蟹的性腺抑制激素。     

中华绒螯蟹不同组织氯霉素药物残留分析

选取经检测氯霉素含量超标的中华绒螯蟹,利用水产品中氯霉素残留测定气相色谱法,对其性腺(蟹黄)、肌肉和肝脏等可食部分分别进行了测定,结果在污染的中华绒螯蟹的性腺(蟹黄)和肌肉中均未检出氯霉素,而在肝脏中检出大量氯霉素.所以在中华绒螯蟹氯霉素检测过程中,样品处理应特别注意肝脏的取舍,同时也提醒人们谨慎食用中华绒螯蟹的肝脏.     

中华绒螯蟹Akt基因的cDNA克隆、序列分析及表达特征

为研究Akt基因在中华绒螯蟹蜕壳前后肌肉生长过程中的功能,应用RACE技术克隆得到编码中华绒螯蟹Akt(命名为Es Akt)的全长为2 200 bp的c DNA序列,包括159 bp的5′非翻译区(5′-UTR)、496 bp的3′非翻译区(3′-UTR)和长度为1545 bp编码514个氨基酸的编码序列。蛋白质结构域分析显示Es Akt含有丝氨酸/苏氨酸蛋白激酶家族的3个特征性保守结构域。多序列比对和系统发育分析显示,Es Akt与中国明对虾、凡纳滨对虾的Akt序列一致性都为0.889,在系统发育树中节肢动物Akt形成一个分支。应用荧光定量RTPCR技术分析Es Akt在性成熟中华绒螯蟹各组织及幼体不同蜕壳时期不同部位肌肉组织中转录水平上表达量的变化。结果显示,Es Akt在性成熟个体的肝胰腺、眼柄、表皮、卵巢、精巢、心脏、螯足、鳃、三角膜等组织中均有表达,其中卵巢、眼柄和精巢中表达量较高,肝胰腺中表达量最低。在幼体不同蜕壳时期不同部位的肌肉中,Es Akt表达量变化不同,步行足肌肉组织中Es Akt m RNA无显著的统计学差异。腹部肌肉组织中Es Akt m RNA水平在蜕壳前晚期D3~D4期表达量显著高于蜕壳后A~B期和蜕皮间期C期。螯足肌肉在蜕壳前晚期D3~D4期急剧下调,蜕壳后A~B期开始表达量显著升高,直至蜕皮间期C期。研究表明,Es Akt在中华绒螯蟹蜕壳过程中不同部位肌肉组织中的表达量变化与蜕壳周期密切相关,说明Es Akt参与中华绒螯蟹蜕壳诱导的肌肉萎缩、生长及重建过程。     

伊乐藻对中华绒螯蟹生长和营养品质的影响

本研究分析了有伊乐藻(Elodea nuttallii)组和对照组(无伊乐藻)中华绒螯蟹(Eriocheir sinensis)在生长、肌肉氨基酸和脂肪酸组成等方面的差异,探讨伊乐藻对中华绒螯蟹生长和营养品质的影响。结果显示,有伊乐藻组中华绒螯蟹体重、壳长和壳宽增长率与肥满度均显著高于无伊乐藻组(P0.05),但肝胰腺指数和性腺指数差异不显著(P0.05)。伊乐藻组中华绒螯蟹肌肉的氨基酸总量、必需氨基酸和鲜味氨基酸含量均显著高于无伊乐藻组(P0.05);伊乐藻组的雌蟹肝胰腺中的鲜味氨基酸含量也显著高于无伊乐藻组(P0.05),而伊乐藻组和无伊乐藻组的雄蟹肝胰腺氨基酸含量差异不显著(P0.05)。伊乐藻组和无伊乐藻组的中华绒螯蟹肌肉脂肪酸组成和含量差异不显著(P0.05),但伊乐藻组中华绒螯蟹的肝胰腺饱和脂肪酸(SFA)、单不饱和脂肪酸(MUFA)和多不饱和脂肪酸(PUFA)含量均显著高于无伊乐藻组(P0.05)。综上所述,伊乐藻不仅有利于中华绒螯蟹的生长,而且能改善中华绒螯蟹的营养品质。     

中华绒螯蟹细胞色素CYP2基因的克隆与表达分析

为研究细胞色素CYP2基因在中华绒螯蟹蜕皮及生长发育中的作用,本试验通过逆转录聚合酶链式RT-PCR反应以及cDNA末端快速扩增RACE技术获得了中华绒螯蟹CYP2基因cDNA全长。用实时荧光定量PCR技术,分析该基因在中华绒螯蟹蜕皮过程中各组织的相对表达量。试验结果显示,中华绒螯蟹CYP2的cDNA全长1772bp,编码491个氨基酸序列,包括一个84bp的5′端非编码区,一个212bp的3′端非编码区、一个1475bp的开放阅读框,经BLAST比对,该核苷酸序列与岸蟹、三疣梭子蟹的相似性分别为66%、64%。实时荧光定量PCR结果显示,中华绒螯蟹蜕皮前,CYP2基因在鳃中的表达量相对最高;在肝胰脏、肌肉中表达量略低于鳃;在Y器官、眼柄、胸神经节、脑神经节、肠中少量表达;在心和胃中表达量最低。中华绒螯蟹在不同的蜕皮时期中,通过荧光定量试验得出,肝胰脏中CYP2基因表达量在蜕皮前期和蜕皮期最高;眼柄中CYP2基因表达量从蜕皮期到蜕皮后期有上升趋势;鳃中CYP2基因表达量在蜕皮前期最高;Y器官、脑神经节和肠中CYP2基因表达量在蜕皮期最高;肌肉中CYP2基因表达量在蜕皮前期最高;胸神经节和胃中CYP2基因表达量在蜕皮后期最高;Y器官中CYP2基因在蜕皮前期表达量高于蜕皮后期和蜕皮期。以上试验结果表明,CYP2对中华绒螯蟹蜕皮生长中的调控可能起到重要作用。     

中华绒螯蟹Smad3(EsSmad3)的cDNA克隆、序列分析及表达特征

为了研究Smad基因在中华绒螯蟹(Eriocheir sinensis)蜕壳前后肌肉生长过程中的功能,应用RACE技术克隆得到编码中华绒螯蟹Smad3(命名为Es Smad3)的cDNA全长序列2021 bp,包括36 bp的5′非翻译区(5′-UTR)、656 bp的3′非翻译区(3′-UTR)和编码442个氨基酸的开放阅读框。蛋白质结构域分析显示EsSmad3含有MH1和MH2两个特征性保守结构域。多序列比对显示,EsSmad3与人、斑马鱼、黑腹果蝇中的同源蛋白序列一致性分别为0.679、0.691、0.619。应用荧光定量RT-PCR技术分析EsSmad3在性成熟中华绒螯蟹各组织及幼体不同蜕壳时期不同部位肌肉组织中转录水平上表达量的变化。结果显示,Es Smad3在性成熟个体的肝胰腺、眼柄、表皮、卵巢、精巢、心脏、螯足、鳃、三角膜等组织中均有表达,其中眼柄和精巢中表达量较高,心脏和肝胰腺中表达量最低。在幼体不同蜕壳时期的不同部位的肌肉中,Es Smad3表达量变化不同:步行足肌肉组织中EsSmad3 mRNA表达在蜕壳间期高于蜕壳前D_(3–4)期和蜕壳后A~B期,但无显著的统计学差异(P0.05)。螯足肌肉在蜕壳前晚期D_(3–4)期急剧下调(P0.05),蜕壳后A~B期开始表达量显著升高(P0.05),直至蜕皮间期C期。腹部肌肉组织中EsSmad3m RNA水平在蜕皮间期C期显著高于蜕壳后A~B期,这种上调一直持续到蜕壳前晚前期D_(3–4)。上述结果表明,Es Smad3在中华绒螯蟹蜕壳过程中不同部位肌肉组织中的表达模式与蜕皮周期密切相关,推测Es Smad3参与了中华绒螯蟹蜕壳诱导的肌肉萎缩、生长及重建过程。     

中华绒螯蟹、日本绒螯蟹及其杂交种性腺发育和生化组成的比较

研究了中华绒螯蟹(Eriocheir sinensis)、日本绒螯蟹(E.japonica)及其杂交种成蟹性腺指数、肝胰腺指数、出肉率、总可食率及其常规生化成分,结果显示:(1)中华绒螯蟹的精巢指数显著高于其它两种群蟹,但其肝胰腺指数、出肉率和总可食率均无显著差异;中华绒螯蟹的卵巢指数也显著高于日本绒螯蟹和杂交蟹,而杂交蟹的肝胰腺指数显著高于其它两种群蟹。(2)中华绒螯蟹、日本绒螯蟹和杂交种精巢中水分、蛋白和脂肪含量分别最低,日本绒螯蟹精巢中碳水化合物含量最高;杂交种肝胰腺蛋白、脂肪和碳水化合物含量均最高,日本绒螯蟹肌肉中脂肪含量显著低于其它两种群。(3)日本绒螯蟹卵巢水分含量显著高于其它两种群蟹,蛋白、脂肪和碳水化合物含量显著低于其它两种群;日本绒螯蟹肝胰腺的水分和蛋白含量最高,但脂肪含量最低,杂交种肝胰腺中脂肪含量最高、水分含量最低;日本绒螯蟹肌肉中的水分含量最高,其它生化成分含量最低;中华绒螯蟹肌肉的水分含量最低,其它生化成分含量最高。整体上,中华绒螯蟹具有早熟特性,不同种群绒螯蟹性腺中的主要生化成分含量可能与其发育阶段有关,杂交蟹肝胰腺中脂肪含量较高,日本绒螯蟹肌肉中脂肪含量较低。     

中华绒螯蟹免疫因子-溶菌酶的初步研究

研究了在自然状态下中华绒螯蟹成蟹血淋巴和大眼幼体组织匀浆上清液中溶菌酶的活性。结果表明,在中华绒螯蟹成蟹血淋巴中均检测出溶菌酶的活性,为(0.0286±0.0081)活力单位(n=30),而在中华绒螯蟹大眼幼体组织匀浆上清液中亦检测到溶菌酶的活性,为(0.0318±0.0073)活力单位(n=4)。     

中华绒螯蟹RXR基因全长cDNA克隆及表达分析

维甲类X受体(retinoid X receptor,RXR),属于核受体超家族(nuclear receptor super family)成员,是一种重要的调控因子,对甲壳动物生长发育和繁殖具有十分重要的作用。本研究根据陆地蟹 RXR基因保守区序列设计上下游引物,采用逆转录聚合酶链反应(RT-PCR)以及cDNA末端快速扩增(RACE)技术克隆中华绒螯蟹(Eriocheir sinensis)RXR基因并进行各组织间的表达分析。序列分析表明中华绒螯蟹RXR cDNA全长1517bp,编码433个氨基酸。经BLASTn和BLASTx分析表明,中华绒螯蟹RXR氨基酸序列与陆地蟹RXR基因的氨基酸序列相似性最高,为96%。系统进化树分析显示,中华绒螯蟹RXR的氨基酸序列与陆地蟹RXR聚为一支。荧光定量PCR结果显示,RXR在所有检测的组织中均有表达,且在中华绒螯蟹Y器官中表达量最高,肝胰腺、鳃和肌肉中有少量表达,心脏、胃、肠、胸神经节和脑神经节中微量表达。在中华绒螯蟹不同蜕皮时期中,Y器官中RXR表达量在D期最高,与AB期、C期呈显著性差异（P＜0.05）;肌肉中RXR在AB期表达量最高,与其他蜕皮时期呈显著性差异（P＜0.05）;肝胰腺和鳃中RXR在AB期表达量最高,E期表达量最低,但各蜕皮时期表达量之间差异不显著。     

绒螯蟹三个种群形态判别比较

对长江水系中华绒螯蟹、莱茵河野生中华绒螯蟹和日本绒螯蟹三个种群的形态特征进行了判别研究。可数性状分析的结果表明,长江水系中华绒螯蟹、莱茵河野生中华绒螯蟹和日本绒螯蟹三个种群,两两之间差异极显著。聚类分析的结果,把长江水系中华绒螯蟹和莱茵河野生中华绒螯蟹划为一组,日本绒螯蟹为另一组。判别分析建立起三者的判别函数,雌雄蟹判别的正确率分别为83．1％、91．9％。     

锯缘青蟹蜕皮抑制激素cDNA的分子克隆及其表达分析

根据近缘种类同源序列设计简并引物,采用逆转录聚合酶链式反应(RT—PCR)技术,首次扩增获得锯缘青蟹(Scylla serrata)眼柄组织中编码蜕皮抑制激素(MIH)成熟肽全长cDNA序列及部分信号肽序列。将该序列克隆到pUCm—T载体上进行序列测定。结果表明,编码锯缘青蟹MIH成熟肽的cDNA由234个碱基组成,由此推测MIH成熟肽含78个氨基酸残基。该序列不仅与其它短尾类甲壳动物的MIH氨基酸序列具有高度的同源性(79％～9l％),还与该激素同一家族的性腺抑制激素、大颚器抑制激素的氨基酸序列具有较高的相似性。RNA斑点杂交结果显示,MIH基因在眼柄神经节及脑组织中均有表达,而在肌肉、中肠腺中不表达。     

Effect of Acclimation Temperature and TemperatureChanges on Molting and Survival of Ablated and Non-ablated Crayfish Orconectes virihs

The effect of acclimation temperatures for producing soft-shell crayfish, and changes upward from acclimation temperature, were evaluated in crayfish Orconectes virilis subjected to either hemilateral or bilateral eyestalk ablation. In each experiment, 60 juvenile O. virilis were reared for 21 d at temperatures ranging from 20 C to 30 C. Equal numbers of male and female crayfish ranging in weight from 5.0 g to 16.0 g were used in each study. In the first three experiments, crayfish were acclimated and maintained at a constant temperature. In the remaining experiments, crayfish were subjected to a step-wise increase in temperature of either 5 or 10 C. Molting was highest in crayfish acclimated to 25 C prior to experimentation at 25 C (experiment 2) and in crayfish acclimated to 14 C prior to experimentation at 20 C (experiment 4). Total molts during these two experiments were 51 (85.0%) and 56 (93.3%), respectively. Bilateral eyestalk ablation resulted in significant reductions in the intermolt period in experiments 1 (acclimation temperature 20 C, experimental temperature 20 C), 3 (acclimation temperature 30 C, experimental temperature 30 C) and 6 (acclimation temperature 15 C, experimental temperature 25 C), while hemilateral eyestalk ablation had no effect on molting when compared to control (non-ablated) crayfish. Bilateral eyestalk ablation resulted in a significant reduction in survival in experiments 5 (acclimation temperature 20 C, experimental temperature 25 C) and 6 (acclimation temperature 15 C, experimental temperature 25 C). Significantly more females molted in one experiment (acclimation temperature 20 C, experimental temperature 25 C), but not in any others. The results of these studies demonstrate that bilateral eyestalk ablation and water temperatures are effective methods of inducing molting in crayfish.     

A rapid non-enzymatic method of DNA extraction for PCR detection of white spot syndrome virus in shrimp

The present study describes a simple method of extraction of white spot syndrome viral DNA (WSSV) from infected shrimp for the polymerase chain reaction (PCR) detection of WSSV. The DNA preparation using this method was found to be free from the host DNA, RNA and protein, and is suitable for different PCR protocols such as single‐step PCR, nested PCR and single‐tube semi‐nested PCR. This method of extraction has worked successfully for extracting the WSSV‐DNA from different organs (haemolymph, eyestalk, carapace, head muscle, heart, gills, appendages, heptopancreas, stomach, intestine, abdominal muscle and tail muscle) of WSSV‐infected adult shrimp, and WSSV‐infected larvae and postlarvae.     

Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone

The present study analyses some biochemical variables in haemolymph proposed as predictive indicators of the maturation capability following eyestalk ablation. Haemolymph of captive females was obtained before and 8 days after eyestalk ablation, and levels of haemocyanin, total proteins, glucose, lactate, cholesterol, triacylglycerides and vitellogenin were determined. Biochemical variables were also analysed in the hepatopancreas at the end of the experiment. Females were grouped as immature (previtellogenic stage) and mature (vitellogenic and cortical stages) based on histological analysis done 8 days after eyestalk ablation. To analyse haemolymph variables before eyestalk ablation in relation to maturation capability, immature females were classified as those with a low maturation capability and mature females as those with a high maturation capability. Females of high maturation capability had significantly higher vitellogenin levels before eyestalk ablation than females of low maturation capability. No significant differences were found for the other biochemical variables. Vitellogenin was also higher in mature than in immature females at the end of the experiment. These results indicate that vitellogenin levels in haemolymph could be used as possible predictive criteria of maturation capability, possibly because they reflect the degree of ovarian development at the time of eyestalk ablation.     

青虾Ran基因的克隆、表达及其在卵巢发育中的功能

本研究应用RACE技术克隆了青虾(Macrobrachium nipponensis)Ran(Ras related nuclear protein,Ras相关核蛋白)基因全长cDNA序列,该基因cDNA全长1191 bp,包括218 bp的5'UTR,648 bp的开放阅读框(ORF),405 bp的3'UTR,编码215个氨基酸。青虾Ran基因属于P-loop-NTPase超级家族,拥有PTZ00132跨结构域,多肽分子量约为24.57 kDa,理论等电点7.13。系统进化树分析表明,在动物界进化中非常保守的青虾Ran多肽与罗氏沼虾(Macrobrachium rosenbergii)聚为一支,具有最近的亲缘关系。使用荧光定量PCR技术检测Ran基因在成体青虾不同组织和卵巢不同发育期的表达差异,结果显示,Ran基因在青虾不同组织中均有表达,其表达量在卵巢中最高,是精巢表达量的7~8倍;随着卵巢的发育,Ran基因的表达水平呈现上升趋势,在卵巢消退期又恢复到较低水平。RNA干扰后,实验组Ran基因表达量显著低于对照组(P0.05),同时卵巢发育关键基因Vg(vitellogenin)在卵巢中的表达量也显著低于对照组(P0.05),推测Ran基因参与雌性卵巢发育过程并对Vg基因的表达起到调控作用。     

眼柄摘除对红螯光壳螯虾生长、性腺发育及体色的影响

为探究眼柄摘除对红螯光壳螯虾(Cherax quadriarinatus)生长、性腺发育及体色的影响,对6月龄红螯光壳螯虾[体质量(47.47±3.48)g]进行了摘除眼柄处理实验。结果显示,4周后摘除双侧眼柄组红螯光壳螯虾体质量为(84.40±13.41)g,显著高于不摘除眼柄组[(49.63±6.47)g]和摘除单侧眼柄组[(51.47±4.08)g](P<0.05);摘除双侧眼柄组红螯光壳螯虾存活率为60.0%±4.8%,显著低于不摘除眼柄组和摘除单侧眼柄组(分别为94.4%±2.3%和97.2%±2.1%);摘除双侧眼柄组红螯光壳螯虾性腺指数为1.23±0.50,显著高于不摘除眼柄组(0.20±0.06)和摘除单侧眼柄组(0.35±0.08)。利用ImageJ软件对红螯光壳螯虾体色RGB值分析表明,摘除双眼柄组红螯光壳螯虾体色G值和B值(分别为99.26±5.23和98.40±3.58)显著高于摘除单侧眼柄组(59.02±3.85和44.07±4.57)(P<0.05)。结果表明,眼柄摘除能促进红螯光壳螯虾生长和性腺发育,影响体色变化。     

Stimulation of ovarian growth by methyl farnesoate and eyestalk ablation in penaeoidean model shrimp,Sicyonia ingentis Burkenroad, 1938

The effect of methyl farnesoate (MF) administration on the vitellogenesis of the penaeoidean shrimp, Sicyonia ingentis, was studied. The short‐ and long‐term treatment effects as well as the effect of two MF injection regimens (0.1 and 1.0 μg MF/injection) were evaluated. The studies were also carried out to understand the pattern of vitellogenesis in eyestalk ablated adult and juvenile shrimps. A combination of endpoints, haemolymph vitellogenin (Vg) levels, gonadosomatic index (GSI) and histology, was used to study the effect of these treatments. The GSI increased in all the MF‐treated shrimp compared with the control shrimps. Although haemolymph Vg levels declined over the experimental period in all the treatments, the Vg levels decreased significantly only in the short‐term treatment with 1.0 μg MF. Similarly, haemolymph protein level also declined over the experimental period in all the treatment groups. However, except in the long‐term treatment with 0.1 μg MF, all treatments showed a significant decrease in haemolymph protein level. Conversely, in all eyestalk ablated adults and juveniles, haemolymph Vg, total protein and GSI increased over the experimental period, all of which were higher than the concurrent control. The discrepancy in the vitellogenic pattern between MF‐treated and eyestalk ablated shrimp was possibly due to the difference in the ovarian phase of the initial control. Although unilateral eyestalk ablation failed to induce vitellogenesis in juveniles, bilateral ablation induced vitellogenesis, which indicates that juveniles are competent to undergo vitellogenesis.     

编码鹰爪虾蜕皮抑制激素基因的cDNA片段的克隆和序列分析

提取鹰爪是眼柄总RNA，以眼柄总RNA为模板，根据日本对虾的具有蜕皮抑制活性的神经肽Pej-SGP-Ⅳ的氨基酸设计兼并引物，进行RT-PCR扩增，在适宜条件下得到一特异性片段。将这一特异性片段克隆到载体中进行测序，测得鹰爪虾的特异性cDNA片段由213个碱基对组成，推测的氨基酸序列由71个氨基酸组成。序列比较发现许多甲壳动物的CHH家庭神经肽与它相似。在所有与该序列推测的氨基酸序列相似的甲壳动物CHH家族神经肽中，MIH与它的相似度最高，表明由鹰爪虾眼柄特异性cDNA片段椎定的氨基酸基酸序列可能是鹰爪虾的MIH片段，它相当于MIH成熟肽的第5至75个氨基酸。