克氏原螯虾丝氨酸蛋白酶类似物部分基因的克隆与鉴定

设计简并引物,PCR扩增了克氏原螯虾(Procam barus clarkii)丝氨酸蛋白酶类似物基因的部分序列,通过与GenBank登录的序列进行比对,结果显示该序列编码的氨基酸与其他物种的丝氨酸蛋白酶类似物氨基酸序列有较高的同源性。SMART软件分析发现其催化三联体结构中的丝氨酸被甘氨酸取代,不具备蛋白酶水解活性。     

cDNA芯片结合抑制性差减杂交技术构建对虾白斑综合征病毒表达基因和螯虾免疫相关基因文库

以克氏原螯虾做为对虾白斑综合征病毒（WSSV）的增殖模型,试验组接种WSSV,对照组接种PBS。分别以试验组为检测组（tester）、对照组为驱逐组（driver）,进行正向抑制性差减杂交;以对照组为tester、试验组为driver,进行反向SSH。将获得的正、反向差减杂交产物克隆入质粒载体PinPointTM Xa1 TVector,再转化大肠杆菌DH5α,共构建了2个分别含1620和400个克隆的正、反向差减文库。经PCR扩增,1514个差减克隆得到产物,其中正向差减克隆1221个,反向差减克隆293个。获得的PCR产物经浓缩、变性处理,用自动点样仪点制于尼龙膜载体上,制成cDNA芯片,用cDNA芯片对SSH获得的差减克隆进行鉴定,共获得差异表达克隆278个,其中攻毒组高表达克隆255个;对照组高表达克隆23个。     

亚硝酸盐对红螯光壳螯虾不同组织免疫相关酶活性及超微结构的影响

应用透射电镜技术,结合生物酶测定,研究了水体中不同浓度亚硝酸盐胁迫下红螯光壳螯虾肝胰腺、鳃和肌肉组织中免疫相关酶的活性变化,以及对肝胰腺和鳃的形态学影响。结果显示,与对照组相比,亚硝酸盐胁迫下,3种组织的ACP、AKP、SOD以及GSH-PX的活性都显著降低(P<0.05);随着亚硝酸盐浓度增加,酶活力呈现降低的趋势;鳃组织Na+,K+-ATPase和Ca2+,Mg2+-ATPase的活性也显示出随亚硝酸盐浓度升高而降低的趋势。超微结构显示,随着亚硝酸盐浓度增加,鳃角质层受损、断裂;上皮细胞排列疏松、空泡化;细胞器变形;鳃腔内也出现空泡化现象,血细胞变形。肝胰腺上皮细胞排列杂乱无章,细胞裂解,空泡化;微绒毛受损、断裂,肝小管间距扩大、结缔组织变得稀薄,血细胞变形;高浓度组R细胞的脂滴减少,核膜解体,细胞膜破裂,空泡化加剧;F细胞的核糖体减少,空泡化加剧,内质网水肿。研究说明亚硝酸盐对红螯光壳螯虾3种组织的免疫相关酶活产生影响,并损伤肝胰腺和鳃的形态学结构,影响其生物学功能。     

红螯螯虾雄性生殖系统组织蛋白酶的初步研究

Cherax quadricarinatus）雄性生殖系统的组织蛋白酶（）进行了定性和定量分析,以探讨其在雄性生殖系统发育、精荚形成和精子释放过程中的作用。福林酚     

中华绒螯蟹免疫相关基因的研究进展

中华绒螯蟹(Eriocheir sinensis)是我国重要的增养殖对象之一,近年来,随着养殖规模的不断扩大和集约化程度的提高,由细菌、病毒和类立克次体等引起的各种疾病日趋严重,给中华绒螯蟹养殖产业造成了巨大损失~[1-3].     

[06]红螯螯虾雄性生殖系统组织蛋白酶的初步研究

用福林酚法和复性电泳对红螯螯虾(Cherax quadricarinatus)雄性生殖系统的组织蛋白酶(Male Reproductive SystemCathepsin,MRSC)进行了定性和定量分析,以探讨其在雄性生殖系统发育、精荚形成和精子释放过程中的作用.福林酚法测定结果显示:仔虾精巢蛋白酶比活力分别在pH6和pH10时出现2个高峰,而成虾精巢只在pH10时出现1个活力较高的峰.成虾雄性生殖系统的各部分在pH10～12的条件下有较强的蛋白酶比活力,于碱性缓冲液中浸泡有助于其后输精管内含物的溶解.复性电泳分离到4个蛋白酶条带,按分子量从大到小依次为:MRSC-A、MRSC-B、MRSC-C和MRSC-D.MRSC-A和MRSC-D广泛分布于雄性生殖系统各部分,其中MRSC-A为酸性蛋白酶,而MRSC-D是碱性蛋白酶;MRSC-C为酸性蛋白酶,只分布于中输精管;MRSC-B活性受pH值影响不大,仅存在于后输精管.在红螯螯虾雄性生殖系统的不同发育阶段,其组织蛋白酶的组成和活性不同.作为精荚的重要组成成分,组织蛋白酶与精荚排出体外后的硬化有直接关系,并促进随后精荚基质的水解以及精子的释放.     

红螯光壳螯虾组织蛋白酶L基因的克隆及维生素C对其表达的影响

为深入了解红螯光壳螯虾组织蛋白酶L基因的表达特性及维生素C对其表达的影响,实验利用RACE-PCR技术及荧光定量PCR技术,从红螯光壳螯虾肝胰腺中克隆得到组织蛋白酶L基因cDNA全长序列,命名为CqCatL(GenBank登录号:KJ913663),同时检测了该基因在红螯光壳螯虾各个组织及添加了不同浓度维生素C的组别中的表达。结果显示,该基因全长1 810 bp,开放阅读框长度为1 026 bp,编码341个氨基酸残基,预测的分子量和等电点(pI)分别为37.63 ku和5.17。同源性分析结果显示,该基因编码的蛋白与其他虾蟹类有较高的相似性,说明组织蛋白酶L基因在甲壳动物具有较高的保守性。组织荧光定量PCR结果显示,CqCatL基因在红螯光壳螯虾的多个组织中均有表达,其中肝胰腺中表达量最高,其次为血细胞,在肠及触角腺中也有一定量的表达。在基础饲料中添加不同水平的维生素C后,CqCatL基因的表达量也存在明显差异,其中维生素C添加量为400 mg/kg的组别中该基因的表达量最高。研究表明,组织蛋白酶L基因在红螯光壳螯虾的生长发育过程中有重要的作用,且其表达量受维生素C的影响。     

红螯螯虾生物学特性的观察及与克氏螯虾的比较

本文描述了红螯螯虾的生物学特性,如分类地位、外形结构、栖息习性、消化器官与食性、呼吸器官及溶氧需求、蜕壳与生长、繁殖等,并与克氏螯虾进行了比较。     

红螯螯虾的生物学特性及养殖技术

红螯螯虾又称淡水龙虾 ,原产于澳大利亚北部的热带地区 ,属甲壳纲、十足目、长尾亚目、拟河虾科 ,是我国新引进的淡水大型虾类之一。其具有个体大 ,食性广、生长快、肉味鲜美 ,可食率高、适应能力强等优点 ,是一种具有推广价值的优良品种。我们于１９９７年从澳大亚利引进了红螯螯虾原种 ,进行了生物学方面研究及养殖试验 ,并将此列入山东省良种产业化开发项目。通过几年的细致研究 ,掌握了其生物学特性及养殖技术 ,为普及该虾在我国的养殖 ,现将有关方面的内容报道如下 :１生物学特性１．１形态特征红螯螯虾体色为褐绿色 ,头胸部与腹部连接…     

氨氮急性胁迫及其毒后恢复对红螯光壳螯虾幼虾相关免疫和代谢指标的影响

应用实时荧光定量PCR技术,结合生物酶和代谢产物测定,研究了氨氮急性胁迫对红螯光壳螯虾幼虾代谢及免疫系统的毒性影响及其毒后恢复情况。实验首先进行3 d的氨氮胁迫,取样后剩余虾移入曝气自来水进行7 d的毒后恢复实验。结果表明,3 d氨氮胁迫后,肌肉ACP、AKP、SOD活性表达均受到显著影响,随着氨氮浓度的升高酶活性分别降低,最高浓度组(16 mg/L)降低为对照组的76%、68%和62%。线粒体MnSOD、胞外Cu/ZnSOD的mRNA表达量也随着氨氮浓度增加而下降,最高浓度组降低至对照组的69%和68%。CAT、GPX活性以及GPX和GST的mRNA表达量变化不显著。肝胰腺中可溶性蛋白和甘油三酯含量随着氨氮浓度升高而降低,最高浓度组分别降低至对照组的72%和59%,AST活性在12 mg/L浓度组显著升高至对照组的134%。7 d恢复期过后,ACP和AKP活性以及各代谢指标恢复到正常水平;而SOD和GPX活性高于对照组。各抗氧化基因的表达量都不同程度高于对照组。实验表明,高浓度氨氮胁迫能抑制部分免疫相关酶的活性及基因表达,对免疫系统造成损害。氨氮胁迫下,红螯光壳螯虾动员蛋白质和脂肪来供能应对胁迫。7 d的恢复时间不足以让红螯光壳螯虾从胁迫中完全恢复,其肌肉仍处于轻度氧化应激状态。     

Innate immune response and related gene expression in red swamp crayfish [Procambarus clarkii (Girard)], induced by selenium‐enriched exopolysaccharide produced by bacterium Enterobacter cloacae Z0206

Selenium‐enriched exopolysaccharide (Se‐ECZ‐EPS) produced by bacterium Enterobacter cloacae Z0206 is an important polysaccharide and has been proved its immunostimulating roles in mice. However, relatively less information is available about the potential role of Se‐ECZ‐EPS in crustaceans. To investigate the modulation of immunity in crustaceans, we examined phenoloxidase (PO) activity, respiratory burst (RB) ability, superoxide dismutase (SOD) level and several immune‐related genes' expression profile in red swamp crayfish [Procambarus clarkii (Girard)] that were individually induced with Se‐ECZ‐EPS at 1 μg g^?1. The protection of crayfish against the white spot syndrome virus (WSSV) by Se‐ECZ‐EPS was also investigated. The results showed that PO, RB and SOD activities of crayfish increased significantly (P<0.05) at 24, 48 and 72 h after being injected with Se‐ECZ‐EPS. Among the genes examined, the mRNA expressions of serine proteinase, HSP70 and Mn‐SOD were significantly up‐regulated at 24 and 48 h after the Se‐ECZ‐EPS treatment, while no significant changes in the expression in proPO were observed from 24 to 72 h. The survival rate of crayfish that received Se‐ECZ‐EPS was significantly higher than that of the control group after 14 days (59.7% and 0% respectively). Taken together, it can be concluded that Se‐ECZ‐EPS is an efficient immunostimulant and can improve the immunity of crayfish.     

白鲢骨骼肌丝氨酸蛋白酶抑制剂的提取、纯化及其特性

从白鲢（Hypophthalmichthys molitrix）骨骼肌中分离纯化到一种丝氨酸蛋白酶抑制剂，该抑制剂的分子晕大约50kD，用PAGE-明胶电泳证明，该抑制剂对胰蛋白酶具有抑制作用；当温度超过60℃时，抑制剂的活性下降70％左右，但在pH3～11的范围内均有活性。用MTT法分析白鲢骨骼肌丝氨酸蛋白酶抑制剂对急性T细胞白血病细胞Jarkat、人骨髓瘤细胞Sp20和人骨瘤细胞MG63生长的影响，结果发现该抑制剂对以上3种肿瘤细胞的增殖都有抑制作用，而且抑制作用呈剂量依赖性。本研究旨为进一步研究白鲢骨骼肌丝氨酸蛋白酶抑制剂的纯化及其抗肿瘤作用提供科学依据。     

复方中草药对克氏原螯虾生长和脱壳的影响

研究了饲料中添加复方中草药(质量分数为1%,2%和4%)对克氏原螯虾(Procambarus clarkii)生长和脱壳的影响。结果显示,添加复方中草药后,虾的脱壳周期缩短,脱壳死亡率和非脱壳死亡率均明显降低(P<0.05),但各中草药添加组间差异不显著(P>0.05)。三个剂量的复方中草药添加组均能不同程度地提高虾的脱壳数、成功脱壳数、硬壳虾重和软壳虾重,与未添加组相比差异显著(P<0.05),其中2%组增加最为显著,分别比对照组高出71.1%、103.2%、20.3%、10.0%。回归分析表明,克氏原螯虾的特定生长率达到最大值时的中草药添加质量分数为2.74%。试验表明克氏螯虾饲料中复方中草药水平以2%左右为宜。     

Effects of simulated precipitation extremes on crayfish (Procambarus clarkii Girard) oviposition in artificial burrows

Mature female Procambarus clarkii were housed individually in artificial burrows simulating conditions in natural burrows. Crayfish were exposed to conditions of simulated temporary flooding dislocation (Experiment 1) or simulated drought (Experiment 2). In Experiment 1, females were dislocated from burrows to simulate 0, 1, 3 or 6 temporary flooding dislocations, and in Experiment 2, females were exposed to the following simulated drought conditions: wet control (no drought), dry control (no free water), month‐long periods without free water during July, August, September and October (1 month drought), two consecutive months without free water (August–September), and three consecutive months without free water (August–October). In Experiment 1, no significant differences in survival or oviposition rate were observed following transfer of individuals from ‘settled/sealed’ burrows to ‘new burrow’ containers, even after six consecutive dislocations. In Experiment 2, simulated drought of 2 or 3 month's duration had a significant negative impact on oviposition. Survival was significantly reduced only among females exposed to no free water during the 6‐month study. No spawning occurred without the presence of free water in the burrows, and when free water was absent during the peak spawning months of September and/or October, oviposition was delayed until free water was returned. These results indicate that brood female P. clarkii can withstand short‐term conditions without free water in the burrow with little effect, but reproduction and survival are significantly reduced by prolonged drought.     

大豆胰蛋白酶抑制剂对鲢丝氨酸蛋白酶的作用

研究了大豆胰蛋白酶抑制剂(STI)对鲢肌肉中肌原纤维结合型丝氨酸蛋白酶(MBSP)引起的肌原纤维蛋白降解作用的影响。大豆胰蛋白酶抑制剂经高度纯化后加入肌原纤维中以判断它对MBSP的抑制效果。在不添加STI的情况下,55℃加热30min即可观察到肌球蛋白重链的分解,延长加热时间则可检测到肌动蛋白及原肌球蛋白的降解。相反,在终浓度为0.75μg·mL-1的STI存在下,包括肌球蛋白重链在内的肌原纤维蛋白降解均能得到有效抑制,表明STI是MBSP的特异性抑制剂。由于肌球蛋白重链及肌动蛋白的完整性是构成鱼糜制品凝胶强度的主要因素,因此,STI的添加有可能提高鲢鱼糜制品的凝胶强度。     

中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal)的重组表达及活性分析

Kazal型丝氨酸蛋白酶抑制剂可以通过精确调控丝氨酸蛋白酶的活力,在生物体的防御应答等众多生物过程中发挥重要作用。以前期克隆的中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal, GenBank注册号为DQ318856)为基础,对其功能结构域进行序列比对和进化分析;组织表达分析结果表明,该基因在血细胞、鳃和淋巴器官等组织中高水平表达,而在眼柄、神经和肌肉中无表达;利用原核表达系统对该基因成熟肽区域成功进行了重组表达,纯化后的目的蛋白最终得率为0.4 g/L培养液;活性分析结果显示,复性后的rFc-Kazal对鳗弧菌、金黄色葡萄球菌、杀鲑气单胞菌、苏云金芽孢杆菌有明显的抑菌作用。     

Growth and survival of Procambarus acutus acutus (Girard, 1852) and P. clarkii (Girard, 1852) in competitive settings

Third‐instar young‐of‐the‐year (YOY) and juvenile Procambarus acutus acutus and P. clarkii stocked in aquaria, rice forage microcosms and a culture pond were evaluated in intraspecific‐ and interspecific‐competitive settings. Procambarus acutus acutus YOY, which were larger than P. clarkii YOY at stocking, grew and survived at significantly greater rates over fall‐, winter‐ and spring‐temperature courses in aquaria. Juvenile P. a. acutus starting an aquarium experiment with a mean total length (TL) size advantage survived at a significantly greater rate than P. clarkii juveniles. Growth rate of similar‐sized stocked P. a. acutus juvenile was significantly faster than that of P. clarkii juveniles in the same microcosms. Survival of juvenile P. clarkii in the microcosms with P. a. acutus was significantly less than when stocked alone. Estimated survival of 2200 P. a. acutus and 2200 P. clarkii YOY in a culture pond over a 174‐day grow‐out period was 84% and 57% respectively. Growth rate of these YOY P. a. acutus and P. clarkii was 0.48 and 0.40 mm TL day^?1. The body‐size advantage at hatching and through the faster growth of P. a. acutus was important in defining competitive interaction with P. clarkii.