刺参血淋巴细胞形态和表面结构观察

通过光镜和扫描电镜观察,较为系统地研究刺参（Apostichopus japonicus）血淋巴细胞的形态和表面结构。根据大小和形态结构,将血淋巴细胞定义为4种类型：小淋巴细胞、吞噬细胞、桑葚细胞和结晶细胞。     

刺参(Apostichopus japonicus)幼参对高温和低盐协同胁迫的生理响应:生长和诱导型热休克蛋白基因表达

全球气候变化背景下的极端高温和强降雨频发天气导致的养殖池塘持续性的高温和低盐环境给刺参池塘养殖带来了严峻的挑战。为了研究刺参对高温和低盐环境的生理响应,本实验分析了高温和低盐协同胁迫对刺参幼参生长及诱导型hsp70和hsp90基因表达的影响。实验设置4个温度梯度（16℃、20℃、24℃和28℃）和3个盐度梯度（22、27和32）,共12个不同的胁迫组。经过40 d的长期胁迫,研究发现,随着温度的升高和盐度的降低,幼参的体重增加量减少,并在高温、低盐组出现体重负增长。长期胁迫提高了刺参幼参的诱导型hsp70和hsp90基因表达量,从而在一定程度上增强了对极端天气的抵抗能力。同时,高温下盐度22处理组刺参幼参诱导型hsp70和hsp90基因的表达量较盐度27条件下低。双因子方差分析结果显示,高温和低盐对幼参特定生长率和诱导型hsp70和hsp90基因表达量的交互作用均不显著,并且盐度对诱导型hsp70和hsp90基因表达量没有显著的影响。因此,相较低盐,高温对刺参幼参的影响更大,可作为刺参育种的选择压力。在高温期应该采取有效措施降低海水温度,并防止极端天气造成的养殖水体温度分层。该研究丰富了刺参生理生态学理论,可为刺参良种选育工作提供思路,并为指导极端天气下刺参生产实践活动提供一定的理论依据。     

Age and Growth of Chub Mackerel （Scomberjaponicus） in the East China and Yellow Seas Using Sectioned Otolith Samples

Although chub mackerel (Scomberjaponicus) is a primary pelagic fish species,we have only limited knowledge on itskey life history processes.The present work studied the age and growth of chub mackerel in the East China and Yellow Seas.Agewas determined by interpreting and counting growth rings on the sagitta otoliths of 252 adult fish caught by the Chinese commercialpurse seine fleet during the period from November 2006 to January 2007 and 150 juveniles from bottom trawl surveys on the spawn-ing ground in May 2006.The difference between the assumed birth date of 1 st April and date of capture was used to adjust the agedetermined from counting the number of complete translucent rings.The parameters of three commonly used growth models,the vonBertalanffy,Logistic and Gompertz models,were estimated using the maximum likelihood method.Based on the Akaike InformationCriterion (AIC),the von Bertalanffy growth model was found to be the most appropriate model.The size-at-age and size-at-maturityvalues were also found to decrease greatly compared with the results achieved in the 1950s,which was caused by heavy exploitationover the last few decades.     

利用引物退火控制技术克隆日本囊对虾性腺差异表达基因

为了解日本囊对虾（Marsupenaeusjaponicus）性腺发育及分化的分子机制,本研究采用改良的引物退火控制技术克隆到了共8个精巢、卵巢的差异表达基因．其中4个为已知基因,分别为凝血酶敏感蛋白基因、皮质棒蛋白基因、增殖细胞核抗原基因和泛素缀合酶基因;4个为未知基因,分别命名为MjACP3-T、MjACP4一T、MjACPl5一T和MjACP4一O基因．采用实时定量PCR方法分析了4个未知基因在性腺发育过程的表达谱,结果表明：MjACP3一T、MjACP4．T和MjACPl5,T基因在日本囊对虾精巢和卵巢中的表达量有显著差异,且在各个时期精巢的表达量均高于卵巢,说明这3个基因可能在精巢的发育中起到某些重要作用．卵巢MjACP4一O基因的表达量变化较大,第5期卵巢的相对表达量达到最高（为1．38）,而其他各个阶段的变化较大,在第4期卵巢的相对表达量最低（为0．03）．在精巢中,MjACP4一O基因的表达量随着精巢逐渐发育成熟呈下降趋势．定量PCR结果与引物退火控制技术的结果有着很好的一致性．研究结果表明,引物退火控制技术是一种简单高效的克隆差异表达基因的技术．采用该技术所克隆到的8个精巢、卵巢的差异表达基因,为进一步了解日本囊对虾性腺发育分化的分子机制提供了基础材料．     

Identification and characterization of a new IgE-binding protein in mackerel (Scomber japonicus) by MALDI-TOF-MS

As fish is one source of the ‘big eight’ food allergens,the prevalence of fish allergy has increased over the past few years.In order to better understand fish allergy,it is necessary to identify fish allergens.Based on the sera from fish-allergenic patients,a 28 kDa protein from local mackerel (Scomber japonicus),which has not been reported as a fish allergen,was found to be reactive with most of the patients’ sera.The 28 kDa protein was analyzed by MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry).Mascot search in NCBI database (Date: 08/07/2010) showed that the top protein matched,i.e.triosephosphate isomerase (TPI) from Xiphophorus maculatus and Poecilia reticulata,had a mowse (molecular weight search) score of 98.In addition,TPI from Epinephelus coioides also matched this mackerel protein with a mowse score of 96.Because TPI is con-sidered as an allergen in other non-fish organisms,such as lychee,wheat,latex,archaeopotamobius (Archaeopotamobius sibiriensis) and crangon (Crangon crangon),we consider that it may also be an allergen in mackerel.     

芽孢杆菌不同使用方式对刺参生长、消化和免疫防御因子的影响

研究了1株芽孢杆菌(Bacillus cereus,BC-02)不同使用方式(活菌、灭活菌和芽孢)对刺参(Apostichopus japonicus)生长、消化和非特异性免疫相关酶活性的影响。实验分低、中、高3个添加梯度,其芽孢杆菌的添加量分别为107、109和1011cfu·kg-1(以活菌量计),以投喂基础饲料组作为对照,实验时间为58d。结果表明:添加中浓度和高浓度的活菌和芽孢粉,不仅能促进刺参生长,还能显著的提高刺参肠道蛋白酶、脂肪酶和淀粉酶的活性,刺参体腔液中的AKP、ACP、T-SOD以及LSZ活性也显著提高(P0.05),而添加灭活后的芽孢杆菌,对刺参的促生长作用不大,在刺参消化酶和体腔液非特异性免疫相关酶活性方面的作用也不及活菌和芽孢粉状态显著,因此综合比较表明,芽孢杆菌BC-02对刺参的适宜使用方式为活菌和芽孢,适宜浓度为(109-1011)cfu·kg-1,可以有效提高刺参的生长、消化酶活性和非特异免疫力。     

Characterization of SNAP-25 gene from marine teleostean, Lateolabrax japonicus

The t-SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) plays an essential role in regulating fusion between the vesicle and plasma membranes during exocytosis. To clone and characterize SNAP-25 gene, the first step in the functional study of SNARE proteins in marine teleostean, was to obtain the cDNA of sea perch SNAP-25 (SPsn25) by RT-PCR and RACE-PCR amplification of a Japanese sea perch. The full-length cDNA of 831bp contains a CDS of 615 bp, coding 204 amino acid residues, and a 5′UTR of 219bp. Bioinformatic analysis revealed that SPsn25 corresponds with SNAP-25a isoform and shares 91.1% identity with SNAP-25a of a goldfish and a zebrafish. The SPsn25 expression in both mRNA and protein levels in the Japanese sea perch had been identified through semi-quantitative RT-PCR and Western Blot assay. Together, these data again confirmed the nerve tissue specificity of the fish SNAP-25 gene expression.     

微卫星标记在刺参(Apostichopus japonicus)人工繁育中亲本识别的应用

选取8个高多态性的微卫星位点, 对240只刺参(Apostichopus japonicus)幼体和14只疑似亲本进行了亲缘关系的鉴定。通过检测, 8个微卫星位点共得到53个等位基因, 平均等位基因数为6.63; 观察杂合度在0.730—0.902; 各位点的多态信息含量为0.696—0.836。通过亲缘关系分析软件CERVUS 3.0分析, 微卫星位点累积排除概率分别为0.9934、0.9997、0.99999。基于排除法个体基因型判定, 有37个子代有1个候选亲本, 194个子代有2个及以上候选亲本; 基于似然法判定, 置信度为80%时, 有198个子代个体找到最似亲本。综合排除法和似然法的亲权鉴定, 成功为229个子代个体找到它们的亲本, 鉴定成功率达95%以上。本研究结果可为刺参育种工作中亲本识别及家系分析提供技术支持和理论依据。     

海洋无脊椎动物组织总RNA提取方法的探讨

RNA提取是分子生物学研究的基础实验,由于海洋无脊椎动物组织结构以及体成分的特殊性,导致现有的RNA提取方法不能完全胜任此工作。作者以刺参(Apostichopus japonicus)、栉孔扇贝(Chlamys farreri)和凡纳滨对虾(Litopenaeus vannamei)为研究对象,对异硫氰酸胍RNA提取方法进行了改进。栉孔扇贝和凡纳滨对虾的肝胰腺富含RNase,刺参体壁结构致密、富含胶原蛋白,而成熟期精巢富含多糖,因此在这些动物组织中提取RNA时,在原有方法基础上增加了瞬时匀浆、增加氯仿及酸性酚的抽提次数,或添加高盐溶液等方法。实验结果显示,改进方法后获得的RNA具有完整的28S、18S和5S r RNA条带,A260nm/A280nm和A260nm/A230nm比值分别为1.89~2.0和1.98~2.11,β-actin基因扩增条带清晰,所获得的组织RNA的纯度和质量符合c DNA合成和基因功能研究的要求。     

Alice台风对东海鲐鱼鱼卵仔鱼的输运和存活率的影响

日本鲐(Scomber japonicus)的补充量受物理环境影响很大。本文基于东海鲐鱼个体的初期生态模型,遴选对东海鲐鱼输运路径影响最大的Alice台风,研究台风导致的物理环境的改变对鲐鱼鱼卵仔鱼的输运分布和补充量的影响。研究发现,Alice台风对鲐鱼仔鱼的输运分布影响不大,原因是Alice台风逆时针旋转的气旋与潮流顺时针旋转的反气旋相互抵消,减少了台风对仔鱼输运区域低频率潮流的影响,使东海鲐鱼的输运路径处在受台风影响最小的区域。台风移动速度快、作用时间短也是造成这个结果的原因之一。台风使鱼卵仔鱼普遍向深水移动,台风过后2.5和5天存活数量分别为4.08×109和3.67×109个,而没有台风影响的分别存活6.64×109和5.93×109个,Alice台风最终导致仔鱼的存活率降低。本文为深入探讨环境变化与鲐鱼资源量的关系提供参考。