Historical demography of southern African patellid limpets: congruence of population expansions,but not phylogeography

Global climatic oscillations have shaped the contemporary genetic structure of marine taxa in different ways. Previous demographic studies have indicated that various intertidal marine species display genetic signatures of demographic expansion that either pre- or postdate the Last Glacial Maximum. Such expansions and the ability of species to colonise new habitats will influence their genetic structure, but the link between scales of larval dispersal and the strength of phylogeographic structure is not always clear. We analysed a fragment of the mitochondrial COI gene of 11 sympatric species of intertidal southern African patellid limpets to investigate how ancient oceanographic dynamics have shaped and maintained their contemporary spatial genetic variation. Our data show that the patellid limpets investigated display congruent evidence of spatial expansion during the Late Pleistocene or Early Holocene, which corresponds with the establishment of the contemporary southern African shoreline. We argue that closely related and co-distributed southern African intertidal invertebrates responded to ancient climatic oscillations as a cohesive group. In contrast, contemporary oceanographic circulation has shaped the phylogeographic patterns of these limpets in different ways. We show close relationships between phylogeography and biogeography for some species, but not for others, despite the similarities in their life histories and exposure to the same climatic changes.     

条石鲷mPRα基因的cDNA克隆和表达模式分析

采用同源性克隆和末端快速扩增(RACE)方法,首次获得全长为1222bp的条石鲷膜孕激素受体α(mPRα)的cDNA序列。其开放阅读框为1060bp,编码了含353个氨基酸的蛋白,N端前16个氨基酸为信号肽。氨基酸序列比较分析发现其存在7个跨膜区域。其预测的蛋白质三级结构中存在着多个蛋白结合位点。同源性比较和进化分析表明,条石鲷mPRα与鲈形目鱼类mPRα聚为一支,进化关系较近,相似度达到95%以上。实时荧光定量PCR方法检测mPRα mRNA表达情况:发现mPRα mRNA在性成熟雌性条石鲷各组织均有表达,并在脑、垂体和卵巢组织中的表达较丰富。在条石鲷繁殖周期的脑和垂体组织中,mPRα mRNA的表达水平在卵巢发育的Ⅳ期达到最大值;在繁殖周期的卵巢组织中,mPRα mRNA的表达水平从卵巢发育Ⅲ期到Ⅴ期持续升高并且在Ⅴ期达到最大值(P0.05)。条石鲷雌鱼血清中雌二醇激素的含量变化在整个繁殖周期中差异显著(P0.05),在性腺发育Ⅲ期含量迅速升高并在Ⅳ期达到峰值。本研究为条石鲷卵巢成熟调控机制研究提供了基础参考资料。     

仿刺参(Apostichopus japonicus)和海地瓜(Acaudina leucoprocta)体壁多肽的响应面法酶解和N末端测序

为充分开发利用海参资源,优化海参体壁酶解工艺,鉴定海参多肽,本文以水解度为指标,设计了3因素(时间、温度和加酶量)3水平的响应面实验,得到海参水解的最优条件是:仿刺参(Apostichopus japonicus)体壁在加酶量1.97%、温度55.7°C、水解136.8min的条件下,水解度为83.39%;海地瓜(Acaudina leucoprocta)体壁在加酶量1.70%、温度55.4°C、水解115.6min的条件下,水解度为63.68%。之后通过使用超滤膜、反相高效液相色谱(RP-HPLC)、基质辅助激光解析电离飞行时间质谱(MALDI-TOF)技术和蛋白测序仪等分析手段从水解产物中分离鉴定出4种多肽。其中,经N端序列鉴定出仿刺参多肽S1、S2氨基酸残基序列分别为Gly-Pro-Val-Gly-Ala-Ser-Gly-Pro-Gln-Gly-ProGln-Gly-Pro-Gln-Gly-Leu-Ser-Ala-Leu和Trp-Pro-Pro-Gly-Asn-Ser-Gly-Ile-Gln-Gly。海地瓜多肽A1和A2氨基酸残基序列分别为Gly-Ala-Asn-Gly-Asn和Trp-Leu-Pro-Gly-Asp-Thr-Gly-Pro-Gln-GlyVal-Thr-Gly-Pro-Val-Gly-Pro-Ala-Gly。     

细胞穿膜肽转导大型海藻及对其光合作用的影响

细胞穿膜肽能够介导外源物质进入细胞,是一种简单高效的分子运输手段。传统的大型海藻外源物质转导方法较为复杂,并且效率偏低。目前尚没有将细胞穿膜肽应用于大型海藻的报道。本实验利用细胞穿膜肽九聚精氨酸(R9)对浒苔、紫菜和海带这三种常见的大型海藻进行转入,观察其转导效果,并测定转导后藻体光合作用效率的变化。实验结果表明,细胞穿膜肽R9能够成功的转入三种大型海藻,转入的效率与藻体细胞壁结构有一定联系;转导后藻体的Fv/Fm、ETR(I)和ETR(II)没有明显变化,说明R9对大型海藻的光合作用没有负面影响。本实验建立了一种新型的大型海藻外源物质转导方法,为今后的大型海藻育种,分子转化以及外源物质转入机制等研究奠定了基础。     

连续降温对大菱鲆(Scophthalmus maximus)成鱼血清生化指标及Wap65-1基因表达的影响

将大菱鲆(Scophthalmus maximus)从正常养殖水温18°C快速连续降温至1°C,并在18、13、8、5、3和1°C共计6个温度点取血采样,分别测定血清中的谷丙转氨酶(ALT)、肌酐(CR)、乳酸脱氢酶(LDH)、血糖(GLU)、皮质醇以及不同组织中Wap65-1基因的表达量。结果发现,随着温度降低,大菱鲆的血清ALT活性呈升高的趋势,且在8—1°C区间内显著高于18—13°C区间(P0.05);血清CR浓度呈升高的趋势,在3°C时最高,且显著高于除1°C之外的其它实验(P0.05);血清中LDH活性呈先升高后降低的趋势,在13—5°C区间内显著高于其它实验组(P0.05);血清GLU浓度呈先升高后降低的趋势,在8—3°C区间内显著高于其它实验组(P0.05);血清皮质醇浓度呈升高的趋势,在1°C时最高,且显著高于其它实验组(P0.05)。连续降温过程中,大菱鲆所有组织Wap65-1基因在特定温度下表达量都有上调(脑、胃、肝脏、头肾、肾脏、脾脏、肌肉、肠道、心脏)。其中,脑、胃、头肾、肾脏和心脏组织中的Wap65-1基因在正常温度下基本不表达,只在降温过程中表达;肝脏、脾脏、肠道和肌肉这4种组织中的Wap65-1基因在正常温度下有所表达,且在降温过程中表达量显著升高(P0.05)。     

泥蚶血红蛋白异源四聚体酶解多肽抗菌活性及其机理研究

采用凝胶层析技术从泥蚶血细胞裂解物中纯化得到两种血红蛋白Tg-HbⅡ(异源四聚体)和TgHbⅠ(同源二聚体),回收率分别为48.15%和24.91%。胰蛋白酶酶解Tg-HbⅡ,酶解液经反相C-18色谱柱分离纯化得7种血红蛋白源多肽F1~F7。通过琼脂扩散法检测酶解多肽抑菌活性,结果发现7种多肽对金黄色葡萄球菌、大肠杆菌、恶臭假单胞菌、枯草芽孢杆菌和坚强芽孢杆菌均无抑菌作用,但多肽F2~F7对副溶血弧菌、溶藻弧菌和哈维氏弧菌具有明显抗菌活性,且多肽F2~F7对副溶血弧菌、溶藻弧菌和哈维氏弧菌的MIC值在0.012~0.200mg/mL范围内。荧光显微镜观察酶解多肽杀菌效果发现多肽杀菌迅速,5min内弧菌细胞几乎全部死亡。利用透射电子显微镜观察正常的和经抗菌肽处理后的溶藻弧菌和哈维氏弧菌细胞的超微结构,结果显示,对照组正常细胞内部结构完整,而经抗菌肽处理过的细胞其细胞壁完整,但因缺乏细胞内容物支持导致局部区域内陷,细胞质膜明显收缩,细胞内物质外流形成许多空泡从而导致细胞死亡。研究结果表明血红蛋白源抗菌肽对泥蚶致病菌弧菌有明显的抗菌活性,其通过破坏细胞内部结构而起到杀菌作用,这些发现为血红蛋白抗菌机制的研究奠定了基础。     

Initiation of two ovarian cell lines from Fugu rubripes (Temminck et. Schlegel)

The ovary is an excellent system for studying stem cell renewal and differentiation, which is under the control of ovarian somatic cells. In order to understand oogenesis in Fugu rubripes (Temminck et. Schlegel) as a marine fish model of aquaculture importance, we established cell lines called TSOC1 and TSOC2 from a juvenile ovary of this organism. TSOC1 is composed of spindle epithelial-like cells, while the other is cobblestone-like cells. Therefore, TSOC1 and TSOC2 appear to consist of ovarian somatic cells. Growth requirement condition was investigated including temperature, concentration of FBS and pH. Significant fluorescent signals were observed after TSOC1 and TSOC2 cells were transfected with pEGFP-N3 vector, indicating its potential utility for genetic manipulation such as gene function studies. It is shown that these cell lines are effective for infection by the turbot reddish body iridovirus and flounder lymphosystis disease virus as evidenced by the appearance of cytopathic effect and virus propagation in the virus-infected cells, and most convincingly, the observation of viral particles by electron microscopy, demonstrating that TSOC1 and TSOC2 are suitable to study interactions between virus and host cells. It is believed that TSOC1 and TSOC2 will be useful tools to study sex-related events and interactions between primordial germ cells and oogonia cells during oogenesis. Therefore, establishment of ovary cell lines from Fugu rubripes seems to be significant for those research areas.     

Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole (Cynoglossus semilaevis)

Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization during Cynoglossus semilaevis early developmental process, eight candidate reference genes (ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S) were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the mature C. semilaevis. EF1A was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages (from oosphere to 90 days old) in this study. However, when divided the C_t value data of the above mentioned early developmental stages into two separate periods (embryo and post-hatching periods), TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.     

花鲈Claudins基因家族的鉴定、进化分析及对环境盐度的表达响应

Claudin (cldn)是细胞间紧密连接蛋白的重要功能和结构组件,通过控制细胞旁通路通透性参与渗透压平衡的调节。本研究对花鲈(Lateolabrax maculatus)cldns基因家族进行了系统的鉴定分析,结果表明:花鲈cldns基因家族共包含55个成员,分布于16条染色体上,其中34个cldns基因在哺乳动物体内有对应直系同源基因,其余21个cldns是硬骨鱼所特有。选择压力分析显示预测的花鲈cldns蛋白的跨膜区域存在14个受正选择作用的氨基酸位点,这些正选择位点可能与家族内基因的多样性和功能分化有关。此外,基于花鲈鳃转录组的基因表达结果表明,至少有24个cldns基因在鳃中表达,其中cldn8c、cldn10b、cldn10c、cldn10d、cldn27a和cldn30b在不同环境盐度处理后表达差异显著,表明其可能是花鲈鳃组织中调节渗透压平衡的候选功能基因。本研究成果为研究花鲈及其他广盐性鱼类cldns基因家族成员的渗透调节机制奠定了理论基础。     

小清河河口邻近海域口虾蛄(Oratosquilla oratoria)COI序列特征及遗传多样性分析

受河流沿岸工农业发展以及人口急剧增加影响,小清河河口及邻近海域受到严重污染。口虾蛄(Oratosquilla oratoria)作为小清河河口及邻近海域重要渔业资源之一,承受着巨大的捕捞和生存压力。为了掌握口虾蛄种群在该区域的种群遗传多样性,本研究于2020年6—10月在小清河河口及邻近海域采集口虾蛄生物样本,进行了基于线粒体COI基因的种群遗传多样性分析。研究结果表明,基于216条长度为655bp的COI基因片段,共定义90个单倍型,63个多态位点,其中包括59个转换位点,1个颠换位点和3个转换与颠换同时存在的位点,核苷酸多样性指数和单倍型多样性分别为0.9700和0.0051。与其他海域相比,小清河河口种群遗传多样性处于相对较高水平;同时遗传结构分析发现黄渤海与东海及南海分布的口虾蛄种群遗传变异较大,但黄渤海海域内遗传变异较小。通过本研究基本掌握了小清河河口及邻近海域口虾蛄种群遗传多样性背景,也可为口虾蛄资源管理以及种质资源库建立等提供科学的基础理论依据。