长江江豚基因组大小测定

本研究采用流式细胞术,以公鸡(Gallusdomesticus)红血细胞DNA含量为标准,测定了23头长江江豚(Neophocaenaphocaenoidesasiaeorientali)的基因组大小(或称C值)。实验过程中采用了保存在3中不同条件下的长江江豚的全血样品,用3种不同的方法提取白细胞。为了获得本实验所用的公鸡红血细胞DNA含量的准确值,首先以人(Homosapiens)的C值为标准,对其进行了校正。然后其C值(2C=2.35pg)用于长江江豚的基因组大小测定。结果发现:长江江豚的单倍体DNA含量为3.27pg/C,由此得出其基因组大小为3.17×109bp;雌性和雄性的C值分别为3.25pg和3.29pg,野外长江江豚和豢养长江江豚的C值分别为3.30pg和3.05pg。对雌雄个体之间以及不同生长条件下长江江豚的C值应用独立样本t检验分析,发现:1)不同性别的长江江豚基因组大小之间没有明显的差异;2)豢养条件下的长江江豚和野生长江江豚之间的基因组大小有明显差异,豢养条件下的长江江豚的基因组明显小于野生条件下的长江江豚。据此推测必要环境因子的变化可能会对长江江豚的基因组DNA含量造成影响。     

长江江豚对人工投食适应过程的观察

作者于1989,1992,2001 和2003 年,从长江铜陵江段共捕获24 头长江江豚,在长江干流水域中,用不同的围养方式做摄食行为的观察,累计88 d。结果表明,野外捕获的长江江豚对人工投喂有3 种适应行为:(1)拒食行为;(2)警戒行为;(3)摄食行为。长江江豚的平均拒食天数为2.4 d,其中成年江豚拒食达3 d,而未成年江豚仅拒食1.5 d,两者差异极为显著(P < 0.01)。在冬季和春季圈养试验中,长江江豚的日食量随时间呈上升趋势,冬春季差异显著(P <0.01)。第2 周后江豚警戒行为所用时间不足3 min,但持续时间较长。根据冬季观察,7 周后长江江豚在40 min 内所摄取的食量不足体重的7% ,推测其摄食行为仍处在适应调整阶段。     

野生与豢养长江江豚血清氨基酸含量比较

应用高效液相色谱(HPLC)技术,首次测定了湖北石首长江天鹅州白豚自然保护区野生长江江豚(Neophocaena phocaenoides asiaeorientalis)和中国科学院水生生物研究所白豚馆人工饲养的长江江豚血清中17种氨基酸的含量.结果表明,除了脯氨酸Pro、蛋氨酸Met和组氨酸His外,人工饲养江豚血清中其余14种氨基酸(天门冬氨酸Asp、谷氨酸Glu、丝氨酸Ser、精氨酸Arg、甘氨酸Gly、苏氨酸Thr、丙氨酸Ala、异亮氨酸Ile、亮氨酸Leu、苯丙氨酸Phe、缬氨酸Val、赖氨酸Lys、酪氨酸Tyr、胱氨酸Cys)的含量显著高于野生长江江豚血清中相应氨基酸的含量.野生江豚和人工饲养江豚的血清氨基酸含量均没有显著的性别差异.野生江豚性成熟个体与未成熟个体之间血清氨基酸含量也没有显著性的差异.在所检测的17种氨基酸中,豢养江豚Glu含量最高,其次为Asp和Lys.野生江豚同样是Glu最高,其次是Lys和Asp.豢养和野生江豚都是Met含量最低.野生和豢养江豚必需氨基酸(EAA)和非必需氨基酸(NEAA)之间的比率分别是0.83和0.92,具有极显著的差异(p <0.01).     

江西水域枯水期长江江豚种群数量和分布特征

研究于2021年11—12月,设置12条考察路线,采用同步目视考察法,对包含长江干流江西段、鄱阳湖和赣江下游的江西水域长江江豚数量和分布开展了考察,结合历史资料,探讨了该水域长江江豚种群数量和分布规律及江湖迁徙关系。结果显示, 11月考察观测到长江江豚217群次、454头次, 12月考察观测到长江江豚236群次、569头次,受水位和天气等环境条件影响,两次考查观察到的长江江豚数量差异显著(t=–2.23, P<0.05)。11月和12月江豚分布规律基本一致:长江江西段,江豚主要分布在湖口石钟山-彭泽三号洲水域;鄱阳湖适宜水深范围水域都有长江江豚分布,其中吴城望湖亭-渚溪河口、都昌船厂-黄金咀-三山-瓢山水域是江豚高密度分布区;赣江下游,长江江豚分布在扬子洲渔业村附近水域。两次考察目击率分别为1.00和1.08次/km,均高于往年调查结果,由此推测,鄱阳湖长江江豚种群数量可能有所增长。鄱阳湖通江水道蛤蟆石-石钟山水域,两次考察分别仅发现3头和2头长江江豚,推测调查期间长江江豚在长江干流和鄱阳湖之间的迁移行为较少,未来需进一步管控江湖迁移水域的人类活动,促进江湖迁移。吴城望湖亭-渚溪...     

基于PAE编码系统构建的半自然条件下长江江豚行为谱

识别和编制动物行为谱是深入研究动物行为及其与环境复杂关系的基础和前提。本研究于2013年8月至2014年4月以栖息在铜陵淡水豚国家级自然保护区半自然水域的8头长江江豚为研究对象,采用焦点动物取样法和随机动物取样法观察记录了动物行为发生的过程、内容和环境,并以"姿势—动作—环境"(posture-act-environment,PAE)为轴心,以行为的功能为依据,构建了长江江豚的PAE行为谱。共分辨记录了半自然水域长江江豚的8种姿势、33种动作和46种行为,并定性描述了不同行为出现频率与年龄组、性别的关系。该行为谱综合了姿势和环境信息,有助于进一步研究长江江豚的生态与行为、促进长江江豚的保护。     

长江江豚脐静脉细胞体外培养的初步研究

长江江豚(Neophocaena phocaenoides asiaeorientalis)属齿鲸亚目鼠海豚科, 是世界上唯一的江豚(Neophocaena phocaenoides)淡水亚种, 主要分布于我国长江中下游干流和洞庭湖、鄱阳湖及其部分支流1—3。       

长江江豚自然保护区建设管理存在的问题及调整建议

长江江豚(Neophocaena asiaeorientalis)是生活在中国长江的珍稀濒危鲸类动物,自然栖息地破坏是造成其种群衰退的重要原因。目前中国已经在长江中下游流域建立了8个长江江豚自然保护区。然而,早期保护区的功能区划随经济发展和生态保护的现实矛盾日渐突出,按照现行法规对保护区难以实行有效管理,迫切需要进行优化调整。文章基于国家政策、长江江豚生物学特点及分布规律,兼顾航运等重大经济发展需求,提出了长江豚类自然保护区调整的原则和建议:(1)将长江江豚喜好的近岸水域及一定范围的重要江滩和洲滩湿地,及江心洲汊江一侧水域划入核心保护区;(2)将中间主航道水域以及长江大堤至核心区边界之间的水域划入一般控制区;(3)兼顾历史遗留问题,细化不同区域的管控措施。该调整方案一方面使保护区功能区划更加符合长江江豚的分布特征和偏好生境,有利于对其自然种群和重要栖息地的有效保护。另一方面也有助于理顺自然保护区管理上存在的法律障碍,为长江江豚自然保护区建设、规划和管理提供新的思路和模式。     

一只长江江豚妊娠期昼间行为及其时间分配

长江江豚(Neophocaena phocaenoides asiaeorientalis Pilleri and Gihr,1972)属于哺乳纲(Mammalia)、鲸目(Cetacea),是仅分布于长江中下游及其附属湖泊中的惟一且相对独立的一个江豚淡水亚种(高安利和周开亚,1995),也是中国水域中三个江豚种群中最为濒危的一个亚种,<中国濒危动物红皮书·兽类>也将其列为濒危级(汪松,1998).     

长江江豚一例腹壁肌间巨型囊肿的组织学分析

正随着白暨豚(Lipotes vexillifer)的"功能性灭绝",江豚(Neophocaena asiaeorientalis)已成为长江目前唯一能见的鲸类物种~[1]。江豚曾被划分为3个亚种,即指名亚种(N.phoconoides phoconoides)、长江亚种(N.phoconoides asiaeo-     

人sTNFR1基因的克隆以及在NIH3T3细胞中的稳定表达

以He1a细胞的总RNA为模板,用RT—PCR方法扩增sTNFR1全编码区基因片段,构建含有目的片段的T载体克隆及真核表达载体pcDNA3．1（-）重组质粒亚克隆,将重组质粒和脂质体共同转染NIH3T3细胞系,G418筛选稳定转染细胞株．经核苷酸序列测序和酶切鉴定,成功构建了pcDNA3．1（-）-sTNFR1真核表达质粒,脂质体法建立了高效表达sTNFRI的稳定转染细胞系,并经RT—PCR和Western Blotting鉴定．人sTNFR1基因能在NIH3T3细胞系中稳定表达,为今后的研究打下了基础．     

Establishment and characterization of fibroblast cell lines from the skin of the Yangtze finless porpoise

The Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), as the sole freshwater subspecies of N. phocaenoides, is endemic to the Yangtze River and its adjacent lakes. Its population has declined significantly over recent decades. In this study, we established a skin-derived finite fibroblast cell line of the Yangtze finless porpoise, named YFP-SF1, using primary cell culture methods, and an immortalized cell line, T-YFP-SF1, through co-transfection (GFP and SV40 T antigens) techniques. YFP-SF1 proliferated continuously with a minimum population doubling time of 31 h and exhibited age-dependent changes in growth rate. T-YFP-SF1 cells exhibited fibroblast morphology and were characterized by a shorter doubling time, higher attachment efficiencies, colony formation at a low seeding density, and growth in low serum concentrations. Anchorage independence and foci formation in the cell monolayer were observed from passage 36. The chromosome number of YFP-SF1 and T-YFP-SF1 remained stable at 2n = 44 in the early passages, and the viability of thawed cells remained above 90% after cryopreservation in liquid nitrogen. Taken together, we have established fibroblast cell lines of Yangtze finless porpoise for the first time, which might assist as an in vitro model for this endangered mammal.     

鲸类微卫星引物对长江江豚的适用性研究

微卫星在长江江豚 (Neophocaenaphocaenoidesasiaeorientalis)中的应用研究还未见报道。本研究采用已发表的来自 6个鲸种的 2 3对微卫星引物对一个长江江豚群体DNA样本进行了微卫星扩增。结果表明其中有 7对引物在此群体中的扩增产物是稳定且多态的 ,序列分析结果表明这 7对引物的扩增产物都具有AC或GT两碱基重复单元 ,从而证明了扩增的有效性。研究结果表明用从其他鲸类分离出的微卫星引物可以快速筛选到适用于长江江豚指纹分析的引物     

Isolation and partial characterization of virus-transformed cell lines representing the A, G and variant complementation groups of xeroderma pigmentosum

We have established viral-transformed, apparently permanent (immortalized) cell lines from diploid fibroblasts representative of normal and xeroderma pigmentosum (XP) A, G and variant individuals. The XP-G and XP-variant cells represent complementation groups not previously available as permanent lines. All the new permanent cell lines exhibit SV40 T-antigen expression. They are also aneuploid and have growth characteristics typical of viral transformants. They have retained the phenotypes of UV sensitivity, reduced repair synthesis or defective 'postreplication repair' appropriate to the XP complementation group they represent. Additionally, the new cell lines are all transfectable with the selectable plasmid pRSVneo. The XP-G and XP-variant cell lines show enhanced transfection with UV-irradiated plasmid DNA; a phenomenon previously reported for normal immortalized cells and for immortalized cells from the A and F complementation groups of XP.     

Herpes-like virus infection in Yangtze finless porpoise (Neophocaena phocaenoides): pathology, ultrastructure and molecular analysis

A moribund juvenile Yangtze finless porpoise (Neophocaena phocaenoides) with skin lesions and ulceration was found in Dongting Lake, China. Pathologic examination, electron microscopy, and polymerase chain reaction of liver tissue revealed widely distributed necrotic lesions, sinusoidal dilatation, congestion and herpes-like virus particles.     

Immortalization of Werner syndrome and progeria fibroblasts.

Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.     

东方田鼠胚胎成纤维细胞永生化细胞系的建立

目的建立东方田鼠胚胎成纤维永生化细胞系,为全面研究东方田鼠抗日本血吸虫机制以及开展不同动物成纤维细胞间比较研究奠定基础和提供细胞实验材料。方法运用脂质体介导的基因转染法将pSV3neo质粒导入第3代东方田鼠胚胎成纤维细胞,经G418筛选抗性克隆并扩大培养,建立永生化细胞系;用PCR检测细胞株中SV40T基因的整合,RT-PCR鉴定SV40T基因在转染细胞中的表达;绘制东方田鼠胚胎成纤维永生化细胞生长曲线。结果阳性细胞克隆已扩大培养并稳定传代50代,经鉴定SV40T抗原已整合到东方田鼠胚胎成纤维细胞中且稳定表达。结论成功建立东方田鼠胚胎成纤维永生化细胞系。     

用微卫星指纹识别天鹅洲保护区长江江豚个体

DNA指纹个体识别技术是保护遗传学研究中的一种非常重要的手段。为了准确地识别天鹅洲保护区中的每一头长江江豚以开展保护遗传学及其它相关研究 ,并实施有效的种群管理 ,本研究应用 4个微卫星座位初步构建了该群体的DNA指纹图谱 ,并利用此图谱成功地对不同时期在保护区捕获的江豚进行了个体识别研究。结果显示微卫星指纹技术是一种适用于长江江豚个体识别研究的可靠手段     

长江江豚微卫星DNA分离的初步研究

为了开发物种特异性微卫星标记,本文采用一种改良的快速微卫星分离法(FIASCO)从长江江豚(Neophocaena phocaenoides asiaeorientalis)的基因组中筛选得到72条微卫星DNA序列。根据重复单元的排列特点,完美型、非完美型及复合型序列所占的比例分别为58.3%、22.2%和19.5%。选择其中30条序列设计PCR扩增引物,并用12个随机选择的长江江豚样品进行多态性筛选。初步结果表明其中14对引物的扩增产物稳定并且具有多态性;在每个座位上获得2-13个等位基因,平均等位基因数为5.87个;14个微卫星座位的平均观察杂合度(Ho)和期望杂合度(He)分别为0.560和0.709。本研究获得了长江江豚的第一批物种特异性微卫星座位及其扩增引物,这些微卫星标记将在后续的保护遗传学研究中发挥重要作用。       

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.