大熊猫和小熊猫粪便DNA提取的简易方法

采集了大熊猫和小熊猫的新鲜粪便样品 ,使用 1 0 0 %乙醇保存。通过重复离心富集研究动物的肠道脱落细胞 ,并使用乙醇和双蒸水洗涤以除去抑制物。用 1 %的SDS快速裂解细胞 ,离心除去残渣后 ,向裂解液中加入蛋白酶进行消化。消化结束后使用等体积的酚 /氯仿抽提 ,乙醇沉淀DNA。用双蒸水溶解粪便DNA后 ,使用PCR产物纯化试剂盒对粪便DNA进行纯化。电泳检测结果显示 ,从乙醇保存的大、小熊猫粪便样品中抽提到高质量的粪便DNA。对线粒体控制区、细胞色素b基因、 1 2SrRNA基因的PCR扩增反应以及测序结果也证实了样品保存方法和DNA抽提方法可靠而高效。此方法使用实验室内常用的分子生物学试剂 ,不仅克服了分子粪便学研究中常见的抑制物粪便DNA微量降解严重等障碍 ,与商业化的粪便抽提试剂盒 (QIAampDNAStoolMiniKit,Qiagen)相比还是一种经济的试验方法 (抽提反应成本为试剂盒的 1 / 5 )。文中还对粪便DNA内细菌基因组等背景DNA可能对分子粪便学试验结果的影响进行了探讨。在基于PCR技术的遗传学研究中 ,对于植食性动物而言 ,粪便内的背景DNA对目标动物DNA片断的扩增和序列测定未见影响 ;但对于肉食性动物 ,则必须考虑被捕食者基因组对试验可能产生的影响 ,应谨慎对待     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm²) and conductance (23-44 µS/cm²) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

圈养繁殖在大熊猫保护生物学中的作用

栖息地的日益恶化、片段化和种群孤岛化已成为保护大熊猫的严重障碍。尽管就地保护濒危物种是最有希望的措施,但对于大熊猫而言,研究表明圈养繁殖也是保护这一极危物和中的有效手段之一,对保护遗传多样性起以了重要作用,是延缓绝灭速率的保证。本文阐述了大圈养繁殖的必要性及其在保护生物学中的作用。     

小熊猫种内遗传及亚种分化研究(食肉目:浣熊科)

到目前为止,多数学者认为小熊猫种内已分指名亚种A.f.fulgens和川西亚种A.f.styani。然而,由于其个体毛色变异较大,一些学者对其亚种分化问题提出质疑。本文采用DNA指纹方法,对小熊猫的种内遗传和亚种分化进行了研究。结果表明,川西亚种所有个体在分子量约为8.4kb处均有一条指名亚种不具有的共有谱带,指名亚种所有个体则在分子量约为1.8kb处具有另外一条川西亚种不具有的共有谱带,且这两条谱带可通过双亲遗传给子代,说明此共有谱带可分别作为区分小熊猫川西亚种和指名亚种的特征带。另外,种内的遗传分化研究表明,川西亚种基因组的多态性强于指名亚种,且川西亚种内各个体之间的遗传变异高于指各亚种,说明小熊猫种内已形成遗传分化。因此,笔者认为以上研究结果为基因水平上进一步证明小熊猫种内已产生遗传分化,并形成两独立亚种,目前的亚种地位成立。     

中国大鲵机械感受器的超微结构

首次以透射电镜研究了大鲵成体（实验材料共两条）皮肤侧线器官中机械受器即表面神经丘和陷器官的超微结构,并在这两种感受器官之间进行了比较。它们都由三种细胞组成：周围的套细胞,底部的支持细胞以及中央的感觉细胞;且感觉细胞的游离面均有一根动纤毛和几十根静纤毛。但这两种器官在大小、各种细胞的数量、形状和排列上下不同,尤其是表面神经丘感觉细胞游离面纤毛具有双向极性,而陷器官体现为多向极性;表面神经丘的突触球集中分布于一个特殊的感觉细胞,而陷器官的每个感觉细胞基部都有一个突触球。     

Hormonal and behavioral relationships during estrus in the giant panda

Nonlactating female giant pandas experience a single estrus during the spring mating season. Although both hormonal and behavioral aspects of estrus have been reported on captive females, little is known about the relationship between them. Data were collected on a single captive female during three successive seasons, revealing a high degree of regularity in both estrogen profiles and the temporal pattern of associated behaviors. The birth of a live cub in the last of the three seasons, using artificial insemination, confirmed the time of ovulation as occurring shortly after a peak in urinary estrogen values. In this study, we examined the occurrence of female scent marking, rear presentations, chirp and bleat vocalizations, and the tail‐up display, and plotted their occurrence relative to the underlying estrogen values. We conclude that the role of estrogen in the expression of these behaviors is neither simple nor directly causal. Temporal associations indicate that a triggering rather than a maintenance function for estrogen is implicated. This analysis provides a clear identification of estrous behaviors, but in both the time and form of occurrence these behaviors most likely vary between females and seasons. Zoo Biol 20:537–543, 2001. © 2002 Wiley‐Liss, Inc.     

Zur Feinstruktur des dorsalen Riesenfasersystems im Bauchmark des Regenwurms

Zusammenfassung Die proximalen Kollateralen der dorsalen Riesenfasern des Regenwurms wurden in Serienschnitten vom Soma bis zum Eintritt in die Riesenfaser verfolgt und im Hinblick auf ihre Feinstruktur und ihre synaptischen Kontakte Untersucht. Es finden sich sowohl chemische als auch elektrische Synapsen. Ihre Feinstruktur wird mit der bekannter Synapsen anderer Wirbellosen und Wirbeltiere verglichen. In beiden Riesenfasersystemen kommen efferente chemische Synapsen mit feinen postsynaptischen Verzweigungen vor, die anscheinend von Bauchmark-Motoneuronen stammen. Das Axon der medianen Riesenfaser weist darüber hinaus nur noch eine elektrische Synapse mit den Rieseninterneuronen auf. Demgegenüber erhalten die Kollateralen der lateralen Riesenfasern zahlreiche Afferenzen, die zum Teil als sensorische Fasern der Epidermis, multisegmentale Fasern der Hauptfaserzüge und Rieseninterneurone identifiziert werden konnten. Weitere Afferenzen stammen vermutlich von unisegmentalen Interneuronen her. Beide lateralen Riesenzellaxone bilden außerdem miteinander eine elektrische Chiasma-Synapse mit besonderen Membraneinfaltungen.

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Ultrastructure of the dorsal giant fibre system in the ventral nerve cord of the earthwormII. Synaptic connections of the proximal collaterals of the giant fibres

Summary The proximal collaterals of the dorsal giant fibres of the earthworm were traced through serial sections from the cell bodies to the giant axons. Their structure and synaptic connections were examined. There are chemical as well as electrical synapses. Their fine structure is compared to that of other known invertebrate and vertebrate synapses. Both giant fibre systems have efferent chemical connections with thin postsynaptic arborizations which probably belong to ventral cord motoneurons. Moreover the median giant axon is connected by an electrical synapse with the giant interneurons. The lateral giant collaterals on the contrary receive many afferences through chemical synapses which were partly identified as sensory fibers from the epidermis, multisegmental axons from the main fibre bundles or giant interneurones. Other afferences probably come from unisegmental interneurones. In addition both lateral giant axons form an electrical chiasma synapse with special membrane folds.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft Gu 117/1.     

Isolation and characterization of 16 tetranucleotide microsatellite loci in the red panda (Ailurus fulgens)

A total of 15 polymorphic tetranucleotide microsatellite loci in the red panda, Ailurus fulgens, were characterized in this study. Based on evaluations of 33 red pandas, the number of observed alleles for each locus ranged from seven to 17 and the expected and observed heterozygosities were 0.412–0.897 and 0.121–0.909, respectively. The mean polymorphic information content was 0.721. These markers would greatly strengthen the utilization of microsatellite tools in genetic variation studies in red panda populations.     

Comparative Analysis and Molecular Characterization of a Gene BANF1 Encoded a DNA-Binding Protein During Mitosis from the Giant Panda and Black Bear

Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.