悦目金蛛和棒络新妇卵袋丝物理化学结构表征及其力学性能研究

蜘蛛丝是一种具有优良机械性能的天然动物蛋白纤维,它特有的结构和性能与其生物学功能密切相关。作者采用氨基酸自动分析仪、傅立叶转换红外光谱仪、扫描电镜和电子单纤强力仪对悦目金蛛（Argiope amoena）和棒络新妇（Nephila clavata）的卵袋丝进行了物理化学结构表征与力学性能的研究,结果表明两种蜘蛛卵袋均由微米级柱状腺丝、大壶状腺丝、亚微米级或纳米级葡萄状腺丝构成。卵袋丝的表面形貌特征、极性氨基酸含量、大侧链与小侧链氨基酸的比值、无定型区、β-折叠结构与结晶结构的含量等氨基酸组成种类与蛋白质二级结构特征,均满足各自生物学功能对断裂强度、延展性、初始模量等力学性能的要求。     

蜘蛛丝的分子结构与力学性能研究

蜘蛛丝尤其是蜘蛛大囊状腺产生的拖丝,具有独特的机械性能,是自然界颇具应用潜力的生物材料。现代分子生物学技术使蜘蛛丝蛋白基因得以克隆,通过高分子物理化学手段方法的利用,有利于揭示蜘蛛丝蛋白质序列、分子结构、以及分子结构和力学性能之间的关系。对不同种类蜘蛛丝蛋白的深入研究,将为基因工程方法人工合成并改造蜘蛛丝成为可能。     

三种类型蜘蛛丝的结构及生物学功能

利用付里叶变换红外光谱仪(FFIR)对棒络新妇(Nephila clavata)、悦目金蛛(Argiope amoena)的大壶状腺丝(拖丝)、悦目金蛛的捕丝(粘性螺旋丝)和卵袋丝这3种不同类型蜘蛛丝的二级结构进行了测试研究。结果表明：蜘蛛丝同时包含无规则卷曲、α-螺旋和β-折叠构象;对这3种蛛丝的红外光谱进行比较表明同一蜘蛛的不同类型蛛丝所含的这3种二级结构的比例不同,这种不同组成的二级结构就赋予了蜘蛛丝不同的特性,这种特性又与其不同的功能相适应。此外,还用扫描电镜(SEM)和光学显微镜对悦目金蛛和小悦目金蛛(A．minuta)的拖丝和捕丝做了形态结构观察。蜘蛛丝这种天然动物蛋白纤维所具有的特殊的形态结构、蛋白质二级结构与其特殊的性能和生物学功能是高度一致的。     

悦目金蛛拖牵丝力学性能的变异

蜘蛛丝作为一种具有优良机械性能的天然动物蛋白纤维,其特有的结构和机械性能与其生物学功能密切相关。由大壶状腺纺出的拖牵丝在蜘蛛的行走、建网、捕食、逃生、繁殖等多种生命活动中均发挥了重要的功能,其机械性能会受到多种内外因素相互作用的影响。本文对在不同体重、不同猎物饲养和不同营养状态3种条件下人工抽出的悦目金蛛(Argiope amoena)拖牵丝与其不同单丝间的力学性能进行了比较研究。结果表明,悦目金蛛拖牵丝的力学性能在组间、组内不同个体,以及同一个体不同丝纤维间变异都较大。随着蜘蛛个体的增大,蛛丝横截面直径逐渐增大,这会使得蛛丝的力学性能更好,便于作为救命索的拖牵丝在遇到危险时承受蜘蛛体重;蜘蛛在经过1个月的饥饿后,蛛丝在屈服点附近的力学性能并未发生显著变化,而断裂点应变和断裂能均显著减小,同时也表明无论对于作为救命索还是网丝,拖牵丝的弹性形变性能在与蛛丝相关的微观进化中要优先于塑性形变。这是蜘蛛在能量摄入受到限制时对拖牵丝的投入权衡的结果。     

棒络新妇和悦目金蛛丝腺形态初步观察

研究比较了结网型蜘蛛棒络新妇Nephila clavata和悦目金蛛Argiope amoena的丝腺形态特征,为国内蜘蛛丝腺蛋白的研究提供原始的丝腺解剖图,同时结合对2种蜘蛛卵袋的解剖、网的特征和室内捕食黄粉虫Tenebrio molitor幼虫行为的观察比较,探讨了2种蜘蛛丝腺的生物学功能与其生存繁殖策略之间的关系。本文分别观察描述了棒络新妇和悦目金蛛的大壶状腺、小壶状腺、鞭状腺、柱状腺、葡萄状腺和梨状腺共6种丝腺。2种蜘蛛丝腺形态特征基本相似;部分丝腺在形态结构和颜色上有些差异;悦目金蛛的葡萄状腺比棒络新妇发达。观察表明2种蜘蛛的网和卵袋特征差异较大,两者捕食策略也不同,棒络新妇采用咬一捆缚（Bit—Wrapping）策略,悦目金蛛则采用捆缚一咬（Wrapping-Bit）策略。棒络新妇和悦目金蛛的网和卵袋特征与丝腺的颜色相一致。同时,其葡萄状腺数量和大小与其各自的捕食策略相关。     

棒络新妇和悦目金蛛拖丝超微结构与力学行为

采用SEM对棒络新妇Nephila clavata腹部向上和向下在水平纱窗上爬行时纺出的拖丝、悦目金蛛Argiope amoena捕食拖丝与垂直向下缓慢纺出的拖丝及其圆网的铆钉丝进行了超微结构观察,采用电子单纤强力仪对棒络新妇拖丝与悦目金蛛圆网铆钉丝进行了力学拉伸试验.结果 表明棒络新妇和悦目金蛛拖丝均呈现出一至多根细丝纤维的多样化超微结构特征,其中悦目金蛛圆网铆钉丝还呈现出"S"形似弹簧的结构.两种蜘蛛丝的力学行为和性能与各自的功能要求相一致.蜘蛛能调节拖丝的超微结构、纤维组成和直径大小以适应其在不同环境条件下对力学性能和功能的瞬时需要.研究结果有助于拓宽和加深人们对蜘蛛丝超微结构、力学性能与生物学功能之间关系的认识和理解.     

3种中型蜘蛛卵袋形态特征与纤维组成结构

采用扫描电镜和氨基酸自动分析仪对球蛛科(Theridiidae)温室拟肥腹蛛(Parasteatodatepidariorum)、肖蛸蛛科(Tetragnathidae)肩斑银鳞蛛(Leucauge blanda)及狼蛛科(Lycosidae)猴马蛛(Hippasa holmerae)3种中型蜘蛛卵袋的超微结构和氨基酸组成进行了观察。形态观察表明,这3种蜘蛛的卵袋形态各异,温室拟肥腹蛛卵袋一头尖,呈梨状;肩斑银鳞蛛卵袋呈扁平状;猴马蛛卵袋呈椭球形。扫描电镜观察表明,温室拟肥腹蛛卵袋外覆盖层仅仅由一种均一直径的柱状腺丝组成,而另外2种蜘蛛卵袋外覆盖层主要由柱状腺丝与少量其他丝腺纺出的丝纤维组成。氨基酸组成分析表明,温室拟肥腹蛛卵袋外覆盖层的丝纤维的氨基酸组成与具有保守性的其他种类蜘蛛柱状腺丝心蛋白的氨基酸组成差异较大,这表明其可能含有新的丝心蛋白家族成员。本文根据氨基酸组成与扫描电镜的结果分析探讨了不同直径丝纤维的丝腺来源。     

悦目金蛛拖丝的超微结构研究

采用非固定的抽丝方法,从一只未麻醉的悦目金蛛(Argiope amoena)的纺器将拖丝抽出,然后用扫描电镜(SEM)对拖丝进行超微结构的观察,结果表明:悦目金蛛拖丝至少具有2根、3根、4根及多根单丝纤维构成的4种不同结构,其中有一种类似弹簧的结构;另外,丝的表面还出现一种小环结构,这两种结构可能是拖丝纤维具有优良机械性能的原因之一。"束状结构"和"小环结构"在文献中未见报道。拖丝的直径范围为0.25~10.77μm;悦目金蛛似乎能调节拖丝的结构和直径,以适应其所面临的即时环境。本文基于上述观察结果并结合前人的研究,提出了蜘蛛拖丝结构-生物学功能多样性假说,对蜘蛛丝的结构与生物学功能之间的关系作了探讨。     

无脊椎动物金属硫蛋白的研究

综述了近几十年来有关无脊椎动物金属硫蛋白（MT）的研究,包括软体动物MT、棘皮动物MT、环节动物MT、节肢动物MT（甲壳纲和昆虫纲）,分析了无脊椎动物MT与哺乳动物MT的异同,在此基础上,提出以下观点：即从无脊椎动物MT到哺乳动物MT的进化来看,动物MT的进化可能是一种趋同进化的模式。     

纺足目昆虫研究进展

纺足目昆虫又被称为足丝蚁，因为其具有独特的纺丝习性而被人所熟知；纺足目是昆虫纲中一个较小的类群，目前只有400余种被描述。本文简述了足丝蚁的形态特征和生物学特性；回顾了足丝蚁现生类群与化石研究历史及进展，概述了近年来足丝蚁的系统发育学研究进展，并提出研究该类群有待解决的问题，展望了纺足目未来的研究方向。     

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring ^1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel ³. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences ⁴. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] ^5,6, tubuliform [synthesizes egg case silk] ^7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] ⁹ and pyriform [produces attachment disc silk] ¹⁰. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.     

From EST sequence to spider silk spinning: identification and molecular characterisation of Nephila senegalensis major ampullate gland peroxidase NsPox

Spider dragline silk is renowned as one of the toughest materials of its kind. In nature, spider silks are spun out of aqueous solutions under environmental conditions. This is in contrast to production of most synthetic fibres, where hazardous solvents, high temperatures and pressure are used. In order to identify some of the chemical processes involved in spider silk spinning, we have produced a collection of cDNA sequences from specific regions of Nephila senegalensis major ampullate gland. We examined in detail the sequence and expression of a putative Nephila senegalensis peroxidase gene (NsPox) from our EST collection. NsPox encodes a protein with similarity to Drosophila melanogaster and Aedes aegypti peroxidases. Northern analysis and in situ localisation experiments revealed that NsPox is expressed in major and minor ampullate glands of the spider where the main components of the dragline silk are produced. We suggest that NsPox plays a role in dragline silk fibre formation and/or processing.     

Characterization and expression of a cDNA encoding a tubuliform silk protein of the golden web spider Nephila antipodiana

Spider silks are renowned for their excellent mechanical properties. Although several spider fibroin genes, mainly from dragline and capture silks, have been identified, there are still many members in the spider fibroin gene family remain uncharacterized. In this study, a novel silk cDNA clone from the golden web spider Nephila antipodiana was isolated. It is serine rich and contains two almost identical fragments with one varied gap region and one conserved spider fibroin-like C-terminal domain. Both in situ hybridization and immunoblot analyses have shown that it is specifically expressed in the tubuliform gland. Thus, it likely encodes the silk fibroin from the tubuliform gland, which supplies the main component of the inner egg case. Unlike other silk proteins, the protein encoded by the novel cDNA in water solution exhibits the characteristic of an alpha-helical protein, which implies the distinct property of the egg case silk, though the fiber of tubuliform silk is mainly composed of beta-sheet structure. Its sequence information facilitates elucidation of the evolutionary history of the araneoid fibroin genes.     

Review the role of terminal domains during storage and assembly of spider silk proteins

Fibrous proteins in nature fulfill a wide variety of functions in different structures ranging from cellular scaffolds to very resilient structures like tendons and even extra-corporal fibers such as silks in spider webs or silkworm cocoons. Despite their different origins and sequence varieties many of these fibrous proteins share a common building principle: they consist of a large repetitive core domain flanked by relatively small non-repetitive terminal domains. Amongst protein fibers, spider dragline silk shows prominent mechanical properties that exceed those of man-made fibers like Kevlar. Spider silk fibers assemble in a spinning process allowing the transformation from an aqueous solution into a solid fiber within milliseconds. Here, we highlight the role of the non-repetitive terminal domains of spider dragline silk proteins during storage in the gland and initiation of the fiber assembly process.     

Strength and structure of spiders’ silks

Spider silks are composite materials with often complex microstructures. They are spun from liquid crystalline dope using a complicated spinning mechanism which gives the animal considerable control. The material properties of finished silk are modified by the effects of water and other solvents, and spiders make use of this to produce fibres with specific qualities. The surprising sophistication of spider silks and spinning technologies makes it imperative for us to understand both material and manufacturing in nature before embarking on the commercialization of biotechnologically modified silk dope.     

利用生物工程技术生产蜘蛛丝的研究进展

蜘蛛丝是一种高分子蛋白纤维,具有高强度、高弹性等许多重要的优良特性,在军事、医学、工业、建筑、纺织等领域具有广泛而巨大的应用。然而蜘蛛的产丝量小,且无法高密度养殖以获取大量的蜘蛛丝,难以满足实际应用的需要。于是人们只能着眼于生物工程方法,即将蜘蛛丝蛋白基因转入其它生物体来表达生产蜘蛛丝蛋白,经过多年的研究,已取得很多重要的进展。对蜘蛛丝蛋白在微生物、植物、哺乳动物及家蚕等不同生物载体中表达的研究进展进行重点阐述,并探讨了已有研究的不足和今后研究展望,为进一步探索和研发蜘蛛丝的规模化生产方法提供借鉴与参考。     

Strength and structure of spiders' silks.

Spider silks are composite materials with often complex microstructures. They are spun from liquid crystalline dope using a complicated spinning mechanism which gives the animal considerable control. The material properties of finished silk are modified by the effects of water and other solvents, and spiders make use of this to produce fibres with specific qualities. The surprising sophistication of spider silks and spinning technologies makes it imperative for us to understand both material and manufacturing in nature before embarking on the commercialization of biotechnologically modified silk dope.     

Spiders spinning electrically charged nano-fibres

Most spider threads are on the micrometre and sub-micrometre scale. Yet, there are some spiders that spin true nano-scale fibres such as the cribellate orb spider, Uloborus plumipes. Here, we analyse the highly specialized capture silk-spinning system of this spider and compare it with the silk extrusion systems of the more standard spider dragline threads. The cribellar silk extrusion system consists of tiny, morphologically basic glands each terminating through exceptionally long and narrow ducts in uniquely shaped silk outlets. Depending on spider size, hundreds to thousands of these outlet spigots cover the cribellum, a phylogenetically ancient spinning plate. We present details on the unique functional design of the cribellate gland–duct–spigot system and discuss design requirements for its specialist fibrils. The spinning of fibres on the nano-scale seems to have been facilitated by the evolution of a highly specialist way of direct spinning, which differs from the aqua-melt silk extrusion set-up more typical for other spiders.     

THE ORIGIN OF THE SPINNING APPARATUS IN SPIDERS

• 1 Previous attempts to explain the evolution of spider silk have relied heavily on conjecture. The formulation of testable historical hypotheses to replace such speculation is discussed.
• 2 The importance of phylogenetic reconstructions and other historical hypotheses for use in generating and testing hypotheses concerning the evolution of specific adaptations is examined. Recent ideas on arachnid phylogeny are reviewed and their relevance to the problem of silk evolution in spiders is explored.
• 3 Evidence from the analysis of three historical problems (origin of spinnerets, origin of silk glands, original selective pressure favouring evolution of silk) is reviewed from three different frames of reference (in-group analysis, out-group analysis, convergence analysis). Several lines of evidence are found which suggest that silk use originated in spiders due to selective pressures associated with reproduction (specifically, the transfer of sperm or the protection of eggs).
• 4 The prevalence of segmental appendages retained for use in manipulating genital products in both arachnids and non-arachnid arthropods and the probable placement of spinnerets near the genital opening in ancestral spiders suggest that spinnerets represent modified gonopods.
• 5 The most primitive types of silk glands are retained in virtually all spiders, in part, for use in the construction of sperm webs and egg sacs. Similar silk glands are found near the genital opening in many male spiders and used in building a portion of the sperm web.
• 6 The silk of adult arthropods other than spiders is used largely in manipulating or protecting sex cells. If there are multiple functions, use in reproduction is typically one of them. Thus, there is evidence for strong selective pressure favouring the evolution of silk for use in reproduction.
• 7 Two hypotheses are proposed which are consistent with the conclusion that silk in spiders evolved for reproductive needs (the spermatophore-sperm web and egg sac hypotheses). Testable predictions of each hypothesis are proposed.