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。     

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.     

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。     

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。     

次壶腹腺丝蛋白功能模块的研究

蜘蛛丝是天然的生物材料,具有潜在的巨大应用价值。研究蜘蛛丝蛋白质的结构与功能,有助于破解蜘蛛丝蛋白质的成丝机理,为制备优良材料学性能的仿生蜘蛛丝纤维提供理论依据。以MiSp蜘蛛丝的重复区和C端非重复区蛋白多肽为研究对象,在不同pH值和离子条件下,在体外研究其二级结构与成丝的关系。CD图谱显示:表达纯化的重组蜘蛛丝蛋白R1R2在pH 7.5、6.5和5.5时二级结构相似,均为无规则卷曲,而R1R2CT则主要呈现为α螺旋构象;扫描电镜结果表明:在以上3种pH条件下,只有pH 5.5时R1R2和R1R2CT才形成重组丝纤维,R1R2CT纤维形态较平整,类似于天然蛛丝纤维形态,而R1R2丝纤维则呈条带状,表面粗糙。另外,氯化钠不利于形成形态平整的丝纤维。该成果为研究蛛丝蛋白的成丝机理奠定基础,也为制备仿生蛛丝蛋白纤维提供理论依据。     

横纹金蛛捕食经验对其丝纤维力学行为与性能的影响

蛛网是蜘蛛的捕食工具,蛛网网丝的结构与性能不仅影响蜘蛛的捕食效率,也关系着蜘蛛的捕食投入。本文利用单纤维电子强力仪研究横纹金蛛(Argiope bruennichi)室内捕食面包虫(Tenebrio molitor)时上前与返回捕食拖丝的力学性能以及捕食经验对圆网半径丝修补前后的力学性能的影响。结果表明,与上前捕食拖丝相比,返回捕食拖丝减小了弹性区的投入,增加了屈服区和加强区的投入,且返回时捕食拖丝更具柔韧性。整体而言,与初始半径丝相比,在未喂食面包虫的条件下,网的半径修补丝增加了力学性能的投入;而在喂食面包虫的条件下,网的半径修补丝减少了力学性能的投入。所测试丝样中出现了两种类型的蛛丝力学行为:一种为典型的蛛丝力学行为;另外一种为似黏流性材料的力学行为,其反映的是满足蛛丝耗散猎物或自身下降时动能的另外一种力学性能的策略。本研究表明蜘蛛能根据其捕食经验遵循Cost-Benefit原则对蜘蛛丝的力学性能进行调节,从而调整捕食投入。     

悦目金蛛3种蛛丝超微结构和理化特性的初步研究

利用扫描电镜、氨基酸分析仪、X-衍射仪和单纤维电子强力仪分别对悦目金蛛Argiopeamoena拖丝、网框丝和卵袋丝的超微结构和理化特性进行了测试和观察。结果表明,悦目金蛛卵袋不是由一种结构均一的丝纤维构成,而是由直径相差悬殊的Ⅰ型卵袋丝和Ⅱ型卵袋丝2种丝纤维共同组成,该结果对卵袋丝仅由管状腺产生的观点提出了疑问。在氨基酸组成上悦目金蛛拖丝和网框丝相似,但其卵袋丝的氨基酸组成与拖丝和网框丝相比差别明显。另外还发现卵袋丝的强度、结晶度大于拖丝和网框丝,而它的延伸性能却不及拖丝和网框丝。     

大腹园蛛主壶腹腺蛛丝蛋白末端结构域的克隆表达

牵引丝(dragline silk)由主壶腹腺蛛丝蛋白(major ampullate spidroin, MaSp)组成,是蜘蛛丝中强度最好的丝,同时具有极佳的生物相容性和可降解性,因此引起研究者的研究热潮。目前关于大腹园蛛MaSp结构和成丝机理方面的研究甚少,限制了其仿生应用。本文以大腹园蛛牵引丝的组成蛋白质之一MaSp1为研究对象,通过锚定PCR的方法首次获取了大腹园蛛MaSp1 NT的完整编码基因,并对其进行了克隆、表达、纯化,产量可达60 mg/L;同时对该MaSp1的CT进行表达纯化,产量可达80 mg/L。另外,通过CD色谱分析了MaSp1 NT和CT的二级结构,结果表明二者的二级结构均以α-螺旋为主。上述结果为大腹园蛛MaSp1的结构和成丝机理研究奠定了基础。     

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。     

4种常见蜘蛛蛛丝光学显微镜观察及机械性能的测定

对4种常见蜘蛛大腹园蛛Araneus ventricosus、迷宫漏斗蛛Agelena labyrinthica、草间钻头蛛Hylyphantes graminicola和棒络新妇Nephila clavata蛛丝的物理特性和机械性能进行了初步研究.结果 表明:捕丝是由2根核心丝构成,4种蛛丝呈现不同的颜色;4种蜘蛛蛛丝的密度略有不同,棒络新妇的蛛丝密度最大,草间钻头蛛的蛛丝密度最小;蛛丝中棒络新妇的断裂伸长率和断裂强度都最大,分别为47.3%和852.6 Nmm-2,草间钻头蛛蛛丝的断裂伸长率和断裂强度都最小,分别为32.1%和652.3 Nmm-2.与其它物理化学材料相比,蛛丝具有优异的综合力学性能,生物学特性与生物学功能具有高度一致性.     

Chimeric spider silk proteins mediated by intein result in artificial hybrid silks

Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K₃PO₄ pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385–392, 2016.     

昆虫的丝和丝腺

文中介绍了吐丝昆虫的种类、昆虫丝的结构与成分、丝与丝腺的类型、吐丝器及泌丝行为等。并简述了丝对泌丝昆虫生活的重要性及近期虫丝的研究动态。     

Expression and characterization of chimeric spidroins from flagelliform-aciniform repetitive modules

Spiders can produce up to seven different types of silks or glues with different mechanical properties. Of these, flagelliform (Flag) silk is the most elastic, and aciniform (AcSp1) silk is the toughest. To produce a chimeric spider silk (spidroin) Flag_R-AcSp1_R, we fused one repetitive module of flagelliform silk from Araneus ventricosus and one repetitive module of aciniform silk from Argiope trifasciata. The recombinant protein expressed in E. coli formed silk-like fibers by manual-drawing. CD analysis showed that the secondary structure of Flag_R-AcSp1_R spidroin remained stable during the gradual reduction of pH from 7.0 to 5.5. The spectrum of FTIR indicated that the secondary structure of Flag_R-AcSp1_R changed from α-helix to β-sheet. The conformation change of Flag_R-AcSp1_R was similar to other spidroins in the fiber formation process. SEM analysis revealed that the mean diameter of the fibers was around 1 ~ 2 μm, and the surface was smooth and uniform. The chimeric fibers exhibited superior toughness (~33.1 MJ/m³) and tensile strength (~261.4 MPa). This study provides new insight into design of chimeric spider silks with high mechanical properties.     

Ultrastructure of spider thread anchorages

Spiders attach silken threads to substrates by means of glue-coated nanofibers (piriform silk), spun into disc-like structures. The organization and ultrastructure of this nano-composite silk are largely unknown, despite their implications for the biomechanical function and material properties of thread anchorages. In this work, the ultrastructure of silken attachment discs was studied in representatives of four spider families with Transmission Electron Microscopy to facilitate a mechanistic understanding of piriform silk function across spiders. Based on previous findings from comparative studies of piriform silk gland morphology, we hypothesized that the fibre-glue proportion of piriform silk differs in different spiders, while the composition of fibre and glue fractions is consistent. Results confirmed large differences in the relative proportion of glue with low amounts in the orb weaver Nephila senegalensis (Araneidae) and the hunting spider Cupiennius salei (Ctenidae), larger amounts in the cobweb spider Parasteatoda tepidariorum (Theridiidae) and a complete reduction of the fibrous component in the haplogyne spider Pholcus phalangioides (Pholcidae). We rejected our hypothesis that glue ultrastructure is consistent. The glue is a colloid with polymeric and fluid fractions that strongly differ in proportions and assembly. We further confirmed that in all species studied both dragline and piriform silk fibres do not make contact with the environmental substrate. Instead, adhesion is established by a thin dense skin layer of the piriform glue. These results advance our understanding of piriform silk function and the interspecific variation of its properties, which is significant for spider biology, web function and the bioengineering of silk.     

Fine structure of sheet-webs of Linyphia triangularis (Clerck) and Microlinyphia pusilla (Sundevall), with remarks on the presence of viscid silk

We examined the webs of Linyphia triangularis (Clerck) and Microlinyphia pusilla (Sundevall) using light and scanning electronic microscopic techniques and compared them with the better known orb‐webs. The linyphiid sheet‐web consists of an unordered meshwork of fibres of different thicknesses. The sheet is connected to the scaffolding by means of attachment discs. Thin threads with globules, which appear similar to the viscid silk droplets of orb‐webs, are present in most webs examined. Webs of M. pusilla had a higher density of these globules than did webs of L. triangularis. Webs of both species possess five types of thread connections and contain no aqueous glue for prey capture. Instead, unlike orb‐webs, the sticky substances produced by the linyphiid aggregate glands cement the different layers and threads of the sheet by drying up after being produced. Due to their function, sheet webs may not require viscid silk, thereby leading to a more economic web. The assumption made in most previous studies, that the globules in linyphiid webs have the same properties and function as viscid silk in orb‐webs, is unfounded.     

蛋白质内含子介导蛛丝功能化平台的建立

为建立高效快捷的蛛丝功能化修饰平台,蛋白质内含子的反式剪接技术被首次应用于重组蛛丝的功能化修饰。在体外通过Ssp Dna B的反式剪接作用,在蛋白质水平上将12 k Da泛素相关修饰蛋白(SUMO)与蛛丝蛋白(W2CT)连接形成功能化蛛丝蛋白SUMOW2CT。修饰后SUMOW2CT与W2CT均能形成纳米至微米级的丝纤维,但SUMOW2CT自动成丝速度明显下降且产量约为W2CT的一半。与W2CT丝纤维(W)相似,SUMOW2CT丝纤维(UW)不具有超收缩能力和对2%SDS不耐受,但机械性能低于W2CT丝纤维。功能化蛋白SUMOW2CT形成的丝纤维中SUMO蛋白仍保持着正确三维结构,可被SUMO蛋白酶酶切。外源功能化蛋白质虽在一定程度上降低了丝的形成速度和机械性能,但修饰上的功能化蛋白仍保持着生物活性,表明断裂蛋白质内含子介导的蛛丝修饰平台成功建立,也为蛛丝的功能化修饰和应用奠定了坚实的技术基础。     

Crystal structure of raw pure Mysore silk fibre based on (Ala-Gly)2-Ser-Gly peptide sequence using Linked-Atom-Least-Squares method

We have carried out crystal structure analysis of raw pure Mysore silk fibers belonging toBombyx mori on the basis of model parameters of Marshet al using Linked-Atom-Least-Squares technique. The intensity of all the reflections were computed employing CCP13 software. We observe that the molecular modification is essentially same as β-pleated structure with antipolar-antiparallel arrangements formed by hydrogen bonds. The essential differences observed in the structure are highlighted and discussed     

原子力显微镜下丝素纤维及丝素蛋白的形态结构研究

利用原子力显微镜(AFM)、透射电镜(TEM)、圆二色谱仪(CD)等手段,研究了家蚕丝素纤维及丝素蛋白的形态结构,并尝试通过改变丝素蛋白溶液的酸碱性来观测其形态变化。结果表明,丝素纤维表面有许多沟槽和条纹,具有原纤结构特征;许多直径为20～50nm的圆形或椭圆形颗粒分子形成丝素蛋白的微观形态。在不同的酸碱条件下,球状颗粒分子具有不同的聚集方式,形成不同的微观形态。     

Characterization and assembly of a GFP‐tagged cylindriform silk into hexameric complexes

Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP‐silk fusion protein is predominantly α‐helical, and that pH can trigger a α‐ to β‐transition resulting in aggregation. Structural analysis by small angle X‐ray scattering suggests that the GFP‐Silk exists in the form of a hexamer in solution. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 378–390, 2014.     

Durability of Simuliid Silk Pads (Simuliidae,Diptera)

A central aspect of simuliid adaption to life in moving water is a silky secretion which they produce in their salivary glands. This secretion is spread on the substrate surface thereby enabling adhesion of the posterior abdominal hooks of the larvae to the substrate surface. Therefore, silk pads are a prerequisit for simuliid filter‐feeding as well as any kind of locomotive activity. If silk pad adhesion is weak, larvae risk drifting off, either directly or during locomotion. Properties of the adhesive (=silk) as well as the substrate surface may cause weak adhesion. A specialist such as S. noelleri which has little chance of surviving after drifting off its lake outlet habitat, should have adaptations to reduce this risk. Such an adaptation could be very durable silk pads, giving larvae the chance to be safely attached for a relatively long time. In this study larval silk pads of S. ornatum and S. noelleri were stained using Giemsa's staining procedures. Changes in silk pad structure and traces of rot after 2–33 days of exposure to stream water were recorded and compared. Silk pads of S. ornatum and S. noelleri showed differences which indicate differences in ageing processes and biochemistry of this secretion.