猕猴白化病分子机制和灵长类的分子进化研究

酪氨酸酶是黑色素合成当中的关键酶。人酪氨酸酶基因包括5个外显子，在染色体11q14-q21位置上占据了约50kb长的区域。对人类眼皮肤型白化病（Oculocutaneous albinism, OCA)的许多研究表明，该病主要是由于酪氨酸酶基因的突变引起的。昆明动物研究所白化猴研究小组数十年来一直从事白化猕猴的培育和研究工作，目前饲养着2只白化猕猴和它们的后代，这提供了我们研究猕猴白化分子机制目的条件。为了弄清猕猴白化病的分子机制，我们根据人酪氨酸酶基因序列设计了5对PCR引物扩增相应的5个外显子，序列分析表明，白化猕猴珍珍酪氨酸酶基因第184个密码子第2位置（外显子1的核苷酸位置551）处发生一个C→A的无义突变，使编码丝氨酸（Ser)的密码子变成了一个终止密码，这样后面1038bp的核苷酸片段（346个氨基酸残基）被截断，导致酪氨酸酶翻译不完全，迄今为止，并没有发现合成黑色素的第二条生化途径，因此由于该酶不能行使正常功能而将导致黑色素不能正常表达。这可能是导致该例猕猴白化病的原因。为了解酪氨酸酶基因序列变异的规律及其与功能的关系，探讨该基因作为系统发育研究中遗传标记的有效性，我们测定了黑猩猩（Pan troglodytes)、倭黑猩猩（Pan paniscus)、大猩猩（Gorilla gorilla)、猩猩(Pongo pygmaeus)、长臂猿（Hylobates lar)、食蟹猴（Macaca fascicularis)、狒狒（Simia cynocephalus)、猕猴（Macaca mulatta)、熊猴（Macaca assamensis)、菲氏叶猴（Presbytis p. crepusculus)、白臀叶猴（Pygathrix nemaeus)、滇金丝猴（Rhinopithecus r. bieti）和蛛猴（Ateles paniscus)13个灵长类中代表种的酪氨酸酶基因全部5个外显子的DNA序列。基于这些序列，用简约法构建了分子系统树。结果表明，人猿超科与旧大陆猴各自形成一单系群。人猿超科各物种和旧大陆猴有明显分化，人与大猩猩的关系比人与黑猩猩的关系近。酪氨酸酶基因在解决灵长类系统发育关系上是一个较有用的基因。为了进一步了解中国猕猴（Macaca mulatta)的亚种分化和不同地理群体间的基因流状况，我们测定了来自中云南、广西、福建、海南、浙江、河南、湖南、湖北、安徽、四川、贵州和越南猕猴共96只个体和一只外群食蟹猴的线粒体DNA控制区576bp的DNA序列，基于这些序列，运用距离法对中国恒河猴的分子进行和遗传多样性进行了分析，我们的研究结果显示，云南、四川和湖南猕猴群体与其它群体存在显著分析，海南群体内遗传多样性最低、四川、广西、浙江、福建和越南群体内遗传多样性较丰富。中国猕猴的分化可能存在三条路线。中国猕猴的遗传多样性较丰富。

柞蚕核型多角体病毒B-ORF6L、ptp、lef-12基因克隆及表达

本研究克隆了柞蚕核型多角体病毒（Antheraea pernyi nucleopolyhedrovirus，ApNPV）基因组pstⅠ-B、pstⅠ-C、pstⅠ-J三个片段，测序分析了pstⅠ-B、pstⅠ-C片段全序列及pstⅠ-J片段一端序列。ApNPV pstⅠ-C片段长6663 bp，包括9个完整ORF及2个不完整ORF；ApNPV pstⅠ-B片段长7406 bp，包括5个完整ORF及2个不完整ORF。ApNPV pstⅠ-J片段末端测定的954 bp序列包括lef-12完整序列及p47和gta部分序列。本研究共鉴定21个ApNPV ORF序列，其中20个属首次报道，占ApNPV已报道基因数的50％。编码ORF同源性分析及克隆片断ORF组成、基因排列顺序分析表明ApNPV与鳞翅目NPV第Ⅰ类群中的OpMNPV、CfMNPV、CfDefNPV、EppoNPV关系较近。 本研究克隆了ApNPV B-ORF6L、ptp-1、ptp-2及lef-12 四个基因，并对这四个基因在柞蚕蛹体内的表达进行了转录分析，结果表明：ApNPV ptp-1、lef-12是早期基因，B-ORF6L、ptp-2是晚期基因。本研究将ApNPV B-ORF6L、ptp-2亚克隆至原核表达载体，并在大肠杆菌中获得高效表达。SDS-PAGE及Western blot分析表明：PTP-2原核表达分子量与预测分子量相符，B-ORF6L融合表达分子量较预测的分子量偏大。以原核表达的B-ORF6L、PTP-2蛋白作为抗原，成功制作了B-ORF6L和PTP-2蛋白兔多克隆抗血清。ApNPV蛋白组分印迹分析表明：B-ORF6L参与包涵体膜及ODV结构组成，是ApNPV结构蛋白；PTP-2不参病毒结构组成。 分子系统发育分析表明，杆状病毒分为4个大的类群，ApNPV属于鳞翅目NPV第Ⅰ类群，与OpMNPV、CfMNPV、CfDefNPV、EppoNPV关系较近，与AcMNPV、RoMNPV、BmNPV关系稍远。

灵长类的减数分裂细胞遗传学研究

I.四种猕猴属(Macaca)动物精母细胞联会复合体的比较研究 本工作采用去污剂微铺展－－硝酸银染色技术比较研究了熊猴、平顶猴、藏酋猴、恒河猴及其亚种毛耳猴的精母细胞联会复合体（SC）核型和SC的结构、形态及其在减数分裂过程的行为。结果表明：1.这几种动物的SC核型及SC的发育过程基本一致。粗线期SC的相对长度和臂比与体细胞染色体的相对长度和臂比具有较好的吻合性。SC的形成开始于线期，成熟于粗线期，解体于双线期。2.在减数分裂前期，性染色体轴表现强烈的嗜银性，配对明显落后于常染色体。根据性染色体的形态和行为，可分为五种类型。此外，本文还对XY染色体的同源性和侧轴加粗等现象进行了讨论。II.食蟹猴（M.fascicularis）和熊猴（M.assamensis）杂种（F1）的细胞遗传学研究 本工作采用染色体显带、组织学观察以及低渗铺张－－硝酸银染色等方法较为详细地研究了食蟹猴和熊猴种间杂种（F1）体细胞染色体的G带、C带、Ag-NOR、精母细胞联会复合体的结构、形态和行为以及精子发生。结果表明，①杂种亲本的染色体级具有高度的同源性；②杂种的精子发生过程完全正常；③食蟹猴和熊猴的种间生殖屏障可能主要是生态隔离。此外，本文还对食蟹猴和熊猴染色体高度同源的原因、二者的分类地位以及杂种细胞中Ag-NOR的多态性等问题进行了分析和讨论。

Wild silkworm cocoon as a natural tough biological composite structure

Silk cocoons are biological composites with intriguing characteristics that have evolved through a long natural selection process. Knowledge of structure-property-function relationship of multilayered composite silk cocoon shells gives insight into the design of next-generation protection materials. The current investigation studied the composite structure and mechanical performance of a wild silkworm cocoon (Chinese tussah silkworm cocoon, Antheraea pernyi) in comparison with the domestic counterpart (Mulberry silkworm cocoon, Bombyx mori). 180º peel and tensile tests were performed on the cocoon walls to understand both their interlaminar and in-plane mechanical properties. The fracture surfaces were investigated under SEM. The wild cocoon showed substantially higher toughness over the domestic cocoon, which explains their unique capability to tackle severe environmental adversaries.

Lessons from silkworm cocoons for future protective materials design

 Evolved over millions of years’ natural selection, very thin and lightweight wild silkworm cocoons can protect silkworms from environmental hazards and physical attacks from predators while supporting their metabolic activity. The knowledge of structure-property-function relationship of multi-layered composite silk cocoon shells gives insight into the design of next-generation protection materials. The mechanical and thermal insulation properties of both domestic (Bombyx mori, or B. moriand Samia. cynthia, or S. cynthia) and wild (Antheraea pernyi and Antheraea mylitta, or A. pernyi and A. mylitta) silkworm cocoons were investigated. The research findings are of relevance to the bio-inspired design of new protective materials and structures.
The 180 degree peel tests and needle penetration tests were used for examining the peel resistance and needle penetration resistance of both domestic and wild silkworm cocoon walls. The temperatures inside and outside of the whole silkworm cocoons under warm, cold and windy conditions were monitored for investigating the cocoon’s thermal insulation function. Computational fluid dynamics (CFD) models were created to simulate the heat transfer through the A. pernyi cocoon wall.
The wild cocoons experienced much higher peeling peak loads than the domestic cocoon. This transfers to a maximum work-of-fracture (WOF) of about 1000 J/m2 from the A. pernyi outer layer, which was 10 times of the B. mori cocoon. The A. pernyi wild cocoon exhibited a maximum penetration force (11 N) that is 70 % higher than a woven aramid fabric. Silk sericin is shown to play a critical role in providing needle penetration resistance of the non-woven composite cocoon structure by restricting the relative motion of fibres, which prevents the sharp tip of the needle from pushing aside fibres and penetrating between them. The wild A. pernyi cocoon exhibits superior thermal buffer over the domestic B. mori cocoon. The unique structure of the A. pernyi cocoon wall with mineral crystals deposited on the cocoon outer surface, can prohibit most of the air from flowing inside of the cocoon structure, which shows strong wind resistance under windy conditions.

The effect of fibrous structural difference on thermal insulation properties of biological composites: silkworm cocoons

As a biological fibrous structure, silkworm cocoon provides multiple protective functionalities to safeguard the silk moth pupa’s metabolic activity. The mechanism of this protection could be adopted in clothing manufacture to provide more comfortable apparel. In this study, the thermal insulation properties of both domestic Bombyx mori (B. mori) and wild Antheraea pernyi (A. pernyi) cocoons were investigated under both warm and cold environmental conditions. Computational fluid dynamics models have been developed to simulate the heat transfer process through both types of cocoon wall structures. The simulation results show that the wild A. pernyi cocoon reduces the intensity of convection and heat flux between the environment and the cocoon interior and has higher wind resistance than its domestic counterpart. Compared with A. pernyi cocoon, the B. mori cocoon facilitates easy air transfer and decreases the temperature lag when the surrounding conditions are changed. The new knowledge has significant implications for developing biomimetic thermal functional materials.

Anti-parasitic and anti-viral immune responses in insects

Insects encounter many microorganisms in nature and to survive they have developed counter measures against the invading pathogens. In Drosophila melanogaster research on insect immunity has mainly been focused on infections by bacteria and fungi. We have explored the immune response against natural infections of the parasite Octosporea muscaedomesticae and the Drosophila C virus as compared to natural infections of bacteria and fungi. By using Affymetrix Drosophila GeneChips, we were able to obtain 48 genes uniquely induced after parasitic infection. It was also clearly shown that natural infections led to different results than when injecting the pathogens. In order to search for the ultimate role of the lepidopteran protein hemolin, we used RNA interference (RNAi). We could show that injection of double stranded RNA (dsRNA) of Hemolin in pupae of Hyalophora cecropia led to embryonic malformation and lethality and that there was a sex specific difference. We continued the RNAi investigation of hemolin in another lepidopteran species, Antheraea pernyi, and discovered that hemolin was induced by dsRNA per se. A similar induction of hemolin was seen after infection with baculovirus and we therefore performed in vivo experiments on baculovirus infected pupae. We could show that a low dose of dsHemolin prolonged the period before the A. pernyi pupae showed any symptoms of infection, while a high dose led to a more rapid onset of symptoms. By performing in silico analysis of the hemolin sequence from A. pernyi in comparison with other Hemolin sequences, it was possible to select a number of sites that either by being strongly conserved or variable could be important targets for future studies of hemolin function.

Directional moisture transfer through a wild silkworm cocoon wall

A silkworm cocoon is a porous biological structure with multiple protective functions. In the current work, the authors have used both experimental and numerical methods to reveal the unique moisture transfer characteristics through a wild Antheraea pernyi silkworm cocoon wall, in comparison with the long-domesticated Bombyx mori silkworm cocoon walls. The water vapor transmission and water vapor permeability (WVP) properties show that the A. pernyi cocoons exhibit directional moisture transfer behavior, with easier moisture transfer from inside out than outside in [e.g., the average WVP is 0.057 g/(h m bar) from inside out and is 0.034 g/(h m bar) from outside in]. Numerical analysis shows that the cubic mineral crystals in the outer section of the A. pernyi cocoon wall create a rough surface that facilitates air turbulence and promotes disturbance amplitude of the flow field, leading to lengthened water vapor transfer path and increased tortuosity of the moist air. It also indicates the vortex of water vapor can be generated in the outer section of cocoon wall, which increases the diffusion distance of water vapor and enhances the turbulence kinetic energy and turbulence eddy dissipation, signifying higher moisture resistance in the outer section. The difference in moisture resistance of the multiple A. pernyi cocoon layers is largely responsible for the unique directional moisture transfer behavior of this wild silkworm cocoon. These findings may inspire a biomimicry approach to develop novel lightweight moisture management materials and structures.