三种两栖类动物皮肤和脑中活性多肽结构、功能与多样性分析

两栖动物是最原始的陆生脊椎动物，分布比较广泛。无尾目两栖动物现有3000 多种，它们皮肤裸露、光滑，为适应广泛的栖息地和生态条件，已进化出各种有效的皮肤防御系统。抗菌肽（Antimicrobial peptides，AMPs）作为两栖类先天防御系统的重要组成部分，在皮肤分泌液中含量异常丰富。我们以来源于云南省普洱市景东县的铃蟾科微蹼铃蟾（Bombina microdeladigitora）和楚雄州双柏县雨蛙科华西雨蛙（Hyla annectans）为实验材料，对其皮肤分泌液中抗菌肽的分子多样性并对其结构和功能进行研究。微蹼铃蟾皮肤抗菌肽多样性非常丰富，我们从单一个体中克隆得到了64 条编码不同抗菌肽的cDNA 序列，其中有两条序列只编码Maximin 一种抗菌肽，其余 62 条均编码Maximin 和Maximin H 两类抗菌肽。这64 条cDNA 序列共编码44 种Maximins 和30 种Maximin Hs，其中有32 种Maximins 和20 种Maximin Hs 为新鉴定的抗菌肽，其余和铃蟾属其它种中发现的抗菌肽具有相同的序列。除了皮肤外，两栖动物的脑也是抗菌肽的丰富资源库。我们分别从微蹼铃蟾和大蹼铃蟾（B. maxima）脑中得到了大量新的抗菌肽cDNA 序列。其中从微蹼铃蟾脑中克隆到21 条新的cDNA序列，共编码16 种Maximins 和10 种Maximin Hs，其中7 种Maximins 和4 种Maximin Hs 为新鉴定的抗菌肽。从大蹼铃蟾脑中克隆到39 条新的cDNA 序列，编码27 种Maximins 和20 种Maximin Hs，其中16 种 Maximins 和12 种Maximin Hs 为新鉴定的抗菌肽。在以上新鉴定的抗菌肽中，Maximins 均为阳离子抗菌肽，Maximin Hs 中除以前鉴定的Maximin H5 外，尚有十余种阴离子抗菌肽。抗菌肽碱基转换/颠换（R=s/v）分析表明，RMaximin<1 而RMaximin H>1，说明这两种抗菌肽碱基转换和颠换发生的几率并不相同，Maximin 间差异主要由碱基颠换引起，而Maximin H 则主要由碱基转换引起。种间进化分析表明，大蹼铃蟾和微蹼铃蟾的遗传距离较近，而它们与欧洲花铃蟾（B. variegata）的遗传距离均较远。种内各部分遗传距离差异较大。与信号肽和酸性间隔肽相比，成熟肽的遗传距离明显增大，其中Maximin 的进化速度比Maximin H 更快。抗菌肽Maximin 和Maximin H 种内、种间均存在正选择（ω>1），而信号肽和酸性间隔肽在分化过程中没有正选择（ω<1），说明Maximin 和Maximin H 经受着达尔文正选择驱动的快速进化，是抗菌肽多样性产生的根本原因。这与抗菌肽参与最终的生物防御功能，从而增加物种对环境的适应是一致的。功能研究发现，有些微蹼铃蟾Maximins 抗菌肽是多功能分子，不但对革兰氏阴性菌、革兰氏阳性菌和真菌起抗菌作用，而且还具有很强的抗氧化功能。脑中抗菌肽基因的大量表达也预示着抗菌肽可能在神经信号传导中起一定作用。用基因克隆方法我们从微蹼铃蟾皮肤得到大量缓激肽前体序列，由1-4 个拷贝的Bombinakinin 或1-4 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成。这与大蹼铃蟾皮肤中缓激肽前体由1-8 个拷贝的Bombinakinin 或1-8 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成有所不同。按同样方法，我们从大蹼铃蟾脑中也得到了三条缓激肽前体序列，其中两条含有6 个 Bombinakinin 拷贝，另一条含2 个Bombinakinin 拷贝。通过比较只含Bombinakinin 和同时含有Bombinakinin 和Bombinakinin-GAP 的前体cDNA 序列后发现， 前者序列中缺失了一段碱基序列TGCGGGTA， 从而导致移码突变， 终止了 Bombinakinin-GAP 的表达。通过生物化学的手段从微蹼铃蟾皮肤分泌液中分离到一种丝氨酸蛋白酶抑制剂BMSI1，与铃蟾属其它种中的胰蛋白酶抑制剂具有很高的相似性。根据已有铃蟾属丝氨酸蛋白酶抑制剂cDNA 序列设计引物，以皮肤cDNA 为模板，扩增丝氨酸蛋白酶抑制剂的基因序列，结果得到两条不同的序列。这两条前体序列与铃蟾属其它两栖动物皮肤中的丝氨酸蛋白酶抑制剂具有高度相似性（>70%），而且它们都含有10 个半胱氨酸残基。BMSI1 对五种丝氨酸蛋白水解发色底物的抑制活性测定表明，BMSI 1 能抑制胰蛋白酶和凝血酶的水解活性，其K(i)分别为0.02 μM 和0.15 μM。通过随机筛选cDNA 文库的方法，我们从大蹼铃蟾脑中得到了一条完整的 Somatostatin（SST）序列，根据该序列，我们在大蹼铃蟾和微蹼铃蟾脑cDNA 文库中筛选到两条变异体序列SST-L(Leu11-SST-14)和SST-R(Arg14-SST-14)。功能研究表明，这两种变异体具有和SST 相似的生物学功能，可抑制肿瘤细胞增殖、抑制细胞因子释放以及具有一定的镇痛作用。从大蹼铃蟾和微蹼铃蟾脑中得到了阿片肽前体POMC 和Proenkephalin 的 cDNA 序列，序列比对发现与东方铃蟾具有较高的同源性。从华西雨蛙皮肤cDNA 中克隆得到两类活性多肽，命名为Annins。其中一类为抗菌肽类似肽，共11 条序列，编码单一的成熟肽序列，其信号肽与雨蛙科信号肽具有很高的同源性，但酸性间隔肽和成熟肽相差较大。其成熟肽由15-17 个氨基酸残基组成，活性分析表明无抗菌和抗氧化作用，但在较高浓度时对部分细菌和多种细胞有促进生长作用，推测可能在使伤口快速愈合方面起重要作用；另一类编码具有2 个拷贝的成熟肽序列，成熟肽由5 个氨基酸残基组成，具有一定的镇痛活性，其镇痛机理可能是拮抗bradykinin 作用。

三种豆科灌木对干旱胁迫的响应与适应

干旱胁迫是全球范围内影响植物生存、生长和分布的重要环境因子。岷江上游干旱河谷区，由于生态环境的脆弱性和长期人类活动的干扰和过度利用，导致植被严重退化，水土流失加剧，山地灾害频繁，干旱化和荒漠化趋势明显。这种趋势若不能遏制，将严重阻碍区域社会经济的快速协调发展，并且威胁成都平原地区的发展和长江中下游地区的生态安全。因而开展干旱河谷生态恢复研究成为解决这些问题的关键。水分匮乏是限制干旱河谷生态恢复的关键因子，在全球气候变化的背景下，干旱胁迫在区域尺度上可能会更加严重，并使干旱河谷的生态环境更加恶化。因此，深入研究干旱河谷乡土植物对干旱胁迫的响应和适应机理，具有非常重要的理论和实践意义。 本论文以岷江上游干旱河谷的三种乡土豆科灌木，白刺花（Sophora davidii）、小马鞍羊蹄甲（Bauhinia faberi var. microphylla）和小雀花（Campylotropics polyantha）理论和实践意义。为研究对象，在人工控制条件下设计了4-5个连续性干旱胁迫处理，系统地研究了灌木幼苗的生长、生物量积累和水分利用效率（WUE）、形态结构和生理过程等对干旱胁迫的反应，揭示了幼苗的干旱适应能力及种间差异。主要研究结论如下： 1) 灌木生长和繁殖对干旱胁迫的反应 在干旱胁迫下，幼苗生长速率显著减小，叶片衰老和脱落比率增大，这些变化随着胁迫强度的增加具有累积效应。叶片比茎对干旱胁迫的反应更敏感。在严重干旱胁迫下，幼苗的有性繁殖被限制，但在中等程度干旱胁迫下，幼苗的有性繁殖能力被提高。 2) 灌木生物量积累及其分配和WUE对干旱胁迫的反应 在干旱胁迫下，灌木各器官的生物量都显著减小，但是生物量的分配侧重于地下部分，使得根茎比在干旱条件下增大。幼苗的耗水量（WU）随着干旱胁迫的增加而显著减少。白刺花和小马鞍羊蹄甲WUE在干旱胁迫下降低；小雀花的WUE在中等干旱胁迫下升高。 3) 灌木叶片结构特征对干旱胁迫反应 白刺花叶片具有较为典型的旱生型结构，而小马鞍羊蹄甲和小雀花则为中生型结构。在1至2年的干旱胁迫下，灌木叶片结构组成未发生本质性的改变，主要是细胞大小的变化。在中等和严重干旱胁迫下，叶肉组织厚度明显减小；并且气孔和表皮细胞面积也显著减小，气孔和表皮细胞密度显著增大；叶肉细胞层数、P/S值、表皮厚度等无显著变化。 4) 灌木对干旱胁迫的生理响应 气体交换参数和叶片相对含水量（RWC）在中等干旱胁迫下发生了明显的改变，而叶绿素荧光参数和光合色素含量在严重干旱胁迫下才发生显著变化。在干旱胁迫下，净光合作用速率（Pn）、气孔导度（gs）和RWC呈下降趋势，而叶片温度（Tl）呈增加趋势，蒸腾速率（Tr）的变化不明显。除了日最大Pn减小以外，干旱胁迫对气体交换参数的日变化无显著影响，但是对光合-光响应曲线有显著的影响，使有效光合时间缩短。在严重干旱胁迫下光系统受到损害而代谢减弱，PSⅡ中心的内禀光能转换效率（Fv/Fm）、量子产量（Yield）、光化学淬灭参数（qP）显著降低，而非光化学淬灭参数（NPQ）明显增加。气孔限制和非气孔限制对Pn的影响与干旱胁迫强度有关。在中度胁迫下，气孔限制起主导作用，在严重胁迫下非气孔限制起主导作用，40% FC水分条件可能是灌木由气孔限制向非气孔限制的转折点。 5) 灌木对干旱胁迫的适应能力及其种间差异 三种灌木对干旱胁迫具有较好的适应能力，即使在20% FC，幼苗未因干旱胁迫III而死亡；80% FC适宜于幼苗生长。白刺花生长速率慢，耗水量较少，具有较强的耐旱和耐贫瘠能力，并具有干旱忍受机制，能够在较干旱的环境中定居和生长。小马鞍羊蹄甲和小雀花，生长快，水分消耗量较大，尤其是小雀花，对干旱胁迫的忍受能力较弱，具有干旱回避机制，因而适宜于在较为湿润的生境中生长。综合分析表明，生长速率较慢的物种抗旱能力较强，其更适宜于作为干旱地区植被恢复物种。 Drought is often a key factor limiting plant establishment, growth and distribution inmany regions of the world. The harsh environmental conditions and long-termanthropogenic disturbance had resulted in habitat destruction in the dry valley ofMinjiang river, southwest China. Recently, it tended to be more severe on the vegetationdegradation, soil erosion and water loss, natural disaster, as well as desertification, whichimpact on regional booming economy and harmonious development, and would be verydangerous to the environmental security in the middle and lower reaches of Yangzi River.Therefore, ecological restoration in the dry valley is one of the vital tasks in China. Waterdeficit is known to affect adversely vegetation restoration in this place. Moreover, in thecontext of climate change, an increased frequency of drought stress might occur at aregional scale in the dry valleys of Minjiang River. The selection of appropriate plantingspecies for vegetation restoration in regard to regional conditions is an important issue atpresent and in further. The research on responses of indigenous species to drought stresscould provide insights into the improvement of the vegetation restoration in the dry valleys of Minjiang River. In this paper, the responses of three indigenous leguminous shrubs, Sophora davidii,Bauhinia faberi var. microphylla and Campylotropics polyantha, to various soil watersupplies were studied in order to assess drought tolerance of seedlings, and to compare interspecific differences in seedlings’ responses to drought stress. The results were as follows: 1 Growth and reproduction of shrubs in response to drought stress Seedling growth reduced significantly while leaf senescence accelerated underdrought stress, the cumulative responses to prolonged drought were found. The capacityfor reproduction was limited by severe drought stress, and improved by moderate droughtstress. Leaf responses were more sensitive than shoot to various water supplies. 2 WUE, biomass production and its partitioning of shrubs in response to drought stress Drought stress reduced significantly the total dry mass and their components ofseedlings, and altered more biomass allocation to root system, showing higher R/S ratiounder drought. Water use (WU) and water-use efficiency (WUE) of both S. davidii and B.faberi var. microphylla declined strongly with drought stress. The WU C. polyantha ofalso declined with drought stress, but WUE improved under moderate drought stress. 3 Anatomical characteristics and ultrastructures of leaves in response to drought stress There were xeromorphic for S. davidii leaves and mesomorphic for B. faberi var.microphylla and C. polyantha at the all water supplies. The foundational changes in leafstructures were not found with drought stress. However, mesophyll thickness, the areas ofstomatal and epidermis reduced slightly while the densities of stomatal and epidermisincreased under severe drought stress. Variations in these parameters could mainly be duoto cell size. Other structures did not displayed significant changes with drought stress. 4 Physiological responses of shrubs to drought stress The gas exchange parameters and leaf relative water content (RWC) were affectedby moderate stress, while chlorophyll fluorescence and chlorophyll content were onlyaffected by severe stress. Drought stress decreased net photosynthesis rate (Pn), stomatalconductance, light-use efficiency and RWC, and increased leaf temperature. Therespiration rates (Tr) were kept within a narrower range than Pn, resulting in aprogressively increased instantaneous water use effiecency (WUEi) under drought stress.Moreover, drought stress also affected the response curve of Pn to RAR, there was adepression light saturation point (Lsat) and maximum Pn (Pnmax) for moderate andsevere stressed seedling. However, diurnal changes of gas exchange parameters did notdiffer among water supplies although maximum daily Pn declined under severe stress.VISevere stress reduced Fv/Fm, Yield and qP while increased NPQ and chlorophyll content.Photosynthetic activity decreased during drought stress period due to stomatal andnon-stomatal limitations. The relative contribution of these limitations was associatedwith the severity of stress. The limitation to Pn was caused mainly by stomatal limitationunder moderate drought stress, and by the predominance of non-stomatal limitation undersevere stress. In this case, 40% FC water supply may be a non-stomatal limitation 5 Interspecific differences in drought tolerance of shrubs Three shrubs exhibited good performance throughout the experiment process, evenif at 20% FC treatment there were no any seedlings died, 80% FC water supply wassuitable for their establishment and growth. S. davidii minimized their water loss byreducing total leaf area and growth rate, as well as maintained higher RWC and Pncompared to the other two species under drought stress, thus they might be more tolerantto the drought stress than the other two species. On the contrary, it was found that C.polyantha and B. faberi var. microphylla had higher water loss because of their stomatalconductance and higher leaf area ratios. They reduced water loss with shedding theirleaves and changing leaf orientation under drought stress. Based on their responses, thestudied species could be categorized into two: (1) S. davidii with a tolerance mechanismin response to drought stress; (2) C. polyantha and B. faberi var. microphylla withdrought avoidance mechanism. These results indicated that slow-growing shrub speciesare better adapted to drought stress than intermediate or fast-growing species in present orpredicted drought conditions. Therefore, selecting rapid-growing species might leavethese seedlings relatively at a risk of extreme drought.

水分梯度上不同灌木生长对干旱河谷土壤理化和生物学性质的影响

岷江上游干旱河谷区水土流失强烈，地质灾害频繁，生态环境十分脆弱，而土壤条件恶劣（水分不足和养分缺乏）是阻碍该区植被恢复的关键因子，因此研究水分和乡土灌木生长对土壤的影响对该区的生态恢复具有指导意义。本文通过定点模拟实验，选取三种优势豆科灌木为研究对象，分别是白刺花（Sophora davidii）、小马鞍羊蹄甲（Bauhinia faberi var. microphylla）和小雀花（Campylotropics polyantha），设置5 个水分梯度，分别为100%、80%、60%、40%和20%田间持水量（FC），对栽种植物与不种植物下土壤理化性质和酶活性进行测定分析，系统比较和研究了不同水分条件和不同乡土灌木生长对干旱河谷区土壤结构、养分循环、酶活性以及微生物量的影响。主要结果如下：1. 无论生长植物与否，土壤的毛管持水量和毛管孔隙度都随着水分含量的减少而降低，最大持水量、总孔隙度和容重变化不大，相应地，土壤中的非毛管孔隙随含水量的减少而升高。各水分条件下，种植植物的毛管持水量和毛管孔隙度低于无植物生长的土壤，非毛管孔隙度相应地高于无植物土壤。土壤含水量在100%－40% FC 时，三种豆科灌木的毛管持水量和毛管孔隙度存在差异，而20% FC 条件下，三种豆科灌木土壤的物理性质基本相同。2. 水分胁迫影响土壤中养分的矿化和积累，主要表现在降低了水溶性碳和铵态氮的含量，中等程度胁迫时（60% FC）促进了有机碳和硝态氮的富集，对速效钾和有效磷没有明显作用。种植豆科灌木后各水分梯度上都增加了有机碳、铵态氮、速效钾和有效磷的积累。增加程度上三种豆科灌木间有一定差异，对于土壤有机碳总量，种植白刺花和小马鞍羊蹄甲明显高于小雀花，同样的情况还出现在铵态氮和速效钾上，但是对于有效磷，种植小雀花后的增加程度则明显高于白刺花和小马鞍羊蹄甲。种植豆科灌木不仅增加了土壤养分的相对含量，也改变了其在水分梯度上的变化趋势及其变化幅度，这种作用主要体现在碳元素和氮元素上。3. 无植物生长时脲酶活性随水分含量的减少而升高，水分胁迫对磷酸酶和过氧化氢酶的作用不显著，蔗糖酶也保持在相对较高的水平。种植植物后，蔗糖酶、磷酸酶活性与无植物时相比有较大幅度的提高，种植白刺花的脲酶活性也升高，其升高的程度在不同水分含量时不同。种植植物还降低了酶活性在水分梯度上的变幅，使之在水分梯度间的差异显著性降低。脲酶活性在指示土壤性质改变方面是较敏感的指标，其它三种酶在不同植物间的差异不明显。4. 在无植物生长时，中等程度的水分胁迫（60% FC）提高了土壤微生物量碳含量，过高或过低的土壤水分均不利于微生物碳的积累。种植小马鞍羊蹄甲后微生物量碳在水分梯度上的变化趋势与无植物生长时一致，而种植白刺花和小雀花后微生物量碳随着水分含量的减少而降低。不同种类植物的微生物量碳在水分梯度上的变化特征也不同，100% FC 条件下三种植物间没有差异，80%和60% FC 条件下小马鞍羊蹄甲显著高于白刺花和小雀花，40%和20% FC 条件下白刺花和小马鞍羊蹄甲也显著高于小雀花，说明不同种类植物随着干旱胁迫程度的加深微生物量碳的降低幅度不同，在极度干旱时，白刺花和小马鞍羊蹄甲土壤依然保持了较高的微生物活性，而小雀花土壤微生物量则明显下降。The dry valley of the upper reaches of the Minjiang River is seriously degradedmountain ecosystem. It was endangered by extremely soil lost and frequentlygeological disaster. Previous studies showed that short of water and nutrients in soilwas the principal limiting factors of vegetation restoration in this area. The typical soiland three dominant leguminous shrubs Sophora davidii, Bauhinia faberi var.microphylla and Campylotropics polyantha in upper reaches of arid Minjiang Rivervalley were considered as experimental material. Two-month old seedlings of eachspecies were exposed to five water supplies (100%, 80%, 60%, 40% and 20% waterfield capacity (FC)) in a temperature and light-controlled greenhouse. Afterthree-month water treatment, soil physiochemical variables and soil microbialactivities were determined by conventional methods. The main results showed that:1. Soil capillary capacity and capillary porosity decreased along water supplyregimes in all treatments, while saturated water capacity, total porosity and bulkdensity kept in a relatively stable level, as a result, the non-capillary porosity andcapacity increased with decrease of water supply. Compared to non-planted soil, theplant-soil systems had a higher non-capillary porosity and capacity, suggestingappropriate oxygen was present in soil to maintain the living of microorganism. Soilof three type shrub species shared the same capillary capacity and capillary porosityunder 20% FC.2. Water soluble carbon and NH4+-N decreased in response to water stress, whiletotal organic carbon and NO3--N promoted by moderate water stress and inhibited by 100% and 20% FC. Total organic carbon, NH4+-N, rapidly available K and availableP increased after the planting of leguminous shrubs in five water supply regimescompared to non-planted soil. For TOC, NH4+-N and rapidly available K, thepromotion effect was higher in S. davidii and B. faberi var. microphylla than C.polyantha planted soil, while available P displayed the opposite side. The planting ofshrubs also reduced the variance of observed traits along water supply gradients.3. Drought stress increased urease activity in non-planted soil, while insignificantdifferences were observed in phosphatase and catalase activity among five watersupply regimes. The planting of leguminous shrubs facilitated the β-glucosidase andphosphatase activity compared to the non-planted soil. It also reduced the variance ofenzyme activity along water supply gradients. Urease was more sensitive to waterstress than other three enzymes.4. Soil water content significantly affected microbial biomass carbon andCmic:Corg. S. davidii and B. faberi var. microphylla showed more drought toleranceability than C. polyantha, attributing not only to their relatively smaller variance ofmicrobial biomass carbon along soil water supply gradients, but also to the highlevel of microbial activity under severe water stress. S. davidii and B. faberi var.microphylla benefited reproduction of soil microorganism at 60%-80% FC, whilesevere drought limited it due to the competition of water and nutrients between plantand soil microorganism.

A new species of Scolelepis (Polychaeta: Spionidae) from sandy beaches in China, with a review of Chinese Scolelepis species

A new species of Spionidae, Scolelepis (Scolelepis) daphoinos sp. nov., is described and illustrated from northern China seas. The species was frequently encountered and highly abundant on sand beaches, but rarely found in subtidal areas. It was formerly misidentified as S. (S.) squamata (Muller, 1806) in China but differed from the latter species in several details in morphology, including the presence of obvious reddish pigmentation patches and the absence of unidentate hooded hooks. Another two Scolelepis species, S. (S.) lingulata Imajima, 1992 and S. (S.) variegata Imajima, 1992, are reported for the first time from Chinese waters. Two species of Scolelepis, S. (S.) globosa Wu & Chen, 1964 and S. (S.) lefebvrei (Gravier, 1905), were reported previously; therefore, five species in this genus are known from China in total. A key to all Scolelepis species from Chinese waters is provided.