羊草生殖生物学研究

羊草 (Leymus chinensis (Trin.) Tzvel. ) ，隶属禾本科赖草属，是欧亚大陆草原区东部的重要草原建群种之一。羊草是牧草之王，属于我国有比较优势的战略性生物资源，对我国北方畜牧业发展以及保护生态环境均具有重要作用。 羊草较弱的有性生殖特性限制了其应用，本文从实验生物学角度，研究了羊草有性生殖的基本特点，并试图通过现代分子生物学手段探讨自交不亲和性的有关机理。本论文的主要结果如下： 1. 实验发现羊草具有自交不亲和性。以6 个羊草居群为材料，测定得知开放授粉时的结实率在6.5% - 56.7%之间，自交时结实率为0.6% - 4.3%，差异极其显著; FDA 染色法检测结果显示羊草成熟花药中有活性的花粉达到92.2% 以上;在发育时间顺序和空间结构上，羊草雌蕊、雄蕊适于异花和自花授粉;花粉柱头亲和性实验表明，自交花粉只有5.5%-11.7% 是亲和的，杂交花粉亲和率达到了60.0%-84.8%，说明自交花粉在柱头上萌发受到抑制，其次，荧光显微镜还观察到“不亲和花粉”在进入柱头后生长缓慢，或停止延伸。 2. 初步确定羊草自交不亲和性具有配子体型遗传特点。以不同居群羊草杂交后的姊妹系作为实验材料，观察到自交组合的亲和率变幅为0 % - 6.9 %，杂交组合的亲和率具有连续性变异和变幅较宽的特点（47.5％ - 96.0 %），且正反交结果具有一定的一致性（88.2％），表现出配子体遗传特性。 3. 羊草居群内结实率存在一定变异。以羊草单株为单位分别进行自交、随机互交和开放授粉，结果显示三者的平均结实率分别为4.6%，18.1% 和35.7%，株间的变异系数分别为33.4%，21.2%和17.1%，这些株间的变异均达到统计上的显著差异;同时羊草自交、杂交和开放授粉之间具有一定的相关性，显示羊草的这种株间差异与株系本身的生理特性相关。 4. 分离了羊草硫氧化还原蛋白 H 基因(ThioLc)并对其功能进行了分析。克隆了ThioLc全长和cDNA序列。序列分析结果显示，DNA全长2257 bp，包括3 个内含子和4 个外显子，与水稻Thio h 的cDNA 序列相比，具有 32.0% 的同源性;Southern 杂交显示 ThioLc 在羊草基因组中是单拷贝;Northern 杂交显示 ThioLc 在羊草根、茎、叶和幼小的雌蕊中没有表达, 在成熟雌蕊和幼小的花粉中微量表达, 在成熟花粉中大量表达，说明分离的羊草硫氧化还原蛋白H 基因具有花粉特异表达特点。 5. 原核表达的ThioLc 蛋白具有较高的催化活性。构建了ThioLc 基因的原核表达载体，检测证明ThioLc基因在大肠杆菌中正常表达;提取表达蛋白，纯化，用胰岛素和二硫苏糖醇反应体系进行硫氧化还原蛋白的催化作用反应，结果表明表达的蛋白具有催化活性。这一结果为进一步搜寻靶向蛋白和研究该蛋白的结构、功能和作用方式奠定了基础。

Aquatic resource conservation - The first Yangtze finless porpoise successfully born in captivity

Background. As the sole freshwater subspecies of finless porpoise (Neophocaena phocaenoides), the Yangtze finless porpoise (N. p. asiaeorientalis) lives only in the middle and lower reaches of the Yangtze River and its appended Poyang and Dongting Lakes. As a result of human activity on the river, including over and illegal fishing, pollution, transportation and dam construction, the population of Yangtze finless porpoises has been steadily and rapidly decreasing during the past several decades, which leads the animal to be endangered. Methods. For saving this unique animal from extinction, three corresponding measures, in situ conservation, ex situ conservation, and intensifying breeding research in captivity, were proposed and have been implemented since the 1980s. Results. After successfully rearing the animals in captivity for almost nine years, the first Yangtze finless porpoise was successfully born in captivity on July 5, 2005. The calf is male, with a body length of 69 cm. This is the first freshwater cetacean ever born in captivity. Conclusion. The successful birth of this calf confirms that it is possible to breed the Yangtze finless porpoise in captivity. Furthermore, this will greatly benefit the conservation efforts, and also greatly bolster our on-going efforts to study the reproductive biology of these animals. Recommendation. More studies and efforts are expected to establish a sustainable, captive colony of the Yangtze finless porpoise, which will not only greatly expand our knowledge about the reproduction biology of this animal, but also help to redeem the wild population through a careful yearly 'soft releasing' process.

鼠尾藻的生理生态和繁殖生物学研究

鼠尾藻 [Sargassum thunbergii (Mert.) O’Kunte] 是北太平洋西部特有的一种暖温性大型经济褐藻，在我国沿海地区北起辽东半岛南至雷州半岛的硇州岛，都有广泛分布。鼠尾藻在食品和医药领域的应用开发在我国有悠久历史。近年来，随着水产动物养殖业迅速发展，其应用领域得到了进一步拓展，发现鼠尾藻是海参、鲍鱼等水产动物的优质天然饵料。 受海参和鲍鱼产业发展影响，鼠尾藻的市场需求量成倍增加。目前鼠尾藻的供应全部依赖自然资源。掠夺式的采收方式导致鼠尾藻的野生资源遭到了很大破坏，种群规模和数量不断减小，生态环境也发生了变化。近年来鼠尾藻供需矛盾已经非常突出，如何保护和有效利用该海藻资源已经成为海藻科研和产业界的一个重要话题。 以往对鼠尾藻的生态学和人工育苗技术方面的比较深入的研究工作很少，许多基础科学问题研究不够，人工繁殖技术没有很好地掌握，已经不能满足产业发展需求并限制了鼠尾藻应用价值的发挥。为此，有必要系统性研究鼠尾藻原生态环境、野生状态下生长发育的年际交替规律、鼠尾藻有性生殖过程、人工育苗方法途径、鼠尾藻吸收无机营养能力、以及在调控藻际菌群能力等基础科学技术问题，带动鼠尾藻饵料规模的良性健康扩大，并进一步推动海参和鲍鱼等水产业的发展。 本文以青岛汇泉角的鼠尾藻为基本实验材料，通过野外实地周年观察并结合实验室工作，对上述问题初步进行了初步探索，主要获得如下结果： （1）在青岛地区海滨岩石地带，全年都可观察到鼠尾藻藻体，藻体的形态大小和生理发育状况随季节变化表现出很大的差别性。夏季藻体黄褐色，初生分枝伸展，有次生分枝、生殖枝和气囊的发育，而冬季的藻体黑褐色，初生分枝粗短，没有次生分枝、生殖枝和气囊的发育。6月鼠尾藻出现有性生殖器官生殖托，到7月生殖托发育成熟，7月藻体最长，平均在50厘米左右，11月可观察到有性生殖和假根产生的幼苗。假根营养繁殖再生苗是鼠尾藻种群新苗源的主体，在维持种群自然繁衍和发展中起主导作用。 （2）有性生殖过程是鼠尾藻个体发育的大事件，精卵结合产生幼苗的潜力巨大，是开展未来规模化人工育苗的希望依托。鼠尾藻初生分枝长度和外形株间差异较大，每株鼠尾藻平均大约有20个初生分枝，约一半初生分枝可发育成熟，充分发育的初生分枝可产生30－50个次生分枝，每个次生分枝上又有大约20－30个生殖枝，每个生殖枝上生殖托数量一般为1－3个，雌托长度大多在3－5mm，雄托长度在10－15mm。生殖托的成熟度由基部向顶端递减。一个雌托排卵量平均为400多粒。以此计算，每株雌性鼠尾藻在生殖季节可产生200多万个卵。 卵从生殖窝孔排出后粘附于雌托表面，雄托排精时间稍晚于雌托挂卵时间，精子游动到卵周围与之结合完成受精过程。受精卵大约在受精后1小时分裂为两个大小相近的细胞，2小时后再进行第二次分裂，其中一个细胞横裂为两个细胞：一个细胞较大，另一个细胞较小，另一个细胞的分裂面与第一次分裂垂直，等分为两个细胞，随后细胞不断分裂。在受精后48小时左右，胚苗一端产生出假根。胚苗从雌托脱落的第一周内，尽管细胞数量增加较快，但新细胞体积减小，使得胚苗的体积增加不多。从第二周开始幼苗的体积出现明显的增加。此时，无机营养盐的有效补充对鼠尾藻幼苗培养长大是十分必要的。下海后的管理是保障幼苗成活的关键，此时常常发生脱苗现象。 （3）鼠尾藻对无机氮、磷都有较强的吸收能力，鼠尾藻从海水中吸收无机氮和无机磷的能力分别为75.1mg/kg/day和36.2mg/kg/day，鼠尾藻对氮、磷吸收的质量浓度比例接近5：1。 （4）鼠尾藻具有调节藻际微生物种群的能力，鼠尾藻对弧菌和总异养菌菌群都有一定程度的抑制作用，研究发现鼠尾藻抑制弧菌的能力存在种的差异，其中对鳗弧菌65#显示的抑制作用最强，鼠尾藻的抑菌能力的强弱还与其生理状态的好坏有显著关系。 通过上述研究结果，我们认为：鼠尾藻通过有性生殖过程培育种苗在理论上具有可行性，发展潜力很大。而解决幼苗后期培养中的脱苗问题、提高苗的成活率是下一步需要研究攻克的关键技术；假根营养繁殖再生苗源是维持该藻自然种群的主要方式，也是目前开展人工养殖获得健壮苗种的重要途径，连根采收方式对鼠尾藻野生资源破坏严重；建议野生鼠尾藻在7月底到8月初（水温27℃左右）采收（此时藻体生物量接近最大、并完成生殖过程），采用剪切方式保留假根和幼小的初生枝收获，该方式还可以应用到收获人工筏式养殖的鼠尾藻中，这对保证假根的完整性，大量获得下一个栽培季节的健壮新苗有实际意义；建议进一步开发鼠尾藻吸收富营养化物质、净化养殖环境水质的能力，以及对弧菌和总异养菌的抑制能力，建立海参、鲍鱼和鼠尾藻生态互补、环境友好的养殖模式，控制病害发生，提高水产品产量和质量。

Adaptive evolution of SCML1 in primates, a gene involved in male reproduction

Background: Genes involved in male reproduction are often the targets of natural and/or sexual selection. SCML1 is a recently identified X-linked gene with preferential expression in testis. To test whether SCML1 is the target of selection in primates, we

New insights to the molecular phylogenetics and generic assessment in the Rhacophoridae (Amphibia: Anura) based on five nuclear and three mitochondrial genes, with comments on the evolution of reproduction

The phylogenetic relationships among 12 genera of treefrogs (Family, Rhacophoridae), were investigated based on a large sequence data set, including five nuclear (brain-derived neurotrophic factor, proopiomelanocortin, recombination activating gene 1, tyr

Seasonal pattern of reproduction of Hizikia fusiformis (Sargassaceae, Phaeophyta) from Nanao Island, Shantou, China

The maturation pattern of sexual reproduction in Hizikia fusiformis (Harvey) Okamura (Sargassaceae, Phaeaophyta) was examined in 2003 at Yunao Bay, Nanao Island, Shantou, China. Maturation began in mid-April (seawater temperature 19-21 degrees C), reached the peak in mid-May (maturation rate ca. 70%, and seawater temperature 23.5-25 degrees C) and finished in late-June (seawater temperature 27.5-30 degrees C). The Hizikia plants continued to gain the length from the beginning of maturation season to reach a maximum mean length of 34.8 cm in mid-May, after which the mean length was reduced drastically due to the senescence and rupture of the larger plants in size. The major portion of the mature plants belonged to the larger plants between April and May, but to the smaller ones in June. It is suggested that the plant must achieve a critical size before reproductive maturation occurred. There was a positive relationship between the number of receptacles (NR), as well as the reproductive allocation (RA), and the plant size of Hizikia population, with the recorded maximum values of NR and RA being 1220 and 64.3% respectively, for a single plant.

Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress

Toxicity of many waterborne organic contaminants to aquatic organisms is mediated through oxidative damages resulting from the production of reactive oxygen species (ROS). Using duroquinone as a model ROS inducer, we carried out in vitro and in vivo experiments to test the hypothesis that reproduction in common carp (Cyprinus carpio) can be impaired through oxidative damage of their spermatozoa. In vitro exposure of fish spermatozoa to 0, 12.5, 25, 50, 100 and 200 mu M duroquinone for 2 h showed a significant increase in the level of ROS in a dose-dependant manner. Sperm motility was significantly reduced in all exposure groups, but lipid peroxidation (LPO) and DNA strand break (measured by comet assay) were only enhanced at 50 mu M and above. A significant decrease in subsequent hatching rate was recorded in all the exposure groups, despite fertilization rate was not affected. In the in vivo experiment, spermatozoa were collected 24 and 72 h after fish received intra-peritoneal injections of 1.0 and 10 mg kg(-1) body weight duroquinone. DNA damage was clearly evident in spermatozoa of all treatment groups after 72 h exposure, and ROS was significantly enhanced in the high concentration group. LPO however, remained unchanged in both treatment groups. The overall results of both our in vitro and in vivo experiments demonstrated that duroquinone can induce ROS production in spermatozoa, which may impair sperm quality and subsequently reproductive success through oxidative stress. (c) 2006 Elsevier B.V. All rights reserved.

Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp (Carassius auratus gibelio bloch) as revealed by RAPD assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp.

Control of reproduction rhythmicity by environmental and endogenous signals in Ulva pseudocurvata (Chlorophyta)

A field population of Ulva pseudocurvata Koeman et C. Hoek (hereafter termed Ulva) at Sylt Island (North Sea, Germany) exhibited biweekly peaks of gametophytic reproduction during the colder seasons and approximately weekly peaks during summer. The reproductive events lasted 1-5 d and were separated from each other by purely vegetative phases. Under constant conditions in the laboratory, a free-running rhythm was observed with reproductive peaks occurring approximately every 7 d. When artificial moonlight was provided every 4 weeks, fewer reproductive events occurred, and the reproductive rhythm became synchronized to the environmental artificial moonlight rhythm. In the laboratory, apical disks were entirely converted into reproductive tissue after 8 d cultivation, while almost all basal disks stayed vegetative, which prevented the entire loss of the vegetative thallus during reproductive events. Seasonal size reduction of the thallus occurred from late autumn onward and was determined to be controlled by a genuine photoperiodic response, since size reduction could be induced from May onward by experimental short-day (SD) treatment but was prevented in a long-day (LD) or night-break regime (NB). A daily fine-tuning occurred with gamete release early in the morning at the first sign of daylight, following an obligatory dark ("night") period of at least 1 h duration. No release took place if the overnight dark phase was replaced by continuous light. Blue, green, or red light all triggered gamete release after a dark phase at an irradiance of 0.1 mu mol photons . m(-2) .s(-1), while 0.001 mu mol photons . m(-2) . s(-1) was equivalent to a dark control.

Cultivation of the brown alga Sargassum horneri: sexual reproduction and seedling production in tank culture under reduced solar irradiance in ambient temperature

As a large conspicuous intertidal brown alga, individuals of Sargassum horneri can reach a length of more than 7 m with a fresh weight of 3 kg along the coasts of the Eastern China Sea. The biomass of this alga as a vital component in coastal water ecology has been well documented. In recent years, a steady disappearance of the algal biomass along the once densely populated coastal areas of the Eastern China Sea has drawn attention in China. Efforts have been made to reconstruct the subtidal algal flora or even to grow the alga by use of long-lines. As part of the efforts to establish an efficient technique for producing seedlings of S. horneri, in this investigation a series of culture experiments were carried out in indoor raceway and rectangular tanks under reduced solar irradiance at ambient temperature in 2007-2008. The investigation demonstrated that: (1) sexual reproduction of S. horneri could be accelerated in elevated temperature and light climates, at least 3 months earlier than in the wild; (2) eggs of S. horneri had the potential to be fertilized up to 48 h, much longer than that of known related species; (3) suspension and fixed culture methods were both effective in growing the seedlings to the long-line cultivation stage; and (4) the life cycle of S. horneri in culture could be shortened to 4.5 months, thus establishing this alga as an appropriate model for investigating sexual reproduction in dieocious species of this genus.

Effects of single and mixed diatom diets on the reproduction of copepod Calanus sinicus

Under laboratory conditions, the potential influence of diatom diets on reproduction of zoo-plankton Calanus sinicus was studied. Four diatom diet ingredients: Skeletonema costatum (SC), Chaetoceros muelleri (CM), Phaeodactylum tricornutum (PT), diatom mixture (MIX) and a control diet: the flagellate Platymonas subordiformis (PS), were used at the same carbon concentrations of 2.0 mu g mL(-1) C. In a period of 17-day laboratory experiment, the effects of these algae diets on egg production and hatching success of the copepod Calanus sinicus were examined. The diets were analyzed for fatty acid content as an indicator of food quality. The results showed that the female survival of all treatments reached more than 80% except PT. Comparing to the initial value, egg production of Calanus sinicus was reduced in diatom diets (PT, CM), but remained in normal level in SC and MIX, indicating that some single diatom diets had a negative effect on the egg production of Calanus sinicus. Feeding with mixed food however can eliminate this negative effect. Among all the treatments, hatching success in filtered seawater was significantly higher than in algal exudates, indicating that not only diatoms but also other phytoplankton in certain concentration can release extracelluar substance that may inhibit eggs from hatching. Fatty acid analysis showed that both egg production rate and hatching success were negatively correlated to the ratio of 20:5 omega 3 and 14:0 in fatty acid composition.

Summer reproduction of the planktonic copepod Calanus sinicus in the Yellow Sea: influences of high surface temperature and cold bottom water

We have observed that Calanus sinicus retreated from neritic areas in the Yellow Sea and concentrated in the Yellow Sea Cold Bottom Water (YSCBW) area in summer. To investigate the summer reproductive strategy of C. sinicus in this situation, effects of high temperature on reproduction and hatching, as well as geographical variation of in situ egg production rate, were studied by onboard incubation in August 2001. Diel vertical migration (DVM) of females was investigated within and outside the YSCBW, respectively. Onboard incubation at 27 degrees C (i.e. surface temperature) resulted in lower fecundities than that at 9.8 and 12 degrees C (i.e. bottom temperature inside and outside the YSCBW) together with decreased hatching rates and increased naupliar malformation. Egg production was more active at stations outside the YSCBW than inside, where chlorophyll-a concentration was also relatively low. Females inside the YSCBW underwent DVM although they rarely entered the surface layer, but DVM was not observed outside the YSCBW. We conclude that surface temperature in summer has deleterious effects on C. sinicus egg production and hatching, and that it cannot reproduce successfully over the whole area. Inside the YSCBW, egg production is depressed by low food availability, while females outside suffer from high temperatures because of strong vertical mixing.

Seasonal variation of reproduction rates and body size of Calanus sinicus in the Southern Yellow Sea, China

Annual variations of egg production rate (EPR) and clutch size of Calanus sinicus, as well as body size of females (prosome length and dry weight), were investigated at a series of stations in the Southern Yellow Sea by onboard incubation. Calanus sinicus was spawning in all the 11 cruises investigated, and the annual variation of EPR was bimodal. Monthly average EPR was highest from May to July, respectively, 5.97, 5.36 and 6.30 eggs female(-1) d(-1), then decreased dramatically to only 1.37 eggs female(-1) d(-1) in August and attained the lowest 1.07 eggs female(-1) d(-1) in October. In November, average EPR increased again to 4.31 eggs female(-1) d(-1). Seasonal variation of clutch size was similar to EPR, except that it decreased gradually after August rather than dramatically as did EPR. Prosome length of females was maximum in May and minimum in October, but dry weight was highest in November. Monthly average EPR correlated better with prosome length than dry weight, while clutch size was rather determined by dry weight of females. It is suggested that egg production of C. sinicus was active during two discontinuous periods when both surface and bottom temperature fell into its favorite range (i.e. 10-23degreesC), and different reproductive strategies were adopted in these two reproductive peaks: other than the highest EPR, longer prosome length was also achieved by C. sinicus from May to July, while females in November developed shorter bodies but accumulated more energy for reproduction.

Spring and autumn reproduction of Calanus sinicus in the Yellow Sea

The fecundity, hatching success and naupliar survival of Calanus sinicus were studied in the Yellow Sea during research cruises in April and October 2006, with emphasis on the regulation of reproduction. During both cruises, the egg production rate (EPR) showed large spatial variations (0 to 25.4 eggs female(-1) d(-1)), generally coinciding with the food availability. In April, the abundant phytoplankton and ciliates in the study area supported active reproduction, which would probably initiate the annual population development. In October, females remained immature in the Yellow Sea Cold Water Mass, likely due to the unfavorable environment (poor food and low temperature). However, reproduction and recruitment were high in the neritic region, which may explain the local population recovery in late autumn. Hatching success varied markedly among stations in April (4 to 85%), whereas it was high overall in the neritic region during October (> 90%). Based on the potential recruitment rates, the spring recruitment would be more important for the annual population dynamics, Female gonad maturity, body size and lipid reserve were examined in relation to fecundity. Regression analyses suggested that the reproductive index (defined as the proportion of females with mature gonads) could be an indicator for the EPR of C. sinicus. Among the regulating factors, external food (ciliates and phytoplankton) seems essential for reproduction, whereas inner lipid reserve may mainly serve metabolic needs. Moreover, fecundity is positively related to body size but independent of temperature, which might exert indirect influences on reproduction.